Recent progress in electrochemical assessment of DNA based on nanostructured sensors

Lue Wang; Waye Zhang

doi:10.1007/s10544-025-00763-0

Recent progress in electrochemical assessment of DNA based on nanostructured sensors

Wang, Lue; Zhang, Waye 2025-07-12 00:00:00 Screening the amount of DNA closely related to early diagnosis of diseases or decoding information in target DNA sequences for biological medicine, infectious identification, or forensic analysis are highly essential in our daily life. This review provides clear understanding of nanostructured sensors (i.e., functionalized electrode-based sensors and nanopores) working for electrochemical assessment of DNA, along with their recent advances and unaddressed issues. Crucial con- stituents for sensor functionalization, electrochemical techniques, and electrodes, used in functionalized electrode-based sensors are briefly introduced, followed by analysis of using this type of sensors for DNA determination and the compari - son of performances such as dynamic ranges and detection limits with other similar works. Subsequently, nanopore sensors including porin-based and solid-state nanopores applied for DNA sequencing are the other interests of discussion in the review. Beyond the achievement of high-resolution DNA sequencing based on porins coupled with enzymatic components, commonly used methods to solid-state nanopore creation, practical use of solid-state nanopores in DNA analysis, and computational modeling for nucleobase pore-threading simulation are depicted in more detail. Finally, conclusions in rela- tion to recent advances and future developments are described. This work offers a powerful guideline for electrochemical assessment of DNA using either functionalized electrode-based sensors or nanopores, enabling scientific groups to have an entire picture upon electrochemical nanodevices used for DNA characterization. Keywords Electrochemical · DNA · Nanosensors 1 Introduction developed. The detection of DNA through electrochemical nanostructured sensors is more promising by virtue of rapid Deoxyribonucleic acid (DNA) molecules are basic constitu- responses, simple manipulation, device portability, cost ent units in most organisms. DNA sequences contain differ - effectiveness, and mass production (Ronkainen et al. 2010). ent but specific biological information, which are used to Electrochemical nanodevices applied for DNA monitor- guide various living activities. Electrochemical assessment ing usually contain functionalized electrode-based sensors of DNA molecules has a close association with genetic and nanopores. For functionalized electrode-based sensors, engineering, genomic studies, and structural prediction of outstanding characteristics of high conductivity and ade- proteins or proteinaceous molecules, as well as medical quate bioactive sites make the sensing interface appropri- diagnoses and foodstuff quality monitoring (Castro-Wal - ate to anchor enough ‘baits’ of target molecules (e.g., probe lace et al. 2017). To make determination more facile, accu- DNA strands), thereby reaching a sensitive detection. After rate, and sensitive, various sensing techniques have been forming double-helix structures with target complements, various impacts upon the electron pathway are immedi- ately triggered, such as electron transfer promotion due to intercalation of electrochemical tags (Fig. 1g, h), or the Waye Zhang other way around, charge transfer resistance (Fig. 1a, b) or [email protected] electrostatic repulsion (Fig. 1a, c) against redox couples, Department of Chemical Engineering, Swansea University, or enabling electrochemical tags away from the electrode Swansea SA1 8EN, UK interface (Fig. 1d, e, f), thereby causing electrical signals School of Pharmaceutical Sciences, Tsinghua University, linearly vary with concentrations of target analytes. As for Beijing 100084, China 1 3 36 Page 2 of 27 Biomedical Microdevices (2025) 27:36 measuring ensembles, the usage of three-electrode system electrode-based sensors. Normally, it contains a work- remains its dominance in concentration determination of ing electrode (e.g., DNA probe covered chips), a counter target molecules after the integration with functionalized electrode, sometimes also called auxiliary electrode (e.g., 1 3 Biomedical Microdevices (2025) 27:36 Page 3 of 27 36 Fig. 1 Functionalized electrode-based sensors for electrochemical base ordering threads through the constriction domain of a assessment of DNA: a electrode surface with DNA probes only, redox nanopore subjected to an electric field force. Many scientific couples move towards against less resistance; b electrode surface with probe-target DNA duplex, increased steric hinderance causes negative or clinical studies have been successfully conducted since correlation between DNA analyte and electrochemical response; c neg- the company of Oxford Nanopore Technologies (ONT) ative correlation caused by electrostatic repulsion between negatively first turned laboratorial DNA nanopore sequencing tech - charged redox couple and DNA sequence; d electrochemical tags niques into commercial applications. Versatile detecting adhere to electrode surface modified with DNA probes only; e negative correlation caused by increased distance between electrochemical tags of microRNA, biomarkers, and small proteinaceous sub- and electrode surface after probe-target DNA duplex formed; f nega- stances via the reading of DNA-barcoded probes is recently tive correlation caused by electrochemical tags away from electrode reported based on the MinION sequencing device, one surface owing to sequence cleavage; g electrode surface with DNA of nanopore products from the ONT, which significantly probes only, providing no room for electrochemical tag intercalation; h electrode surface with probe-target DNA duplex, electrochemical tags expands the practical use of nanopore technique (Koch et intercalated into structural gaps leading to positive correlation between al. 2023). To reach clear-cut ionic current signals, Mereuta DNA analyte and electrochemical response; i Porin-based sequencing, et al., reported a novel method to ssDNA sequencing based DNA sequence threads through nanopore at lower speed controlled by on the α-hemolysin (α-HL) nanopore, where negatively motor protein; j ssDNA passes through solid-state nanopore charged AuNPs coupled to charge neutral peptide nucleic platinum) and a reference electrode (e.g., Ag/AgCl). Latest acids (PNA) were applied to specifically recognize target advances about functionalized electrode-based sensors for ssDNA strands (Mereuta et al. 2020). Another approach to the electrochemical DNA assessment reveal that two-dimen- improving the signal fidelity of DNA is to develop porin- sional (2D) nanomaterials such as graphene oxide nanorib- based nanopores with dual constriction (Verren et al. bons (GONR) (Pareek et al. 2023), transition metal carbides 2020). However, only DNA homopolymer sequences were (e.g., MXene Ti C ) (Duan et al. 2023) are often introduced assessed through the system. Conventional DNA sequenc- 3 2 onto the electrode surface, aiming to offer tremendous room ing based on porins always introduces motor proteins such for DNA probe immobilization because of the increased as DNA polymerase or helicase to enable the DNA strand to surface-area-to-volume ratio. Furthermore, electrochemi- move at the pace of individual base when threading through cal sensors coupled with enzymatic components such as the nanopore. However, enzymatic components are limited clustered regularly interspaced short palindromic repeats by various ambient factors such as temperature, solution associated proteins (CRISPR-Cas 12a) (Duan et al. 2023; pH, or osmotic pressure, which are not very suitable for You et al. 2023), restriction endonuclease EcoRI (Meftah all environments. Facing this challenge, Qing and Bayley et al. 2023) have been receiving more interest owing to the reported a method to DNA identification via chemical step - remarkable enhancement of sensitivity. Although function- ping instead of enzyme-based sequencing (Qing and Bayley alized electrode-based sensors for electrochemical DNA 2021). Since the end of 2019, the COVID-19 pandemics determination are mostly used in fields of cancer biomarker have caused serious health threat to the people all over the monitoring, environmental protection, and food security, world, making the technique of DNA sequencing highly the monotonous function of concentration quantification useful for virus infection confirmation (Stüder et al. 2021). hinders this type of sensor further development. Beyond this application, nanopore-based DNA sequencing It is noteworthy that studies on the use of nanopores for can be also applied in analysis of single-molecule methyla- assessment of biological molecules also belong to the field of tion of cell-free DNA for early warning of cancer, or various electrochemical detection, however studies on porin-based classes of DNA methylation from microbiomes and bacteria nanopores themselves are often defined as electrophysiol - (Lau et al. 2023; Tourancheau et al. 2021). Although, up to ogy. The review pays more attention to actual application date, high resolution DNA sequencing has only been real- of nanopores rather than focusing on nanopores themselves. ized on porin-based nanopores (Fig. 1i), there are still a cou- Simple nanochannels coupled with electrophoretic forces ple of issues often occurring during the formation of lipid always make nanopores exhibiting stronger versatility over membrane. Like enzymes, the form of double-layered lipid other nanodevices. Many substances in fact can be electro- membrane also requires rigorous conditions such as appro- chemically detected using nanopores such as DNA (Laszlo priate temperature and humidity, while unproper parameters et al. 2014), ribonucleic acid (RNA) (Soneson et al. 2019), employed prone to result in failure of membrane formation. small molecules (Nestorovich et al. 2002; Fan et al. 2024; Additionally, porin-based nanopores are almost all trans- Sun et al. 2025), and peptides (i.e., precursors of proteins) membrane proteins, which periodically require complicated (Wanunu 2022). Sensing of DNA molecules based on nano- protein expression and purification for one-batch mass pro - pores, in most cases, is described as a more pertinent term as duction, leaving lengthy pre-preparation prior to the formal “DNA sequencing” since nucleobase-dependent ionic cur- sequencing assay. Solid-state nanopores, someday, may rent drops are created when a sequence of DNA with random replace porin-based nanopores owing to unique advantages 1 3 36 Page 4 of 27 Biomedical Microdevices (2025) 27:36 of low cost, simple fabrication, excellent reusability, good 1.1 Electrochemical assessment of DNA using stability, and free design on nanopore sizes (Fig. 1j) (Xue et functionalized electrode-based sensors al. 2020). The sophisticated porin preparation, lipid mem- brane coverage on a micro-aperture-containing support, and Functionalized electrode-based sensors are commonly used porin embedment are no longer required when using solid- in quantitative analyses of target DNA analytes either in a state nanopores, thus avoiding the risk of membrane bro- buffered saline or a real biological solution (Table 1). ken at any unexpected moment. Notwithstanding, the poor signal-to-noise ratio (SNR), lack of active sites for assigned 1.2 Functional or bio-functional modification on anchoring of biological components nearby a solid-state sensor surfaces nanopore, and difficulty in fabrication of solid-state nano - pores with atomic-grade consistency, all make this type of The functional or bio-functional modification with nano - nanopore not feasible yet for high-resolution DNA sequenc- structures or nanocomposites on electrode surfaces is con- ing. Scientific studies in relation to DNA sequencing based sidered as an indispensable fabrication process since it on solid-state nanopores currently concentrate on machine helps in enlarging the surface area for accommodation of learning or computer-assisted modeling to understand or bioactive sites, leading to improved sensitivity and speci- provide interpretation on DNA translocation through a solid- ficity in electrochemical sensing. Functional monomers state nanopore (Jena et al. 2022). Breakthroughs regarding such as 4-aminobenzoic acid (4-ABA) (Wan et al. 2023), practical use of solid-state nanopores are not too much. melamine (Mel), and glutamic acid (GA) (Ali et al. 2023), The nanopore developed by pulling a quartz capillary has are often introduced on the surface of electrode, creating received an increasing amount of attention in recent years a biocompatible coverage via an electro-polymerization due to cost effectiveness, simple fabrication, and potential for immobilization of DNA probes. As functional layers, for large-scale production. However, sectional diameters of self-assembled monolayers (SAMs) play a vital role in fab- these artificially conical nanopores are about 3 ~ 5 nm, or rication of electrochemical sensors as their multiple prop- even more, which are double- or multiple- fold wider than erties including hybridization promotion, non-specific site general ssDNA strands (Chen et al. 2021, 2023). Choud- blocking, parallel orientation of DNA strands, and electrode hary et al., created a nanofluidic system consisting of dual lifetime improvement (Rasanang et al. 2022; Bahner et al. nanopores for single-molecular DNA sequencing, aiming to 2018; Ting et al. 2009; Nguyen and Minteer 2015; Lee et al. study behaviors of DNA capturing and threading with high 2014). Commonly used SAMs include 11-mercaptoundeca- fidelity (Choudhary et al. 2020). While for high-resolution noic acid (MUA) (Garcia-Melo et al. 2023; Han et al. 2023) reading of DNA, the setup requires further development in and 6 carbon mercaptan (MCH) (Han et al. 2023). Carbon the pore size reduction from current 10 nm to 1 nm. nanomaterials are another class of important functional lay- Interestingly, the discrimination of target DNA from other ers for electrodes. Graphene oxide nanoribbons (GONR) interferents can be realized via both classes of nanostruc- (Pareek et al. 2023) are used for increasing the surface-area- tured sensors. Functionalized electrode-based sensors allow to-volume ratio are often covered on the electrode surface target DNA molecules to specifically bind to an electrode through an electrodeposition method. Functionalized carbon surface, yielding distinctively electrochemical responses black (f-CB) is a class of CB that contains organic groups that are always several orders of magnitude higher or lower (e.g., carboxylic group (Aamri et al. 2023)), which is usu- than those of other interferents. While nanopores monitor ally prepared into a dispersed solution drop-casted on sur- base-dependent ionic current blockages as DNA sequences face of electrodes serving as a highly conductive underlayer transverse through a nanopore, achieving target DNA rec- friendly to biomolecule binding. Similarly, oxidized gra- ognition by virtue of the unique readout different from other phene dispersion is also required to undergo a drop-casting interfering sequences. This review mainly focuses on two process to form a uniform layer on glassy carbon electrodes types of DNA nanostructured sensors (i.e., functionalized (GCE), offering a good environment for immobilization of electrode-based sensors or nanopores) in combination with biomolecules or other nanostructures (Bo et al. 2011). electrochemical techniques. Miscellaneous nanodevices In some cases, DNA probes are not able to directly attach prepared, along with their sensing performances such as to the functionalized electrode surface unless there is a dynamic ranges and detection limits are carefully analyzed, cross-linking agent involved such as 1-ethyl-3-(3-dimethyl with further development or prospects for two types of DNA aminopropyl) carbodiimide (EDC) coupled to N-hydroxy nanostructured sensors provided at the end of the review. succinimide (NHS) (Wan et al. 2023), disuccinimidyl suber- This work aims to offer study groups or companies a power - ate (DSS) (Ali et al. 2023), and 3,3’-dithiopropionic acid ful scientific support in electrochemical sensing based on (DPA) (Hushiarian et al. 2015). Multiplex linkers are also functionalized electrode-based sensors or nanopores. used in some studies allowing electrode surfaces to have 1 3 Biomedical Microdevices (2025) 27:36 Page 5 of 27 36 Table 1 Functionalized electrode-based sensors used for electrochemical assessment of DNA DNA analytes Electrode modification Electro- Dynamic ranges and Purposes for electrochemi- Refs chemical LODs (pM) cal assessment techniques ARGs GCE/4-ABA/EDC/NHS/probe DNA EIS 21.6 ~ 477.12 and 21.6 Pollutants monitoring Wan et al. 2023) cDNA LIG/C15 pDNA/23 random DNA SWV 0.1 ~ 10 and 0.057 DNA hybridization Bahri et al. quantification 2023) –3 4 –4 Target DNA AuNWE/MCH/HS-DNA SWV 10 ~ 10 and 4.8 × 10 Target DNA quantification Hua et al. 2018) 5 –3 Target DNA Graphene microfiber/ZnO NWs/probe EIS 0.01 ~ 10 and 3.3 × 10 DNA hybridization Zhang et al. DNA quantification 2019) Target DNA GCE/GO/PANIw/probe DNA DPV 2.12 ~ 2.12 × 10 and DNA-based diagnosis Bo et al. 2011) 0.325 Genomic AuE/PEDOT-PSS/AuNPs/DPA/DNA DPV 3,130 ~ 10 and 0.054 Detection of white rot Hushiarian et DNA reporter fungus al. 2015) RASSF1A SPCE/PT/anti-5mC antibody/methyl- DPV 0.01 ~ 5,000 and 0.002 DNA methylation screening Daneshpour et tumor sup- ated target strand al. 2016) pressor gene HPV-16 SPCE/Fe O /AuNPs/thiolated probe DPV 0.1 ~ 10 and 100 Early warning of cervical Rasouli et al. 3 4 DNA cancer 2023) –4 6 –4 HPV-16 ITO/GONR/Ag@AuNPs/probe DNA LSV and 10 ~ 10 and 10 Early warning of cervical Pareek et al. EIS cancer 2023) HPV-18 SPCE/MXene Ti C /AuNPs/ssDNA SWV 10 ~ 5 × 10 and 1.95 Early warning of cervical Duan et al. 3 2 cancer 2023) IL6 and SPCE/AuNPs/thiolated ODN probes DPV IL6: 0.1 ~ 10 and 0.048 Early warning of ovarian Meftah et al. TGFβ1 TGFβ1: 0.05 ~ 100 and cancer 2023) 0.017 –8 6 –8 Lung cancer AnE/SnO -QD-Au/ssDNA EIS 10 ~ 10 and 3.2 × 10 Early warning of lung Richard et al. biomarker cancer 2023) DNA p53 gene MGCE/Fe O /α-Fe O @Au/HS-PNA DPV 1 ~ 10 and 0.85 Early warning of human Liu et al. 2023) 3 4 2 3 cancer p53 gene Au/MUC/ssDNA CV 0.001 ~ 100 and Early warning of human Garcia-Melo et –4 1.75 × 10 cancer al. 2023) –9 6 –10 S. flexneri ITO/P-Mel/PGA/DSS/CP DPV 10 ~ 10 and 7.4 × 10 Food quality evaluation Ali et al. 2023) DNA –7 –2 stx1 and stx2 Si-Au IDEs/AuNPs/Cht-Au/probe SWV stx1: 10 ~ 10 and Food quality evaluation Wasiewska et –7 DNA 10 al. 2023) –7 –1 stx2: 10 ~ 10 and –7 5 6 Base G and A N/P/S doped graphene paper/dsDNA CV G: 4 × 10 ~ 4 × 10 and Exceptional biomarker Jagannathan et 1.5 × 10 detection al. 2023) 5 6 A: 4.4 × 10 ~ 4.4 × 10 and 2.38 × 10 5 7 Base G and A SPCE/f-CB/glass microbead with DPV G: 5 × 10 ~ 10 and Exceptional biomarker Aamri et al. probe DNA 2.8 × 10 detection 2023) 5 7 A: 5 × 10 ~ 2 × 10 and 1.78 × 10 4 7 4 r60 Au/MCH/MUA/NHS/DANP/probe EIS 10 ~ 10 and 10 Target DNA quantification Han et al. 2023) DNA –6 –5 KRAS G12D SPE/probe DNA CV 10 ~ 1 and 10 Target DNA quantification You et al. 2023) M. Smegmatis C223AT-SPE/MCP/16S rRNA probe EIS and - Detection of antibiotic Güzel et al. DNA target SWV susceptibility 2021) specific affinity for DNA probes such as NHS coupled (e.g., graphene) or other inorganic nanomaterials (Bahri with 2, 7-diamino-1, 8-naphthyridine (DANP) (Han et al. et al. 2023). Nanoparticles, another class of frequently 2023) that prefers adsorption of cytosine bulge structures. utilized nanostructures, are regarded as one of key com- Moreover, poly-cytosine (poly-C) DNA strands (e.g., ponents in surface biofunctionalization of electrochemi- C15 pDNA) are sometimes applied as a special bio-linker cal nanosensors. Gold nanoparticles (AuNPs) are mostly owing to the strong adsorption to carbon nanostructures used nanoparticles capable of introducing various bonds 1 3 36 Page 6 of 27 Biomedical Microdevices (2025) 27:36 such as the gold-thiol (Hua et al. 2018) or gold-streptavidin biochemical events as they usually comprise of a real part (Daneshpour et al. 2016) conjugation for covalently bond- (i.e., charge transfer resistance) and an imaginary part (i.e., ing with DNA probes. Interestingly, AuNPs in combina- capacitance). Electrochemical techniques for assessment of tion with other metal or metal oxide nanoparticles such as DNA on functionalized electrode-based sensors are mostly silver and ferroferric oxide nanoparticles (Fe O NPs) can voltammetric measurements such as cyclic voltammetry 3 4 form nanoparticle-based composites as silver coated gold (CV) (You et al. 2023; Garcia-Melo et al. 2023; Jagan- nanoparticles (Ag@AuNPs) (Pareek et al. 2023) and Fe O - nathan et al. 2023), differential pulse voltammetry (DPV) 3 4 Au core–shell nanoparticles (Rasouli et al. 2023), respec- (Meftah et al. 2023; Bo et al. 2011; Hushiarian et al. 2015; tively. The increased surface area allows more DNA probes Daneshpour et al. 2016; Rasouli et al. 2023; Liu et al. 2023; anchoring on the electrode functionalized with nanoparti- Ali et al. 2023; Aamri et al. 2023), square wave voltam- cle-based composites, compared with that of untreated elec- metry (SWV) (Duan et al. 2023; Bahri et al. 2023; Hua et trode surfaces. Owning a strong magnetic feature, ferroferric al. 2018; Wasiewska et al. 2023), and linear sweep voltam- oxide nanoparticles (Fe O NPs) can be also integrated with metry (LSV) (Pareek et al. 2023). As the typical technique 3 4 DNA probes such as magnetic nanoparticle-capture probes of impedimetric measurements, electrochemical impedance (MNP-Capture probes) (Hushiarian et al. 2015), which can spectroscopy (EIS) is another effective approach to charac - be used for target DNA enrichment. Quantum dots (QD) terizing DNA at trace amount (Pareek et al. 2023; Wan et such as SnO -QD (Richard et al. 2023), belong to a spe- al. 2023; Zhang et al. 2019; Richard et al. 2023; Han et al. cial class of nanoparticles with geometrical size less than 2023). The use of EIS recent years has received a growing 100 nm. They have been recently applied in the process of number of attentions owing to powerful properties of simple electrode fabrication to achieve a downsized device with program setting, rapid analysis, high sensitivity, and setup higher sensitivity and selectivity (Kolhe et al. 2023; Hansen miniaturization. Amperometric measurements in contrast et al. 2006). Nanowire-based structures play essential roles are less reported in the determination of DNA. This is prob- in electrochemical characterization of DNA molecules. ably due to the mechanism not suitable for low concentra- Sensing devices with polyaniline nanowires (PANIws) are tion monitoring. of particular interest, since polyaniline possesses strengths Electrodes are also the important component in electro- including straightforward fabrication, large surface area, chemical sensing systems as they play a role in electron and tunable conductivity. (Maynor et al. 2002; Wu and Bein transportation from bioactive interfaces to signal transfor- 1994; Noll et al. 1998) Zinc oxide nanowires (ZnO NWs), mation devices. For functionalized electrode-based sen- a nanomaterial with a broad band gap of 3.37 eV, recently sors, screen-printed carbon electrodes (SPCE) (Duan et al. has been applied in sensor fabrication (Cao et al. 2016; 2023; Meftah et al. 2023; Daneshpour et al. 2016; Rasouli Kong et al. 2009; Tereshchenko et al. 2016). The growth of et al. 2023; Aamri et al. 2023) or gold electrodes (Hua et al. ZnO NWs on various substrates can be easily achieved via 2018; Hushiarian et al. 2015; Richard et al. 2023; Garcia- a hydrothermal method (Zhang et al. 2019). Additionally, Melo et al. 2023; Han et al. 2023) are mostly used electrodes the positively charged surface is of non-negligible charac- for electrochemical detection of DNA. SPCEs are usually teristic upon ZnO NWs, which directly attaches negatively designed as “three route” pattern catering for the three- charged probe DNA strands to the surface through electro- electrode system, of which the sensing window is always static interaction (Zhang et al. 2019). with a concentric shape to maximumly increase the effective contacting area. Other than SPCEs, the screen-printing tech- 1.3 Electrochemical techniques and electrodes nique can be also utilized in fabrication of electrodes with gold, forming screen-printed gold electrodes (SPGE) (You Electrochemical techniques suitable for functionalized elec- et al. 2023). Polydimethylsiloxane (PDMS) films are some - trode-based sensors consist of voltammetric, amperometric times used to cover around the sensing window of SPCEs and impedimetric categories. Voltammetric measurements to form a cylindrical chamber avoiding the testing solution provide information of current variation over an assigned to spread out of the electrode edge (Fig. 2a) (Yuan et al. range of potential, where peak currents normally represent 2023). In comparison to simple gold electrodes, interdigi- oxidation or reduction level of signal indicators. Ampero- tated gold microelectrodes engraved at the surface of silicon metric measurements offer a picture of current variation chip (Si-Au IDEs) have sophisticated patterns capable of versus time, and information of tested concentrations can simultaneously detecting multiple DNA sequences (Fig. 2b) be directly indicated according to a series of current levels. (Wasiewska et al. 2023). A gold electrode coupled with a Impedimetric measurements can convert the actual micro- novel 2D material of graphitic carbon nitride (g-C N ) is 3 4 environment of electrode-solution interface into an equiva- recently reported for real-time determination of 5-hydroxy- lent circuit, of which impedance signals vividly reflect the methylcytosine (5hmC) in genomic DNA molecules. The 1 3 Biomedical Microdevices (2025) 27:36 Page 7 of 27 36 Fig. 2 Various electrodes used for electrochemical assessment of c AuE attached with g-C3N4. Image reprinted with permission from DNA: a SPCE electrode coupled with a PDMS cover forming a con- Imran et al. (2023); d LIG electrode integrated with a three-electrode fined sensing window. Image reprinted with permission from Yuan et electrochemical sensing system. Image reprinted with permission from al. (2023); b structure of Si-Au IDE along with the chip inserted in a Bahri et al. (2023) holder. Image reprinted with permission from Wasiewska et al. (2023); Fig. 3 Flexible electrodes used for electrochemical analysis of DNA: of wax-on-PET chips, along with its mass production and high flex - a preparation workflow of graphene paper electrodes. Image reprinted ibility. Image reprinted with permission from Das et al. (2023) with permission from Jagannathan et al. (2023); b fabrication process use of g-C N makes the sensor having highly selective terephthalate (PET) substrates subjected to wax patterning 3 4 “antennas” capturing target DNA sequences via hydrogen are made as an underlayer for deposition of AuNPs and gra- bonding (Fig. 2c) (Imran et al. 2023). Graphene-based elec- phene oxide (GO), yielding a multiplexed electrode for pep- trodes are another commonly used electrodes applied for tide nucleic acid (PNA) adsorption. The wax-on-PET chip electrochemical assessment of DNA. Laser-induced gra- presents good flexibility and can be massively produced phene (LIG) electrodes possess similar designs like SPCEs, from batch to batch (Fig. 3b) (Das et al. 2023). Other soft which are fabricated through a photothermal pyrolysis substrates such as indium tin oxide (ITO) (Ali et al. 2023) sourced from laser pulses (Fig. 2d) (Bahri et al. 2023). can also be made as flexible electrodes, however, ITO elec - Flexible electrodes recently have earned a great amount trodes used for electrochemical analysis of DNA are less of interest owing to their light weight, disposability, and reported because of the high price of the material. Interest- low cost. Heteroatom doped graphene electrodes coated ingly, there are also only a few reports on electrochemi- on a paper substrate have been used for rapid DNA detec- cal determination of DNA using glassy carbon electrodes tion, which the cost-effective preparation starts from corn (GCE), while GCEs are top-preferred electrodes in other cobs (Fig. 3a) (Jagannathan et al. 2023). Polyethylene 1 3 36 Page 8 of 27 Biomedical Microdevices (2025) 27:36 sensing applications (Wan et al. 2023; Bo et al. 2011; Liu a versatile nanoelectrode used for label-free monitoring et al. 2023). of lung cancer DNA biomarkers (Fig. 4b) (Richard et al. 2023). The use of AuNPs enables the electrode surface to 1.4 Negative correlation between electrochemical have a uniform coverage of DNA probes, compared with responses and target DNA concentrations the surface without AuNPs. Impedimetric responses pro- duced by the sensor were observed in an increasing behav- There are many studies in which resulting electrochemical ior as more target DNA molecules captured, reaching an –8 6 outcomes present a negative correlation with concentra- extremely wide detection range of 10 ~ 10 pM, coupled –10 tions of target DNA sequences. Such inversely proportional with an ultralow LOD of 7.4 × 10 pM. The probe-target relationship can be explained as the electrostatic resistance DNA combination presented the highest charge transfer or steric hinderance sourced from the probe-target DNA resistance over other groups including SnO -QD-Au only, hybridization against the charge transfer of redox ion pairs. probe, non-complementary, and single base mismatch Wan et al., prepared an electrochemical DNA sensor DNA, providing evidence of good selectivity. The sensor for sensitive determination of antibiotic-resistant bacteria also exhibited outstanding electrode reproducibility in the (ARG) or resistance genes (ARGs) (Fig. 4a) (Wan et al. charge transfer measurement for both single-stranded DNA 2023). An increasing impedance against the electron trans- (ssDNA) and double-stranded DNA (dsDNA) cases, along 3−/4− fer of [Fe(CN) ] was observed as more target genes with a long-term stability of up to 21 days. Although target were introduced and hybridized with DNA probes. The sen- analytes were not always the biomarker for lung cancer in sor presented a linear behavior towards ARGs with different other studies in regard of cancer biomarker detection using concentrations ranging from 21.6 to 477.12 pM, reaching a electrochemical sensors, this work verified the technique detection limit of 21.6 pM. The strong environmental adapt- of EIS sometimes can reach an ultrasensitive detection ability makes the sensor successful in detecting the target towards DNA strands. Beyond this, the sensor displayed gene in actual sewage or river water specimens even inter- an extra function of detecting anti-p53 antibody. Neverthe- fering with ions and other pollutants. Richard et al. reported less, it worked only at the perfect dsDNA coverage. Liu Fig. 4 Schematic illustration of functionalized electrode-based sensors biomarker and anti-p53 antibody. Image reprinted with permission used for assessing DNA molecules, where electrochemical results are from Richard et al. (2023); c A special “one-pot” sensing strategy used caused by steric hinderance sourced from double-stranded structures: for detection of p53 genes. Image reprinted with permission from Liu a Impedimetric DNA sensor prepared for determination of ARGs. et al. (2023); d Gold based electrochemical genosensor applied for Image reprinted with permission from Wan et al. (2023); b EIS based detection of p53 gene mutation. Image reprinted with permission from electrochemical DNA sensor used for detection of lung cancer DNA Garcia-Melo et al. (2023) 1 3 Biomedical Microdevices (2025) 27:36 Page 9 of 27 36 et al. depicted a special “one-pot” sensing process for the al. 2023). In this case, the resistance against the charge 3−/4− detection of p53 gene. The Fe O /α-Fe O nanocomposite transfer of [Fe(CN) ] consists of two parts: one is the 3 4 2 3 6 tethered with gold nanoparticles served as a substrate for intense electrostatic repulsion between the redox couple 3−/4− immobilization of thiolated peptide nucleic acid (SH-PNA) of [Fe(CN) ] and the hybridized probe-target DNA probes and subsequent hybridization with target DNA, all complex, the other is the steric hinderance caused by the in a single centrifuge tube (Fig. 4c) (Liu et al. 2023). 6-mer- probe-target DNA hybridization. The sensor provided a lin- captohexanol (MCH) was also involved and attached to the ear response towards the concentration of target genes of nanocomposite for blocking non-specific binding sites or HPV-16 from 0.1 to 10 pM (Fig. 5b), along with a LOD pinholes. The resulting solution was finally cast onto the of 100 pM. Selectivity analysis was performed by compar- electrode surface to increase the steric hinderance against the ing the peak current value of DNA probe control, non-com- redox couple, causing reduced DPV signals. The dynamic plementary, three-base mismatch, one-base mismatch, and range regarding the p53 gene detection based on the sen- complementary DNA. Results revealed the sensor presented sor was 1 ~ 10 pM, with a LOD of 0.85 pM. The superior less difference in the peak current between the detection of selectivity of the sensor made it capable of accurately differ - complementary DNA and mismatch sequences. No repro- entiating single-, double-, and triple- base mismatched DNA ducibility and real sample testing were presented in their sequences. The good reproducibility was demonstrated by work. However, these results could be further improved by testing seven electrodes with the target DNA, yielding cur- another HPV-16 sensor made by Pareek et al. (Fig. 5c), both rent responses with a negligible relative standard deviation LSV and EIS characterizations were performed, reaching a –4 6 (RSD). Sensor stability was also studied, with the response wider dynamic range of 10 ~ 10 pM (Fig. 5d) and a LOD –4 at the twelfth day only presenting a 16.8% drop of the initial of 10 pM (Pareek et al. 2023). The selectivity showed the response. Furthermore, the target p53 gene was spiked into highest impedance against the target DNA but also presented human serum samples to assess the practicability of the sen- non-negligible responses after hybridization with non- sor. The ultraviolent (UV) absorbance method was used to complementary sequences. Two concentrations of HPV-16 provide a double verification that the sensor is reliable in were spiked into real samples in this work to evaluate the real clinical applications. Garcia-Melo et al. constructed a feasibility for practical usage, and reasonable recovery val- simple electrochemical genosensor for sensitive detection ues were obtained. The sensors fabricated by Rasouli et al. of the 175p2 mutation of the p53 gene (Fig. 4d) (Garcia- and Pareek et al., were also compared with other recently Melo et al. 2023). In comparison to the detection without reported electrochemical biosensors used for detection of doxorubicin (Dox), the LOD was lowered by one order HPV, showing a broader dynamic range and a competi- of magnitude in the presence of Dox, based on a detec- tive LOD. Han et al. reported an on-demand EIS sensor for tion range of 0.001 ~ 100 pM. Three non-complementary detection of the r60 DNA (Fig. 5e, f) (Han et al. 2023). The and the target DNA probes were tested using the sensor use of mixed SAMs containing MCH and MUA aimed to to evaluate the selectivity and only the target DNA probes form a dense and uniform layer for connection with organic presented reduced current signals after the hybridization linkers (e.g., NHS coupled with DANP). DNA probes fea- for both cases of with or without Dox. Nonetheless, results turing hairpin structures contain the cytosine bulge that can on reproducibility, stability, and real sample testing were covalently bind to the ligand of DANP. The charge transfer not discussed in the work. The proposed sensor was also resistance (R ) increased as more target r60 strands were ct 4 7 compared to electrochemical nanodevices reported in other captured, with linear response shown in a range of 10 ~ 10 studies, confirming a relatively wide detection range and a and a LOD of 10 pM. Zhang et al. developed a DNA lower LOD. The use of MB in the work of Meftah et al. nanosensor where the growth of ZnO NWs on the graph- (Meftah et al. 2023) results in greater currents measured as ite fiber surface was achieved via a hydrothermal method higher concentrations of target DNA are introduced, while (Fig. 5g) (Zhang et al. 2019). The EIS was used to assess decreased current reponses were observed when the other the concentration of target DNA ranging from 0.01 ~ 10 pM signal indicator of Dox is applied in this work. A relatively (Fig. 5h). Increased impedance signals were observed as reasonable explanation is the introduction of Dox causes the higher concentrations of target DNA were introduced. compaction of the superficial layer which alters the spatial After the demonstration with good signal reproducibility structure of double-helix complexes near the electrode sur- and stability, the selectivity of the sensor was also assessed face (Malanina et al. 2022). by testing non-complementary DNA (non-cDNA), single- Rasouli et al., created an electrochemical DNA nanosen- mismatched DNA, and cDNA, all at same concentration of sor for sensitive detection of human papillomavirus type 10 pM. The highest resistance of charge transfer (i.e., 250 3−/4− 16 (HPV-16), aiming to provide a reliable sensing platform Ω) against the redox ion pair of [Fe(CN) ] was recorded for early warning of cervical cancer (Fig. 5a) (Rasouli et as the target cDNA was added, while the hybridization with 1 3 36 Page 10 of 27 Biomedical Microdevices (2025) 27:36 Fig. 5 Functionalized electrode-based sensors used for electrochemi- NHS, and DANP for detection of target r60 DNA. Image reprinted cal detection of DNA, where redox ion pairs are usually suffered from with permission from Han et al. (2023); f Nyquist plot revealing steric hinderance and negative-negative charge repulsion: a SPCE impedance increased after Cb-HP probe sequence attachment and tar- coated with Fe O -Au core shell nanocomposites used for detection of get DNA hybridization. Image reprinted with permission from Han et 3 4 HPV-16. Image reprinted with permission from Rasouli et al. (2023); al. (2023); g Graphite fiber wrapped with ZnO NWs for monitoring of b DPV results with concentrations of HPV-16 ranging from 0.1 to target DNA sequences. Image reprinted with permission from Zhang 10 pM. Image reprinted with permission from Rasouli et al. (2023); c et al. (2019); h EIS measurements with concentrations of target DNA ITO electrode attached with GONR/Ag@AuNPs for determination of sequence ranging from 0.01 to 10 pM. Image reprinted with permis- HPV-16. Image reprinted with permission from Pareek et al. (2023); d sion from Zhang et al. (2019); i CRISPR-Cas 12a dependent HRCA –4 EIS measurements with concentrations of HPV-16 ranging from 10 in combination with electrochemical sensing system for detection of to 10 pM. Image reprinted with permission from Pareek et al. (2023); KRAS G12D. Image reprinted with permission from You et al. (2023) e On-demand gold-based EIS sensor functionalized with MCH, MUA, 1 3 Biomedical Microdevices (2025) 27:36 Page 11 of 27 36 single-mismatched DNA resulted in a lower resistance of electrode surface since non-target DNA sequences failed 125 Ω, and the case of the non-cDNA provided with almost to induce the trans-cleavage activity, leaving obvious cur- no resistance. The decrease of resistance is accordance with rent signals. The CRISPR-Cas 12a along with the program- the synergistic effect of steric hinderance and negatively ming CRISPR-RNA (crRNA) triggers the trans-cleavage charged repulsion. Moreover, the electrode after the probe activity once target DNA sequences hybridize with the MB DNA immobilization presented strong reusability of up to conjugated ssDNA, causing reduced peak currents. The pro- ten cycles. Compared with LODs shown in other works, a posed sensor exhibited excellent selectivity for both HPV- –3 value of 3.3 × 10 pM yielded from the sensor is considered 16 and HPV-18, while had no response towards other HPV as a highly competitive LOD, which implies that the imped- subtypes. Based on the good performance in the real urine imetric methods can offer ultrasensitive detection over other sample testing, human immunoglobulin G (IgG) and bovine electrochemical techniques particularly with redox couples serum albumin (BSA) were tested in both urinal and stan- suffered from dual hinderance. You et al., introduced the dard buffered samples to evaluate the sensor selectivity for CRISPR-Cas 12a dependent hyperbranched rolling circle non-target proteins, with negligible differences observed amplification (HRCA) into the sensing system for the elec - in current signals. Beyond a highly competitive detection trochemical detection of Kristen rat sarcoma viral onco- range of 10 ~ 500,000 pM and a LOD of 1.95 pM, the sensor gene homolog (KRAS G12D) (Fig. 5i) (You et al. 2023). also equipped with good stability and less response time, A variety of bioactive enzymes including ligase and poly- compared to other DNA detections based on CRISPR-Cas merases (e.g., phi29 and Bst 2.0) were used contributing to 12a powered electrochemical sensors. amplification of target sequences from both sense and anti- DNA probes used are mostly ssDNA strands. However, sense direction. The resulting target strands then bound to other biological sequences complementary to DNA targets the probe DNA, leading to increased spatial resistance and such as RNA are also applied for electrochemical detection more intense repulsion force against the redox couple. The of DNA. Güzel et al., developed an effective electrochemi - –6 sensor enabled a wide range of detection ranging from 10 cal sensor for detection of antibiotic susceptibility, where the –5 to 1 pM, along with a LOD of 10 pM. The good selectiv- surface of the C223AT-SPE was uniformly functionalized ity of the sensor was demonstrated by detecting the KRAS with 16S rRNA probes for recognizing M. Smegmatis DNA G12D mixed with other DNA interferents, and only the tar- targets (Fig. 7b) (Güzel et al. 2021). The backfilling solu - get owned the highest current over 300 μA. tion containing 3-mercapto-1-propanol (MCP) and tris(2- Hybridization between complementary DNA (cDNA) carboxyethyl) phosphine (TCEP), along with the probe and probe DNA (pDNA) molecules was quantified by solution was prepared for the formation of SAM. The tar- means of a LIG based electrochemical sensor (Fig. 6a) geted fragment DNA was obtained after the culture of bac- (Bahri et al. 2023). Square wave voltammogram showed a teria coupled with a series of centrifugation (Fig. 7a). Two weakened variation of peak current after series of concen- different electrochemical techniques (i.e., EIS and SWV) trations of cDNA from 0.1 to 100,000 pM were measured. were applied to achieve the detection of the M. Smegmatis Ferrocene (Fc) was used as an electrochemical tag aiming DNA target. Current values in the SWV measurement nega- to yield obvious current signals. Decreased peak currents tively varied after the introduction of DNA targets (Fig. 7d), are recorded due to Fc molecules lifted from the electrode which was in accordance with the mechanism that increased surface after the hybridization, extending the distance of electrostatic resistance or steric hinderance could debilitate electron transfer towards the electrode. A femto-level LOD the electron transfer of redox couples. While EIS results was eventually obtained using this sensing strategy. Non- presented the trend of variation in an opposite behavior complementary DNA (ncDNA) and several mismatch DNA (Fig. 7c). The impedance drastically increased after stages strands with a concentration of 100,000 pM were involved from “pre probe” and “past probe” to “past target”, imply- to evaluate the selectivity of the sensor, yielding negligible ing the positive correlation between electrochemical signals signals compared to that of the cDNA. Good reproducibility and DNA analyte concentrations. More studies revealing was investigated by the determination of cDNA at 1,000 pM this positive correlation can be found below. on three individual sensors. The sensor was also tested in tenfold diluted human serum, verifying a good adaptabil- 1.5 Positive correlation between electrochemical ity in real sample measurement. Based on a similar sens- responses and target DNA concentrations ing mechanism, Duan et al., provided an electrochemical DNA sensor integrated with a biological component of Electrochemical responses recorded have a positive corre- CRISPR-Cas 12a for highly sensitive detection of HPV- lation with target DNA concentrations, which are mainly 18 (Fig. 6b) (Duan et al. 2023). Serving as a signal indica- attributed to the use of redox tags intercalated in probe- tor, methylene blue (MB) molecules are confined near the target DNA complex. A restriction endonuclease EcoRI 1 3 36 Page 12 of 27 Biomedical Microdevices (2025) 27:36 Fig. 6 Functionalized electrode-based sensors used for electrochemi- modified with MXene Ti C and AuNPs for target DNA determination. 3 2 cal assessment of DNA, from which signal variations are sourced from Amount of MB molecules attached at electrode surface are controlled increased distance between electrochemical tags and electron transfer via target activated trans-cleavage, thus causing different electrochem - interface: a LIG based electrochemical sensor for detection of cDNA. ical signals. Image reprinted with permission from Duan et al. (2023) Image reprinted with permission from Bahri et al. (2023); b SPCE assisted bio-platform prepared by Meftah et al. was used for IL6 and TGFβ1, respectively. The selectivity towards to determine polymorphisms in both interleukin-6 (IL6) two genes was assessed by mixing with their 1-, 2- mis- and transforming growth factor β1 (TGFβ1) genes from match, and non-complementary sequences and only tar- patients suffered from ovarian cancer (Fig. 8a) (Meftah et get IL6 and TGFβ1 genes feedbacked a significant high al. 2023). MCH acting as a blocking agent was applied to response even the concentration used was 30 times lower the electrode surface to avoid any non-specific adsorption at than those of rest interferents. Moreover, the sensor was uti- active sites. MB molecules used as a signal indicator prom- lized in real samples containing TT, TG/TC, and GG/CC ised an increased peak current once they intercalated into genotypes, and achieved good results on the discrimina- the double-stranded oligonucleotide sequences (ODN). The tion in heterozygous and homozygous genes. Sensing per- sensor presented wide linear ranges of 0.1 ~ 100,000 and formances regarding the proposed sensor were compared 0.05 ~ 100 pM, along with LODs of 0.048 and 0.017 pM, with those from other reported electrochemical nanosensors 1 3 Biomedical Microdevices (2025) 27:36 Page 13 of 27 36 Fig. 7 16S rRNA probe functionalized sensors for electrochemical M. Smegmatis DNA target hybridization. Image reprinted with permis- detection of M. Smegmatis DNA target: a Preparation of M. Smegma- sion from Güzel et al. (2021); c Positively varied EIS responses after tis DNA target from bacteria culture. Image reprinted with permission a sensing process. Image reprinted with permission from Güzel et al. from Güzel et al. (2021); b Detection of M. Smegmatis DNA target (2021); d Negatively varied SWV responses after a sensing process. using C223AT-SPE, including immobilization of 16S rRNA probe and Image reprinted with permission from Güzel et al. (2021) for DNA polymorephism detection. However, target genes reaching a positive correlation between DPV responses and restriction endonuclease applied were not very consis- and concentrations of the target DNA, probably due to the tent as the current study, which were not able to provide accommodation of the AQMS in the LT-RP double-stranded convincible results in the literature comparison. Ali et al. structure. The sensor presented an extremely wide detection –9 6 developed an electrochemical DNA nanosensor for moni- range from 10 to 10 pM, along with an ultralow LOD –10 toring genes extracted from Shigella flexneri ( S. flexneri ) of 7.4 × 10 pM. The developed nanodevice can be uti- bacteria (Fig. 8b) (Ali et al. 2023). Capture probes (CP) lized for determination of other target sequences by easily were introduced forming a uniform coverage for immobili- changing the CP, LT, and RP, revealing a strong applica- zation of linear targets (LT). Report probes (RP) were also tion versatility. The largest current response was produced involved in hybridizing with the LT. The anthraquinone- by the hybridization with LT, compared with those yielded 2-sulfonic acid monohydrate sodium salt (AQMS) played from mismatched and non-complementary LT sequences. In as a role of signal indicator and was applied in the detection, detection of pathogenic bacteria, S. flexneri series exhibited 1 3 36 Page 14 of 27 Biomedical Microdevices (2025) 27:36 Fig. 8 Schematic illustration of electrochemical sensors used for (2023); c Multiplex silicon supported DNA nanosensor for simultane- assessing DNA molecules, where electrochemical results are caused ous detection of stx1 and stx2 genes. Image reprinted with permis- by electron transfer promotion in presence of signal indicators inter- sion from Wasiewska et al. (2023); d Paper-based electrochemical calated in double-stranded structures: a EcoRI assisted electrochemi- sensor with nanostructures of N-doped graphene used for nucleobase cal bio-platform prepared for determination of polymorphisms in IL6 detection of A and G from calf thymus DNA. Image reprinted with and TGFβ1 genes. Image reprinted with permission from Meftah et permission from Jagannathan et al. (2023); e f-CB/SPE based electro- al. (2023); b Flexible ITO based nanosensor used for monitoring of chemical sensor used for determination of A and G released from DNA S. flexneri genes. Image reprinted with permission from Ali et al. hydrolysis. Image reprinted with permission from Aamri et al. (2023) –7 –1 higher current signals over other categories. These demon- 10 to 10 pM, for stx1 and stx2 genes respectively, caus- strate high selectivity of the sensor for both DNA and bac- ing increase of square wave voltammetry (SWV) responses –7 teria detection. Moreover, the eminent stability makes the and an ultralow LOD of 10 pM. The nanosensor was also sensor still provide measurable signals even after very long- tested after simultaneously introducing two types of probe term storage (e.g., up to 8 weeks). The reusability was also sequences complementary to target genes, showing strong tested by breaking the DNA duplex and rebinding with the discrimination ability particularly with presence of the MB. LT several times, and results showed that not much appar- In comparison to previously reported DNA sensors used ent signal decreasing was recorded within 5 ~ 6 regeneration for detection of genes from the STEC, the sensor presented cycles. The target S. flexneri DNA added real food samples the lowest LOD. However, target genes compared were not were eventually analyzed, with recovery values ranging always stx1 and stx2. Jagannathan et al., developed a type from 90 ~ 103%, evidencing good practicability of the sen- of paper-based electrodes functionalized with nanostruc- sor. Wasiewska et al., prepared a multiplex silicon supported tures of N-, P-, and S- doped graphene and used to detect DNA nanoelectrode for simultaneous determination of stx1 calf thymus DNA (Jagannathan et al. 2023). The cathodic and stx2 genes from the Shiga toxin-producing E. coli cyclic voltammetry was applied throughout the detec- (STEC) (Fig. 8c) (Wasiewska et al. 2023). The robust func- tion towards guanine (G) and adenine (A). Results yielded tional layer with very good conductivity was achieved by from the N-doped graphene electrode present characteriz- electrodepositing AuNPs and the chitosan-gold nanocom- able current signals for both G and A, compared with other posite (Cht-Au) on the gold interdigitated electrode (Au- heteroatom doped (e.g., P-, S- doped) or untreated ones IDE), providing biocompatible circumstance for attachment that can only detect G with less discrimination (Fig. 8d). of probe DNA sequences (Wasiewska et al. 2022). MB mol- The sensor eventually presented a concentration range of 5 6 6 ecules increasingly intercalated into the probe-target DNA 4 × 10 ~ 4 × 10 pM, and a LOD of 1.5 × 10 pM for detection –7 –2 5 6 duplex as concentrations detected from 10 to 10 pM and of G, and a concentration range of 4.4 × 10 ~ 4.4 × 10 pM, 1 3 Biomedical Microdevices (2025) 27:36 Page 15 of 27 36 and a LOD of 2.38 × 10 pM for detection of A. Aamri et apparently improved. Additionally, there are many reports al., performed a similar work of determining both G and on determination of G and A, or thymine (T), based on vari- A released from DNA hydrolysis based on a f-CB assisted ous electrochemical sensors, yielding more broad ranges of electrochemical sensor (Fig. 8e) (Aamri et al. 2023). Glass determination along with several order-of-magnitude lower microbeads were used for immobilization of probe DNA LODs, compared with the current two works, indicating strands or probe-target DNA duplexes. The reaction was further development upon the sensor is needed to improve allowed to be carried out in a tube, from which the resulting sensing performances for detection of DNA bases. solution was drop-casted onto the f-CB layered electrode In the work of Bo et al. (Bo et al. 2011), the surface of surface, followed by a DPV measurement to evaluate the oxidized graphene coupled with PANIws was elaborately amount of released G or A molecules. The proposed DNA fabricated to provide an adequate, biocompatible, and highly nanosensor exhibited good selectivity towards target strands, conductive room for immobilization of probe sequences since electrochemical responses of the target are remarkably (Fig. 9a). A series of linear DPV signals were observed after higher than those of the probe only or non-complementary a wide dynamic range of 2.12 ~ 2.12 × 10 pM was tested, sequences. The repeatability was also assessed by testing where reduction peaks became stronger with the incre- three equally prepared sensors incubated with the target of ment of target DNA concentration (Fig. 9b). Daunomycin microRNA-21 at different concentrations, yielding rela - was used as the electroactive signal indicator, of which the tively consistent results with a RSD of 5.6%. In comparison reduction was achieved via transfer of electrons after the to the work of Jagannathan et al., the sensor presented in formation of probe-target double helix structure. The pro- this study enables a slightly wider detection range towards posed nanosensor demonstrated that the electrode surface both G and A, although LOD values obtained have not been modified with probe-target DNA duplexes presented the Fig. 9 a GCE modified with GO and PANIws used for detection of ful hybridization. Image reprinted with permission from Hushiarian target DNA molecules. Image reprinted with permission from Bo et et al. (2015); f DPV responses in relation to different concentrations al. (2011); b DPV responses in relation to different concentrations of of methylated target strands ranging from 0.01 to 5,000 pM. Image target DNA from g to a representing a range of 2.12 ~ 2.12 × 10 pM. reprinted with permission from Daneshpour et al. (2016); g DPV Image reprinted with permission from Bo et al. (2011); c AuNWE responses in relation to different concentrations of target probes rang - covered with MCH and thiolated DNA probes for electrochemical ing from 3,130 to 10. pM (a to g). Image reprinted with permission analysis of DNA. Image reprinted with permission from Hua et al. from Hushiarian et al. (2015); h SPCE covered with PT and anti-5mC (2018); d T4 DNA ligase involved DNA sensing mechanism, along antibody used for immobilization of methylated target strands. Nano- with presence of MNP for target sequence enrichment. Image reprinted composites of Fe3O4/TMC/Au/streptavidin hybridized with biotin- with permission from Hushiarian et al. (2015); e Gold-based biosensor labeled probe sequences forming a “sandwich” sensing strategy after coupled with PEDOT-PSS, AuNPs, and DPA used for sensitive detec- they recognize methylated target strands. Image reprinted with permis- tion of genomic DNA, an image with more details revealing success- sion from Daneshpour et al. (2016) 1 3 36 Page 16 of 27 Biomedical Microdevices (2025) 27:36 highest current response in the presence of daunomycin, only (Fig. 9d). Ru(dppz), another redox complex, was compared to cases of base mismatched sequences and non- applied in this work to produce sensitive signals after they complementary DNA. Other than the low detection limit of inserted into double-stranded structures. A positive cor- 0.325 pM, the reproducibility and repeatability were also relation between DPV signals and target DNA concentra- studied on seven sensors, with a small RSD of 1.15%. The tions was observed (Fig. 9g), yielding a LOD of 0.054 pM. storability was evaluated by placing the electrode in 0.1 M This work employs magnetic nanoparticles and DNA ligase phosphate buffered saline (PBS), pH 7.3, at 4 °C for over for separation of target sequences from non-specific sub - seven days, and current signals with a slight decrease were stances, which presents great significance in the detection observed. The proposed sensor owns a wider linear range of genomic DNA with various types, although the value coupled with a lower LOD in comparison to values shown in of detection limit is not as low as that shown in their pre- other reports. Hua et al., allowed MB molecules to undergo viously reported paper (Dutse et al. 2014). A “sandwich” a reduction into methylene white (LB) on a single gold sensing strategy for the electrochemical assessment of RAS nanowire electrode (AuNWE), serving as indicator of SWV association domain family (RASSF) 1 A tumor suppressor signals for electrochemical analysis of DNA (Fig. 9c). (Hua DNA methylation was reported by Daneshpour et al., where et al. 2018) Probe DNA sequences were fixed on the sensor the electrode was functionalized with polythiophene (PT) surface via the gold-thiol bonding, followed by the hybrid- and the anti-5-methylcytosin (anti-5mC) monoclonal anti- ization with target DNA strands to enable MB molecules body (Fig. 9h) (Daneshpour et al. 2016). Fe O nanospheres 3 4 to intercalate into the probe-target DNA duplex through the coated with N-trimethyl chitosan (TMC) are preferable for intense electrostatic effect (Dai et al. 2012; Yau et al. 2003). attachment of AuNPs, forming Fe O /TMC/Au nanocom- 3 4 However, the mismatched hybridization led to the failure posites to anchor streptavidin molecules. Biotin-labeled of MB intercalation, thus providing no reduction signal. probe sequences subsequently grew at the surface of the The nanosensor presented a linear response over concentra- nanosphere owing to the strong affinity between streptavi - –3 4 tions ranging from 10 ~ 10 pM, yielding a detection limit din and biotin. Methylated target DNA capable of being cap- –4 of 4.8 × 10 pM. The LOD was three orders of magnitude tured by the anti-5mC antibody bound to probe sequences lower than that presented in the work of Bo et al. (Bo et dragging the Fe O /TMC/Au nanocomposite nearby the 3 4 al. 2011), two orders of magnitude lower than that obtained electrode surface, forming a “sandwich” configuration for from the GCE modified with AuNPs, reduced graphene electron transfer of AuNPs (Fig. 9h). The DPV characteriza- oxide (rGO), and Adriamycin (Zhang and Jiang 2012), as tion evidenced that the genosensor had an extremely wide well as lower than results in other MB-assisted electro- concentration range of methylated DNA ranging from 0.01 –4 chemical sensors. An even lower LOD of 1.1 × 10 pM to 5,000 pM. The reduction peak current varied in a posi- was produced by Wang et al. (Wang et al. 2014), through a tive correlation with the concentration of target sequences simple GCE covered with electropolymerized Eriochrome (Fig. 9f). A reasonable explanation is the more methylated Black T (pEBT). Nevertheless, the detection range was target DNA strands are introduced, the more Fe O /TMC/ 3 4 –3 4 very narrow compared with this work (i.e., 10 ~ 10 pM). Au nanocomposites land at the electrode surface, caus- In addition, the sensor stability was also tested, however, ing reduction peak current signals with higher intensity as current signals recorded were with non-negligible decreases increased amount of gold ions are reduced into the elemental only after 6 days. A gold-based biosensor modified with gold. An estimated LOD of 0.002 pM was presented in their poly(3,4-ethyllenedioxythiophen)-poly(styrenesulfonate) work and was proved lower than LODs yielded from other (PEDOT-PSS) and AuNPs were constructed for detection relative studies. The specificity of the sensor was evaluated of the genomic DNA in white rot fungus, Ganoderma bon- by analyzing signals of methylated sequences, unmethylated ienese (Fig. 9e) (Hushiarian et al. 2015). Initially, MNP- sequences, and sheared genomic DNA, at concentrations of Capture probes bound to the complementary template and 500 and 1,000 pM, and only the methylated sequence pro- intended to link with the target DNA sequence in the pres- vided measurable responses. The excellent signal reproduc- ence of T4 DNA ligase, yielding extended DNA strands ibility and repeatable electrode fabrication make the work (35-mer). The 35-mer DNA fragment then dissociated from highly useful in many practical applications. The proposed the template after a heating treatment and was isolated from sensor was tested in real plasma solutions containing five other undesired DNA sequences under an applied magnetic different concentrations of the methylated DNA, reaching a field, followed by hybridization with DNA reporter probes good recovery range with a relatively small RSD. Addition- that were immobilized on the electrode surface by means ally, the sensor also displayed acceptable long-term stability of a self-assembled DPA monolayer (Fig. 9d). While the with a slight decrease of peak reduction current obtained use of non-complementary template resulted in the failure from electrodes already stored for 1 to 8 weeks at 4 °C. of probe-target hybridization, leaving MNP-Capture probes 1 3 Biomedical Microdevices (2025) 27:36 Page 17 of 27 36 1.6 Electrochemical assessment of DNA using membrane. Blockages against ions are created while bio- nanopore-based sensors logical molecules (e.g., DNA) pass through the pore, which are recorded via a real-time signal transformation setup. Nanopores, another class of prevalently advocated sensing Current responses with different magnitudes, shapes, and devices, have been widely developed for high-resolution dwell times, revealing information of tested sequences, are assessment of DNA molecules. In a scheme of nanopore- deemed as evaluation criteria for DNA sequencing (Deamer based sensing, ssDNA molecules thread through a nano- et al. 2016). An excellent overview reported by Crnkovic ́ dimensional channel under an applied potential, causing et al., mainly focuses on commonly used porin-based nano- blockage of ions and thus yielding current signals varied pores, outlining information about geometric dimensions with time. By virtue of rapid data recording, excellent regarding proteinaceous nanochannels (Crnković et al. precision, and cost-effectiveness, nanopores have been 2021). Among them, α-hemolysin (α-HL) and Mycobacte- employed in characterization of various biological ana- rium smegmatis porin A (MspA) are commonly reported lytes such as ssDNA or double-stranded DNA (dsDNA) porin-based nanopores, which can be massively produced sequences, aptamers (i.e., short-length of ssDNA or RNA), through a mature process of Escherichia coli ( E. coli) peptides, proteins, and DNA-peptide conjugates (Ven- induced protein expression. Although it is complicated and katesan et al. 2009). DNA translocation events are usually time-consuming, this method ensures porin-based nano- measured through ionic current when a DNA sequence pores, prepared from batch to batch, are with an identical threading through a nanopore. Other measuring protocols dimension at a molecular level. Up to now, porin-based such as tunnelling current is also used in nanopore-based nanopores have been developed into various mutated forms assessments of DNA, although there is no successful report catering for high-resolution DNA and protein sequencing. in identifying DNA bases using tunneling current (Ivanov DNA oligonucleotides with different sequences were et al. 2011). Nanopores can be largely classified into two successfully discriminated through the α-HL nanopore with subtypes namely biological nanopores and solid-state nano- a probe DNA sequence anchored at the Cys mutation site pores. Biological nanopores can be made through a vari- (Fig. 10a) (Howorka et al. 2001). Target sequences hybrid- ety of materials including toxins (Sarangi and Basu 2018), izing with the probe caused physical hindrance against ion porins (Wang et al. 2021), pore-forming proteins (Parker flows, yielding signals with a lowered current level. They and Feil 2005), and substrate-specific channels such as DNA were subsequently torn apart from its complementary domains (Langecker et al. 2012). However, the diameter of sequence due to the electric field force and threaded through channels within most of them is too large to detect DNA. the β-barrel region of the engineered α-HL nanopore, caus- Some of the porins where the size of constriction zone can ing unique downward spike-shaped signals. Nevertheless, be down to 1 ~ 2 nm are considered as terrific nanopore can - mismatched sequences (e.g., single-base altered DNA) didates for DNA sequencing. Therefore, this review mainly directly passed through the pore without any obstruction focuses on the porin-based nanopores rather than other owing to the absence of base-to-base hybridization, present- biological counterparts. Also, breakthroughs particularly ing downward spikes merely (Fig. 10b). This strategy is for ssDNA sequencing at individual-nucleobase resolution useful in recognizing target DNA sequences in hodgepodge facilitate porin-based nanopores to be developed into more DNA samples but may not be very suitable for continu- promising nanodevices suitable for high-throughput bio- ous DNA sequencing as lack of a component serving as a logical analyses or medical diagnoses. As another crucial ‘brake’ for rapid DNA translocation. Moreover, the protein nanopore subtype, solid-state nanopores are discussed at the mutation involved in this work makes the nanopore fabrica- later stage of the review. tion more complicated and increases time cost. Facing the challenge of high translocation speed of DNA, some enzy- 1.7 Porin-based nanopores for DNA sequencing matic components specific to DNA strands such as DNA polymerases or helicases were introduced to slow down the Porin-based nanopores, also named transmembrane pro- translocation event. Nanopore sensing integrated with DNA teins, are the initial elaboration of scientific groups aiming polymerases, as so-called sequencing by synthesis (SBS), for continuous DNA sequencing. The double-layer phos- has been regarded as one of routes to DNA sequencing at pholipid membrane prefers to cover onto a chip surface a single-molecule level. Tagged nucleotides are assembled containing an artificial aperture normally with a diameter into a DNA strand in the presence of DNA polymerases, of several micrometers. Driven by the electrophoretic force while tags indicative of different nucleobases dissociates (i.e., cathode in the cis- compartment and anode in the from nucleotides and are sequentially move through the trans- compartment), transmembrane proteins then move nanopore, leading to base-corresponding current signals towards the membrane and ultimately embed within the (Fig. 10c). In Kumar’s work, detectable tags containing a 1 3 36 Page 18 of 27 Biomedical Microdevices (2025) 27:36 Fig. 10 a α-HL nanopore with a DNA oligonucleotide anchored at polymerase immobilized α-HL nanopore, along with different current mutated site of Cys 17. Image reprinted with permission from Howorka blockages revealing four nucleobases. Image reprinted with permis- et al. (2001); b Probe oligo-1 hybridized with complementary oligo- sion from Kumar et al. (2012); d Statistical study on tags passing 2G sequences or one-base mutated oligo-2C, leading to different through nanopore to achieve discrimination of bases. Image reprinted threading signals. Image reprinted with permission from Howorka et with permission from Kumar et al. (2012) al. (2001); c Schematic illustration of tags threading through a DNA small molecule of coumarin coupled to polyethylene glycol A sensing ensemble of MspA nanopore integrated with (PEG) in different lengths including PEG , PEG , PEG , phi29 DNA polymerase (DNAP) was reported to suc- 16 20 24 and PEG , were prepared reflecting nucleobases of thymine cessfully realize a clear and continuous decoding of DNA (T), guanine (G), cytosine (C), and adenine (A), respectively sequences (Laszlo et al. 2014). Target analytes were elabo- (Kumar et al. 2012). Current drops with various magnitudes rately designed into a phi29 DNAP attached short length of caused by blockage of tags were statistically recorded, ssDNA linking to the target double stranded DNA (dsDNA) revealing information of four nucleobases (Fig. 10d). The molecule coupled with a second adaptor for sequence proposed method verifies an indirect DNA sequencing rely - rereading (Fig. 11a). Complementary sequences attach to ing on base-related tags. However, the preparation of tags the lipid bilayer due to presence of cholesterol ends, aim- lengthens the experimental procedure before the nanopore ing for the analyte enrichment. The ssDNA, under the force sensing. According to results, signals with respect to bases of electric field, passed through the MspA pore first, drag - of T, G, and C present a close magnitude except the base of ging the phi29 DNAP and dsDNA towards the pore as well. A, which to some extent, may increase errors in base identi- The phi29 DNAP was then blocked at the cis- entrance of fication. A possible interpretation is use of PEG chains with the MspA pore owing to the oversized proteinaceous con- similar lengths (i.e., PEG , PEG , and PEG ) for indicat- formation, conducted polymerization at a pace of single 16 20 24 ing bases of T, G, and C, while a relatively longer chain of nucleobase to reduce the speed of DNA translocation, PEG represents the base of A. It is more worth noting that yielding continuous current signals varied with clear stairs current blockages on bases shown in this study are not rig- (Fig. 11b). Comparisons in relation to ionic current read- orously consistent with outcomes presented in other works outs between the sequence measured and reference values (Stranges et al. 2016; Fuller et al. 2016), as it may be due predicted by the de Brujin quadromer map were evalu- to different chemical structures or lengths of tags that cause ated (Fig. 11b), along with the analysis of false alignment spatial blockages in different forms. based on 91 reads of phi X 174 genomic DNA aiming to 1 3 Biomedical Microdevices (2025) 27:36 Page 19 of 27 36 Fig. 11 a phi29 DNA polymerase assisted DNA sequencing based on rithm and alignment between measured current blockages and pre- MspA nanopore and continuous recording of target DNA sequences. dicted values; d Analysis regarding false alignments based on 91 reads b Prediction on current signals of previously unevaluated sequence of phi X 174 genomic DNA. Image reprinted with permission from using de Brujin quadromer map, along with the comparison with phi Laszlo et al. (2014) X 174 consensus; c Data processing by means of a level-finding algo - enhance the reliability of nanopore sequencing (Fig. 11c,d). Next development for the work was to apply a reference 1.8 Solid-state nanopores for DNA sequencing sequence to calibrate amplitude of ion current signals for the de novo DNA sequencing. Also, some locations within Solid-state nanopores for DNA sequencing can be made a piece of current signal recording were in the lack of tran- using either “top-down” or “bottom-up” methods. “Top- sient pore-threading information probably due to the phi29 down” methods such as focused ion beam (FIB) and focused DNAP movement or mathematical errors in calculation of electron beam (FEB) allow high-energy particles (e.g., gal- the level-defining algorithm (Laszlo et al. 2014). The use lium (Miles et al. 2013) and helium (Marshall et al. 2012) of DNA helicases was depicted as a future attempt to sig- ions, or electrons (Chen et al. 2017)) to penetrate the surface nificantly improve DNA sequencing because of the monoto - of thin film, creating in-plane defects with various geom - nous moving nature of helicases upon DNA strands (Laszlo etries. System temperature, beam duration, or intensity are et al. 2014). key parameters in the process of beam-induced nanopore fabrication. Altering them or one of them can yield solid- state nanopores with a very small diameter. Interestingly, electron beams acting at 2D material surfaces for a longer 1 3 36 Page 20 of 27 Biomedical Microdevices (2025) 27:36 duration also result in smaller nanopores. (Li et al. 2001) current blockages observed are not caused by a single base Reactive ion etching (RIE) applies chemically to reactive or amino acid. Nanopore contraction techniques also belong plasma (e.g., O plasma) to engrave treated surfaces, leav- to “Bottom-up” methods. Among them, metal oxide deposi- ing cavities especially for thick wafers (Bai et al. 2014), tion induced nanopore contractions are realized by introduc- which is deemed as another effective method for nanopore ing an ultrathin layer of metal oxides such as Al O , TiO , 2 3 2 creation. “Bottom-up” methods, sometimes called self- and HfO through the atomic layer deposition (Chen and Liu assembly methods to nanopore fabrication, often enable 2019). However, this technique often leads to an increase of nanopore-containing films to be constructed based on mate - membrane thickness, causing length nanopore channels that rial building blocks. In contrast with traditional nanopores are not suitable for high-resolution DNA reading. in silicon nitride substrate, there are also substantial stud- DNA sequencing based on solid-state nanopores usually ies focusing on production of hybrid nanopores. They are faces the challenge of drilling nanopores with a diameter at a class of special solid-state nanopores that are always round 1 nm. The first solid-state nanopore was successfully functionalized with bio-components for overcoming the produced within a thin layer of Si N membrane containing 3 4 drawbacks of bare solid-state nanopores including less bio- a bowl-shaped depression on the other side, via the ion-beam compatibility and difficulty of active site creation (Yusko sculpting (Fig. 12a) (Li et al. 2001). The blockage against et al. 2011; Bell et al. 2012). Yusko, et al., created hybrid ion current was verified when dsDNA threaded through the nanopores of which the surface of silicon nitride nanopore Si N nanopore with a diameter of 5 nm (Fig. 12b). This 3 4 was coated with lipid membrane. Compared with untreated work opens a new path for characterization of DNA mol- silicon nitride nanopores, the lipid-coated one can allow an ecules based on inorganic nanodevices. Nanopores with individual protein molecule translocating through the chan- further reduced diameters (i.e., 0.8 ~ 2 nm) were fabricated nel without clogging (Yusko et al. 2011). Hybrid nanopores by drilling the thin layer of SiNx (thickness of 5 ~ 8 nm) have versatile functions, however, the fabrication process coated on the Si/SiO2 chip (Fig. 12c), using as nanosensors may be time-consuming with a higher cost. Moreover, the for assessing DNA molecules (Fig. 12d). (Venta et al. 2013) length of the channel is not radically changed as most of The constriction domain of the SiNx nanopore featuring a them are still limited by the thick solid substrate, leading to mean diameter of 1.2 nm and a length of 1.7 nm is highly Fig. 12 Solid-state nanopores used for DNA sequencing: a Schematic among dA , dC , and dT . Image reprinted with permission from (30) (30) (30) illustration of a Si3N4 nanopore via argon ion-beam sculpting. Image Venta et al. (2013); f Schematic diagram of DNA sequencing on a gra- reprinted with permission from Li et al. (2001); b dsDNA translocation phene nanopore. Image at right-hand side reveals a piece of graphene event recorded. Image reprinted with permission from Li et al. (2001); layer transferred onto a silicon nitride chip containing a 5 μm aper- c SiN nanopore fabricated on a large aperture-containing Si/SiO ture. Image reprinted with permission from Schneider et al. (2010); x 2 substrate. Image reprinted with permission from Venta et al. (2013); g Translocation signals upon drilled graphene nanopore, along with d ssDNA threading through the SiN nanopore. Image reprinted with DNA translocation events with different spatial conformation and cor - permission from Venta et al. (2013); e DNA homopolymers translo- responding statistical analyses. Image reprinted with permission from cate through prepared SiN nanopore and comparison of current levels Schneider et al. (2010) 1 3 Biomedical Microdevices (2025) 27:36 Page 21 of 27 36 comparable to those in commonly used porin-based nano- the capture radius (R ) of the MoS pore (Fig. 13a). The use c 2 pores. DNA homopolymers that contain 30 bases, includ- of BmimPF6 can effectively slow down the DNA translo - ing poly (dA), poly(dC), and poly(dT), were tested using cation since the highly viscous ionic liquids provide DNA the nanopore, followed by a statistical analysis on numer- strands with intense Stokes dragging force and keep them ous threading signals to achieve the base discrimination as an entwined configuration before passing through the (Fig. 12e). However, the presented open-pore baseline is nanopore (Feng et al. 2015). Two classes of DNA analytes thick probably due to intrinsic large background noise of including DNA homopolymers (30-mers) and deoxynucleo- solid-state nanopores. Moreover, only the signal of poly(dA) tides (monophosphates) were tested using the MoS nano- is differentiable from other homopolymers according to the pore, yielding downward spikes with different amplitudes to magnitude of current blockage as there is no apparent differ - identify the four nucleobases (Fig. 13b, d). The dwell time, ence observed between the signal of poly(dC) and poly(dT). another significant nature in nanopore sensing, was also Nanodevice fabrication based on two-dimensional (2D) assessed using deoxynucleotides tested, from which results materials has been becoming a hot topic since Novoselov yielded (e.g., current drop versus dwell time) were plotted and Geim first obtained a clean graphene sheet peeled off in both merged and separate ways (Fig. 13d). Separated from a bulky graphite using the ‘scotch tape’ method in plotting contains points with different densities revealing 2004 (Novoselov et al. 2004). Garaj et al., then claimed gra- the reappearing frequency of current blockages related to phene can be applied as a trans-electrode membrane with each deoxynucleotide. This work is a successful example a thickness below 1 nm (Garaj et al. 2010). By drilling a of the use of thin film nanopores to determine biological nanopore (diameter of 22 nm) within a single layer of gra- molecules. However, there are still several issues required phene on a Si/SiO chip containing a 5 μm aperture, Schnei- to be addressed before reaching the destination of continu- der et al., aimed to provide proof of concept the possibility ous DNA sequencing at high resolution: 1) nanopores with of DNA sequencing could be realized on nanopores within reduced diameters of 1 ~ 1.2 nm are more suitable for high atomically thick membranes (Fig. 12f) (Schneider et al. resolution DNA reading, while the size of the nanopore fab- 2010). Up to 1,222 events of DNA translocation presented ricated within the MoS membrane shown in this work is as conductance signals were statistically recorded revealing 5 nm; 2) there is lack of attempts on successive reading of a different behaviors, involving the open-pore (i.e., blank), strand with random base sequence, although the ionic liquid non-, partial-, and entire- folding (Fig. 12g). Same as the of BmimPF6 is applied to remarkably reduce the transloca- work of Venta et al., there was no effort on the reduction tion speed of DNA at the current work; 3) the current-drop of nanopore noise, resulting in a thick open-pore baseline values, either for 30-mer homopolymers (e.g., A30, T30, (Fig. 12g). Merchant et al. reported a graphene single layer C30, and G30) or for deoxynucleotides (e.g., dAMP, dTMP, supported on a tandem SiN/SiO /Si composite, along with a dCMP, and dGMP), are discriminated depending on count- nanopore drilled in the graphene plane for DNA transloca- ing peaks. However, from a view of comparison, signals tion (Merchant et al. 2010). Diameters of nanopores created in relation to these two types of biomolecules are not very were about 5 ~ 10 nm, they were suitable for DNA translo- consistent, which are hard to realize reasonable identifica - cation while maybe not very appropriate for reading DNA tion on current blockages reflecting each base particularly sequences. However, these works are still regarded as a pio- in random base sequencing (Fig. 13c, d). neering attempt in solid-state nanopore based DNA sequenc- With the development of computer science, an increas- ing. For future development, enzymatic components such as ing number of study groups start to use computer-aided DNA polymerase or helicase can be introduced to overcome simulation or mathematical modeling to current signals and the challenge in reading sequences under an extremely high potential interactions yielded when DNA sequences thread translocation speed. through solid-state nanopores, especially for ones created Aleksandra Radenovic et al. reported progress on solid- within 2D thin films. Wells et al. theoretically studied ssDNA state nanopore sensing regarding discrimination of four molecules passing through a graphene nanopore based on types of nucleotides via a nanopore manufactured in a all-atom molecular dynamics and atomic-resolution Brown- MoS membrane (Fig. 13a) (Feng et al. 2015). The detec- ian dynamics (Wells et al. 2012). Likewise, Zhang et al., tion pool was separated by the MoS membrane, having the used molecular dynamic simulations to investigate DNA ‘cis’ compartment loaded with viscous room-temperature strands threading through the nanopore created within a ionic liquid (RTILs) of 1-butyl-3-methylimidazolium hexa- monolayer of hexagonal boron nitride (h-BN) for the single- fluorophosphate (BmimPF ), while the ‘trans’ compart- base identification (Zhang and Wang 2016). Moreover, all- ment loaded with KCl aqueous solution. Electrophoretic atom steered molecular dynamics (SMD) simulations can and electroosmotic forces offered a synergistic ‘push-like’ provide information about effects of nanopore geometries effect upon DNA molecules once they were trapped within within a graphene film on DNA sequencing. (Zhang et al. 1 3 36 Page 22 of 27 Biomedical Microdevices (2025) 27:36 Fig. 13 DNA sequencing based on a single layer of MoS : a Detection tion events of DNA homopolymers; c Statistical evaluation upon cur- pool separated by a MoS membrane containing a nanopore, where rent blockages of A30, T30, C30 and G30; d Statistical analysis upon cis- area is loaded with an ionic liquid of BmimPF and trans- area dAMP, dTMP, dCMP, and dGMP translocation events, of which data is loaded with KCl aqueous solution, along with a TEM image of revealing current drops versus dwell times are presented in both inte- 5 nm nanopore created within the monolayer of MoS ; b Transloca- grated and individual ways 2014). Characterizations on intrinsic properties of a novel 2 Conclusions and prospects layer with atomic thickness such as graphdiyne (GDY) and status of four types of nucleobases threading through its As the review presents, functionalized electrode-based sen- in-plane nanopore can be achieved by the first-principles sors and nanopores are two primary nanodevices used for density functional theory (Kumawat and Pathak 2021). Fur- electrochemical assessment of DNA molecules. Functional- thermore, this method can be also applied to evaluate dif- ized electrode-based sensors are often equipped with func- ferent spatial conformations of adenine (A), cytosine (C), tional layers highly effective to adsorption of DNA probe guanine (G), thymine (T) and uracil (U), when they pass sequences for sensitive detection of target DNA, providing through a graphene nanopore, as well as their effects on outcomes of electrochemical responses varied with analyte ssDNA or RNA sequencing (Nelson et al. 2010). Nonethe- concentrations. Voltammetric (e.g., DPV) and impedimetric less, the transformation from computational analyses to (e.g., EIS) measurements are mostly used for trace amount practical applications (e.g., high-resolution DNA sequenc- detection of DNA molecules, while other electrochemi- ing) for solid-state nanopores is still the common aspiration cal techniques like CV, SWV, and LSV are also frequently of scientific researchers all over the world, although the reported. Detection results reveal both positive and negative detection of DNA molecules via theoretical studies have correlation between concentrations of target DNA sequences realized in-depth development. and electrochemical signals owing to use of different sensing 3−/4− mechanisms. Redox couples (e.g., [Fe(CN) ] ) against a synergistic resistive effect made of the probe-target steric hinderance and negative charge repulsion usually lead to a typical negative correlation, while electroactive indicators 1 3 Biomedical Microdevices (2025) 27:36 Page 23 of 27 36 (e.g., MB) intercalating into structural intervals formed after in current signals being hard to differentiate and analyze. the probe-target hybridization promote the electron transfer, For example, the challenge of high background noise or low resulting in a positive correlation. SNR mainly sourced from large total capacitance of entire Although functionalized electrode-based sensors in com- nanopore-containing chips or in-plane lattice vibration of bination with electrochemical techniques often achieve out- solid materials appears almost in all types of solid-state standing performances on determination of DNA such as nanopores. A probably feasible method worth performing wide dynamic range, ultralow LOD, high selectivity, good is introducing an extra dielectric layer since successive lay- reproducibility, long storability, and reliable real sample ers can mimic tandem capacitance connection left a reduced detection, majority of them merely exhibit a single function total capacitance. Fragasso et al., offered a nice review on of concentration quantification. Multiplexed electrodes that the comparison of current noise in biological and solid- can simultaneously detect two or even more DNA analytes state nanopores (Fragasso et al. 2020). Other strategies and versatile sensors capable of screening not only DNA for lowering the background noise of nanopores are pre- strands but also macromolecules (e.g., proteins) are prob- sented in their work. Moreover, unlike porin-based nano- ably in large demand in the future. Also, flexible electrodes pores that always can couple with DNA-related enzymes based on inexpensive soft substrates and electrodes inte- to slower the transverse speed of DNA, there is extremely grated with novel 2D materials (e.g., MXenes) have potent hard to anchor such enzymatic components precisely nearby advantages of low cost, mass production, large surface area solid-state nanopores. Nanopores created within functional and light weight, which are competitive alternations that group-containing materials (e.g., graphene oxide) or metal replace conventional hard electrodes (e.g., GCE) in near organic frames (MOFs) may somehow address this issue someday. since functional groups or active metal cores exhibit strong Nanopores are a class of special electrochemical nanode- affinity towards enzymatic biomolecules via either covalent vices that are able to decipher bioinformation relying or non-covalent adsorption. Furthermore, the dimension of on nucleobase-dependent current blockages when DNA solid-state nanopores obtained from current pore-making sequences thread through the nanopore driven by an elec- techniques are unable to achieve an atomic-grade accuracy trical field force applied. Up to date, high-resolution DNA like porin-based nanopores due to the sub-nano-level atom sequencing is only realized on porin-based nanopores. α-HL arrangement of solid materials. This is currently considered and MspA nanopores as well as their mutated variants are as one of the most difficult technical bottlenecks. Creating mostly used transmembrane channels in comparison to other a pore-diameter of about or sub 1 nm within a single 2D porins. Continuous reading of a DNA strand with clear “stair- membrane is possibly accessible, but creating solid-state like” current signals can be obtained from these porin-based nanopores with configurational identity at atomic level is nanopores coupled with enzymatic translocation speed con- extremely hard based on commonly used pore-drilling trollers (e.g., DNA polymerases or helicases). However, in techniques. This drawback can be overcome through a actual studies, double-layered phospholipid membranes are computational algorithm or an alignment with consensus highly influenced by temperature, humidity, and solution sequences to rectify current-blocking levels of nucleobases osmatic pressure. Extremely thick phospholipid membrane from batch-to-batch measurements. However, the work of often leads to failure of porin embedding, while ultrathin signal collection and data analysis would be substantially phospholipid membrane easily causes break-up or multiple increased. Following the trend of nanopore studies, porin- porin embedding under a certain voltage. Polymeric mem- based nanopores still dominate the currently laboratory and branes recently have been attracting a great amount of atten- commercialized DNA sequencing technologies. Besides, tion owing to strengths of good stability, high durability, and there are increasing number of studies on peptide or organic membrane-forming efficiency, which may be employed as small molecules sequencing based on porin-based nano- the next generation membrane for porin-based nanopores. pores. Unlike negatively charged DNA molecules, electron Several studies have made great effort to recognize DNA carriage of tested peptides becomes more complicated. homopolymers or deoxynucleotides through solid-state Some scientific groups made great achievements on design - nanopores, other studies also have achieved evaluation ing different mutation sites near the constriction area of pro - on structural variations or motion behaviors of individual tein nanopores aiming to increase the signal resolution by nucleobases as they pass through 2D membrane-based nano- introducing the electroosmotic flow (EOF) (Yu et al. 2023; pores using mathematical modeling and computer-assisted Martin-Baniandres et al. 2023; Sauciuc et al. 2024; Liu et al. machine learning, there is hitherto no report of a milestone 2022). Porin-based nanopores in the future would be with on continuous sequencing of DNA at single-base resolution more various modifications catering for different purposes, making use of solid-state nanopores. This is largely attrib- while solid-state nanopores are still on the way to address uted to inherent issues upon solid-state nanopores that result 1 3 36 Page 24 of 27 Biomedical Microdevices (2025) 27:36 technical bottlenecks before they are deemed as a counter- area for high-throughput DNA sequencing, thus reducing part of porin-based nanopores someday. the cost of sample preparation and testing. According to In a view of comparison, functionalized electrode-based recent advances and future perspectives, next generations of sensors are more suitable for quantitative analysis of DNA either functionalized electrode-based sensors or nanopores than nanopores. While nanopores exhibit more strengths require to be developed into more effective, cheaper and than functionalized electrode-based sensors when they are smarter forms to meet growingly higher demands of human used to decipher information of bases in a DNA strand. beings. Functionalized electrode-based sensors have advantages of Acknowledgements We would like to thank the Shuimu Tsinghua cost effectiveness, easy to use, less-time responses, setup Scholar Program (2023SM195) and EPSRC International Doctoral miniaturization, and onsite application. However, most of Scholars—IDS grant NCZ1080-EP/W524694/1-2323187 for the sup- them are only with a single function of target quantifica - port of this work. tion reflecting concentration results after recognized with Author contribution L.W. wrote the main manuscript. W.Z. reviewed target-specific DNA probes. Nanopores instead can dis - and revised the the manuscript. criminate multiple targets via base-dependent current lev- els. However, the target quantification based on nanopore Data availability No datasets were generated or analysed during the technologies is somehow less effective than functionalized current study. electrode-based sensors since “spike-like” signals obtained from the nonenzymatic pore-threading are unable to accu- Declarations rately reveal the amount of DNA strands passing through a nanopore within a fixed duration. Moreover, the constric - Competing interests The authors declare no competing interests. tion area of porin-based nanopores only allow biomolecules Open Access This article is licensed under a Creative Commons with cross-sectional dimensions about or less than 1 nm to Attribution 4.0 International License, which permits use, sharing, pass through, which may be more friendly to “chain-like” adaptation, distribution and reproduction in any medium or format, molecules without secondary or even more complicated as long as you give appropriate credit to the original author(s) and the structures. source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this Functionalized electrode-based sensors integrated with article are included in the article’s Creative Commons licence, unless microfluidic systems achieving multiplex target monitoring indicated otherwise in a credit line to the material. If material is not may be in high demand in the future (Ghorbanpoor et al. included in the article’s Creative Commons licence and your intended 2022). Equipped with increasingly higher sensitivity, this use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright type of sensor may be very valuable in early diagnosis of holder. To view a copy of this licence, visit h t t p : //c r e a t i v e c o m m o n s. o nucleic acid-related diseases and determination of toxins r g / l i c e n s es / b y / 4 . 0 / . in water or soil specimens from natural environments. To further reduce the fabrication cost, soft substrates may be preferred in future fabrication of functionalized electrode- References based sensors though a massively produceable printing process. Moreover, majority of soft substrates own proper- M.R. Ali, M.S. Bacchu, S. Das, S. Akter, M.M. Rahman, M.A.S. 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“ DNA Translocation Through Graphene Nanopores

Publisher: Springer Journals
Copyright: Copyright © The Author(s) 2025
ISSN: 1387-2176
eISSN: 1572-8781
DOI: 10.1007/s10544-025-00763-0
Publisher site: See Article on Publisher Site

Abstract

Screening the amount of DNA closely related to early diagnosis of diseases or decoding information in target DNA sequences for biological medicine, infectious identification, or forensic analysis are highly essential in our daily life. This review provides clear understanding of nanostructured sensors (i.e., functionalized electrode-based sensors and nanopores) working for electrochemical assessment of DNA, along with their recent advances and unaddressed issues. Crucial con- stituents for sensor functionalization, electrochemical techniques, and electrodes, used in functionalized electrode-based sensors are briefly introduced, followed by analysis of using this type of sensors for DNA determination and the compari - son of performances such as dynamic ranges and detection limits with other similar works. Subsequently, nanopore sensors including porin-based and solid-state nanopores applied for DNA sequencing are the other interests of discussion in the review. Beyond the achievement of high-resolution DNA sequencing based on porins coupled with enzymatic components, commonly used methods to solid-state nanopore creation, practical use of solid-state nanopores in DNA analysis, and computational modeling for nucleobase pore-threading simulation are depicted in more detail. Finally, conclusions in rela- tion to recent advances and future developments are described. This work offers a powerful guideline for electrochemical assessment of DNA using either functionalized electrode-based sensors or nanopores, enabling scientific groups to have an entire picture upon electrochemical nanodevices used for DNA characterization. Keywords Electrochemical · DNA · Nanosensors 1 Introduction developed. The detection of DNA through electrochemical nanostructured sensors is more promising by virtue of rapid Deoxyribonucleic acid (DNA) molecules are basic constitu- responses, simple manipulation, device portability, cost ent units in most organisms. DNA sequences contain differ - effectiveness, and mass production (Ronkainen et al. 2010). ent but specific biological information, which are used to Electrochemical nanodevices applied for DNA monitor- guide various living activities. Electrochemical assessment ing usually contain functionalized electrode-based sensors of DNA molecules has a close association with genetic and nanopores. For functionalized electrode-based sensors, engineering, genomic studies, and structural prediction of outstanding characteristics of high conductivity and ade- proteins or proteinaceous molecules, as well as medical quate bioactive sites make the sensing interface appropri- diagnoses and foodstuff quality monitoring (Castro-Wal - ate to anchor enough ‘baits’ of target molecules (e.g., probe lace et al. 2017). To make determination more facile, accu- DNA strands), thereby reaching a sensitive detection. After rate, and sensitive, various sensing techniques have been forming double-helix structures with target complements, various impacts upon the electron pathway are immedi- ately triggered, such as electron transfer promotion due to intercalation of electrochemical tags (Fig. 1g, h), or the Waye Zhang other way around, charge transfer resistance (Fig. 1a, b) or [email protected] electrostatic repulsion (Fig. 1a, c) against redox couples, Department of Chemical Engineering, Swansea University, or enabling electrochemical tags away from the electrode Swansea SA1 8EN, UK interface (Fig. 1d, e, f), thereby causing electrical signals School of Pharmaceutical Sciences, Tsinghua University, linearly vary with concentrations of target analytes. As for Beijing 100084, China 1 3 36 Page 2 of 27 Biomedical Microdevices (2025) 27:36 measuring ensembles, the usage of three-electrode system electrode-based sensors. Normally, it contains a work- remains its dominance in concentration determination of ing electrode (e.g., DNA probe covered chips), a counter target molecules after the integration with functionalized electrode, sometimes also called auxiliary electrode (e.g., 1 3 Biomedical Microdevices (2025) 27:36 Page 3 of 27 36 Fig. 1 Functionalized electrode-based sensors for electrochemical base ordering threads through the constriction domain of a assessment of DNA: a electrode surface with DNA probes only, redox nanopore subjected to an electric field force. Many scientific couples move towards against less resistance; b electrode surface with probe-target DNA duplex, increased steric hinderance causes negative or clinical studies have been successfully conducted since correlation between DNA analyte and electrochemical response; c neg- the company of Oxford Nanopore Technologies (ONT) ative correlation caused by electrostatic repulsion between negatively first turned laboratorial DNA nanopore sequencing tech - charged redox couple and DNA sequence; d electrochemical tags niques into commercial applications. Versatile detecting adhere to electrode surface modified with DNA probes only; e negative correlation caused by increased distance between electrochemical tags of microRNA, biomarkers, and small proteinaceous sub- and electrode surface after probe-target DNA duplex formed; f nega- stances via the reading of DNA-barcoded probes is recently tive correlation caused by electrochemical tags away from electrode reported based on the MinION sequencing device, one surface owing to sequence cleavage; g electrode surface with DNA of nanopore products from the ONT, which significantly probes only, providing no room for electrochemical tag intercalation; h electrode surface with probe-target DNA duplex, electrochemical tags expands the practical use of nanopore technique (Koch et intercalated into structural gaps leading to positive correlation between al. 2023). To reach clear-cut ionic current signals, Mereuta DNA analyte and electrochemical response; i Porin-based sequencing, et al., reported a novel method to ssDNA sequencing based DNA sequence threads through nanopore at lower speed controlled by on the α-hemolysin (α-HL) nanopore, where negatively motor protein; j ssDNA passes through solid-state nanopore charged AuNPs coupled to charge neutral peptide nucleic platinum) and a reference electrode (e.g., Ag/AgCl). Latest acids (PNA) were applied to specifically recognize target advances about functionalized electrode-based sensors for ssDNA strands (Mereuta et al. 2020). Another approach to the electrochemical DNA assessment reveal that two-dimen- improving the signal fidelity of DNA is to develop porin- sional (2D) nanomaterials such as graphene oxide nanorib- based nanopores with dual constriction (Verren et al. bons (GONR) (Pareek et al. 2023), transition metal carbides 2020). However, only DNA homopolymer sequences were (e.g., MXene Ti C ) (Duan et al. 2023) are often introduced assessed through the system. Conventional DNA sequenc- 3 2 onto the electrode surface, aiming to offer tremendous room ing based on porins always introduces motor proteins such for DNA probe immobilization because of the increased as DNA polymerase or helicase to enable the DNA strand to surface-area-to-volume ratio. Furthermore, electrochemi- move at the pace of individual base when threading through cal sensors coupled with enzymatic components such as the nanopore. However, enzymatic components are limited clustered regularly interspaced short palindromic repeats by various ambient factors such as temperature, solution associated proteins (CRISPR-Cas 12a) (Duan et al. 2023; pH, or osmotic pressure, which are not very suitable for You et al. 2023), restriction endonuclease EcoRI (Meftah all environments. Facing this challenge, Qing and Bayley et al. 2023) have been receiving more interest owing to the reported a method to DNA identification via chemical step - remarkable enhancement of sensitivity. Although function- ping instead of enzyme-based sequencing (Qing and Bayley alized electrode-based sensors for electrochemical DNA 2021). Since the end of 2019, the COVID-19 pandemics determination are mostly used in fields of cancer biomarker have caused serious health threat to the people all over the monitoring, environmental protection, and food security, world, making the technique of DNA sequencing highly the monotonous function of concentration quantification useful for virus infection confirmation (Stüder et al. 2021). hinders this type of sensor further development. Beyond this application, nanopore-based DNA sequencing It is noteworthy that studies on the use of nanopores for can be also applied in analysis of single-molecule methyla- assessment of biological molecules also belong to the field of tion of cell-free DNA for early warning of cancer, or various electrochemical detection, however studies on porin-based classes of DNA methylation from microbiomes and bacteria nanopores themselves are often defined as electrophysiol - (Lau et al. 2023; Tourancheau et al. 2021). Although, up to ogy. The review pays more attention to actual application date, high resolution DNA sequencing has only been real- of nanopores rather than focusing on nanopores themselves. ized on porin-based nanopores (Fig. 1i), there are still a cou- Simple nanochannels coupled with electrophoretic forces ple of issues often occurring during the formation of lipid always make nanopores exhibiting stronger versatility over membrane. Like enzymes, the form of double-layered lipid other nanodevices. Many substances in fact can be electro- membrane also requires rigorous conditions such as appro- chemically detected using nanopores such as DNA (Laszlo priate temperature and humidity, while unproper parameters et al. 2014), ribonucleic acid (RNA) (Soneson et al. 2019), employed prone to result in failure of membrane formation. small molecules (Nestorovich et al. 2002; Fan et al. 2024; Additionally, porin-based nanopores are almost all trans- Sun et al. 2025), and peptides (i.e., precursors of proteins) membrane proteins, which periodically require complicated (Wanunu 2022). Sensing of DNA molecules based on nano- protein expression and purification for one-batch mass pro - pores, in most cases, is described as a more pertinent term as duction, leaving lengthy pre-preparation prior to the formal “DNA sequencing” since nucleobase-dependent ionic cur- sequencing assay. Solid-state nanopores, someday, may rent drops are created when a sequence of DNA with random replace porin-based nanopores owing to unique advantages 1 3 36 Page 4 of 27 Biomedical Microdevices (2025) 27:36 of low cost, simple fabrication, excellent reusability, good 1.1 Electrochemical assessment of DNA using stability, and free design on nanopore sizes (Fig. 1j) (Xue et functionalized electrode-based sensors al. 2020). The sophisticated porin preparation, lipid mem- brane coverage on a micro-aperture-containing support, and Functionalized electrode-based sensors are commonly used porin embedment are no longer required when using solid- in quantitative analyses of target DNA analytes either in a state nanopores, thus avoiding the risk of membrane bro- buffered saline or a real biological solution (Table 1). ken at any unexpected moment. Notwithstanding, the poor signal-to-noise ratio (SNR), lack of active sites for assigned 1.2 Functional or bio-functional modification on anchoring of biological components nearby a solid-state sensor surfaces nanopore, and difficulty in fabrication of solid-state nano - pores with atomic-grade consistency, all make this type of The functional or bio-functional modification with nano - nanopore not feasible yet for high-resolution DNA sequenc- structures or nanocomposites on electrode surfaces is con- ing. Scientific studies in relation to DNA sequencing based sidered as an indispensable fabrication process since it on solid-state nanopores currently concentrate on machine helps in enlarging the surface area for accommodation of learning or computer-assisted modeling to understand or bioactive sites, leading to improved sensitivity and speci- provide interpretation on DNA translocation through a solid- ficity in electrochemical sensing. Functional monomers state nanopore (Jena et al. 2022). Breakthroughs regarding such as 4-aminobenzoic acid (4-ABA) (Wan et al. 2023), practical use of solid-state nanopores are not too much. melamine (Mel), and glutamic acid (GA) (Ali et al. 2023), The nanopore developed by pulling a quartz capillary has are often introduced on the surface of electrode, creating received an increasing amount of attention in recent years a biocompatible coverage via an electro-polymerization due to cost effectiveness, simple fabrication, and potential for immobilization of DNA probes. As functional layers, for large-scale production. However, sectional diameters of self-assembled monolayers (SAMs) play a vital role in fab- these artificially conical nanopores are about 3 ~ 5 nm, or rication of electrochemical sensors as their multiple prop- even more, which are double- or multiple- fold wider than erties including hybridization promotion, non-specific site general ssDNA strands (Chen et al. 2021, 2023). Choud- blocking, parallel orientation of DNA strands, and electrode hary et al., created a nanofluidic system consisting of dual lifetime improvement (Rasanang et al. 2022; Bahner et al. nanopores for single-molecular DNA sequencing, aiming to 2018; Ting et al. 2009; Nguyen and Minteer 2015; Lee et al. study behaviors of DNA capturing and threading with high 2014). Commonly used SAMs include 11-mercaptoundeca- fidelity (Choudhary et al. 2020). While for high-resolution noic acid (MUA) (Garcia-Melo et al. 2023; Han et al. 2023) reading of DNA, the setup requires further development in and 6 carbon mercaptan (MCH) (Han et al. 2023). Carbon the pore size reduction from current 10 nm to 1 nm. nanomaterials are another class of important functional lay- Interestingly, the discrimination of target DNA from other ers for electrodes. Graphene oxide nanoribbons (GONR) interferents can be realized via both classes of nanostruc- (Pareek et al. 2023) are used for increasing the surface-area- tured sensors. Functionalized electrode-based sensors allow to-volume ratio are often covered on the electrode surface target DNA molecules to specifically bind to an electrode through an electrodeposition method. Functionalized carbon surface, yielding distinctively electrochemical responses black (f-CB) is a class of CB that contains organic groups that are always several orders of magnitude higher or lower (e.g., carboxylic group (Aamri et al. 2023)), which is usu- than those of other interferents. While nanopores monitor ally prepared into a dispersed solution drop-casted on sur- base-dependent ionic current blockages as DNA sequences face of electrodes serving as a highly conductive underlayer transverse through a nanopore, achieving target DNA rec- friendly to biomolecule binding. Similarly, oxidized gra- ognition by virtue of the unique readout different from other phene dispersion is also required to undergo a drop-casting interfering sequences. This review mainly focuses on two process to form a uniform layer on glassy carbon electrodes types of DNA nanostructured sensors (i.e., functionalized (GCE), offering a good environment for immobilization of electrode-based sensors or nanopores) in combination with biomolecules or other nanostructures (Bo et al. 2011). electrochemical techniques. Miscellaneous nanodevices In some cases, DNA probes are not able to directly attach prepared, along with their sensing performances such as to the functionalized electrode surface unless there is a dynamic ranges and detection limits are carefully analyzed, cross-linking agent involved such as 1-ethyl-3-(3-dimethyl with further development or prospects for two types of DNA aminopropyl) carbodiimide (EDC) coupled to N-hydroxy nanostructured sensors provided at the end of the review. succinimide (NHS) (Wan et al. 2023), disuccinimidyl suber- This work aims to offer study groups or companies a power - ate (DSS) (Ali et al. 2023), and 3,3’-dithiopropionic acid ful scientific support in electrochemical sensing based on (DPA) (Hushiarian et al. 2015). Multiplex linkers are also functionalized electrode-based sensors or nanopores. used in some studies allowing electrode surfaces to have 1 3 Biomedical Microdevices (2025) 27:36 Page 5 of 27 36 Table 1 Functionalized electrode-based sensors used for electrochemical assessment of DNA DNA analytes Electrode modification Electro- Dynamic ranges and Purposes for electrochemi- Refs chemical LODs (pM) cal assessment techniques ARGs GCE/4-ABA/EDC/NHS/probe DNA EIS 21.6 ~ 477.12 and 21.6 Pollutants monitoring Wan et al. 2023) cDNA LIG/C15 pDNA/23 random DNA SWV 0.1 ~ 10 and 0.057 DNA hybridization Bahri et al. quantification 2023) –3 4 –4 Target DNA AuNWE/MCH/HS-DNA SWV 10 ~ 10 and 4.8 × 10 Target DNA quantification Hua et al. 2018) 5 –3 Target DNA Graphene microfiber/ZnO NWs/probe EIS 0.01 ~ 10 and 3.3 × 10 DNA hybridization Zhang et al. DNA quantification 2019) Target DNA GCE/GO/PANIw/probe DNA DPV 2.12 ~ 2.12 × 10 and DNA-based diagnosis Bo et al. 2011) 0.325 Genomic AuE/PEDOT-PSS/AuNPs/DPA/DNA DPV 3,130 ~ 10 and 0.054 Detection of white rot Hushiarian et DNA reporter fungus al. 2015) RASSF1A SPCE/PT/anti-5mC antibody/methyl- DPV 0.01 ~ 5,000 and 0.002 DNA methylation screening Daneshpour et tumor sup- ated target strand al. 2016) pressor gene HPV-16 SPCE/Fe O /AuNPs/thiolated probe DPV 0.1 ~ 10 and 100 Early warning of cervical Rasouli et al. 3 4 DNA cancer 2023) –4 6 –4 HPV-16 ITO/GONR/Ag@AuNPs/probe DNA LSV and 10 ~ 10 and 10 Early warning of cervical Pareek et al. EIS cancer 2023) HPV-18 SPCE/MXene Ti C /AuNPs/ssDNA SWV 10 ~ 5 × 10 and 1.95 Early warning of cervical Duan et al. 3 2 cancer 2023) IL6 and SPCE/AuNPs/thiolated ODN probes DPV IL6: 0.1 ~ 10 and 0.048 Early warning of ovarian Meftah et al. TGFβ1 TGFβ1: 0.05 ~ 100 and cancer 2023) 0.017 –8 6 –8 Lung cancer AnE/SnO -QD-Au/ssDNA EIS 10 ~ 10 and 3.2 × 10 Early warning of lung Richard et al. biomarker cancer 2023) DNA p53 gene MGCE/Fe O /α-Fe O @Au/HS-PNA DPV 1 ~ 10 and 0.85 Early warning of human Liu et al. 2023) 3 4 2 3 cancer p53 gene Au/MUC/ssDNA CV 0.001 ~ 100 and Early warning of human Garcia-Melo et –4 1.75 × 10 cancer al. 2023) –9 6 –10 S. flexneri ITO/P-Mel/PGA/DSS/CP DPV 10 ~ 10 and 7.4 × 10 Food quality evaluation Ali et al. 2023) DNA –7 –2 stx1 and stx2 Si-Au IDEs/AuNPs/Cht-Au/probe SWV stx1: 10 ~ 10 and Food quality evaluation Wasiewska et –7 DNA 10 al. 2023) –7 –1 stx2: 10 ~ 10 and –7 5 6 Base G and A N/P/S doped graphene paper/dsDNA CV G: 4 × 10 ~ 4 × 10 and Exceptional biomarker Jagannathan et 1.5 × 10 detection al. 2023) 5 6 A: 4.4 × 10 ~ 4.4 × 10 and 2.38 × 10 5 7 Base G and A SPCE/f-CB/glass microbead with DPV G: 5 × 10 ~ 10 and Exceptional biomarker Aamri et al. probe DNA 2.8 × 10 detection 2023) 5 7 A: 5 × 10 ~ 2 × 10 and 1.78 × 10 4 7 4 r60 Au/MCH/MUA/NHS/DANP/probe EIS 10 ~ 10 and 10 Target DNA quantification Han et al. 2023) DNA –6 –5 KRAS G12D SPE/probe DNA CV 10 ~ 1 and 10 Target DNA quantification You et al. 2023) M. Smegmatis C223AT-SPE/MCP/16S rRNA probe EIS and - Detection of antibiotic Güzel et al. DNA target SWV susceptibility 2021) specific affinity for DNA probes such as NHS coupled (e.g., graphene) or other inorganic nanomaterials (Bahri with 2, 7-diamino-1, 8-naphthyridine (DANP) (Han et al. et al. 2023). Nanoparticles, another class of frequently 2023) that prefers adsorption of cytosine bulge structures. utilized nanostructures, are regarded as one of key com- Moreover, poly-cytosine (poly-C) DNA strands (e.g., ponents in surface biofunctionalization of electrochemi- C15 pDNA) are sometimes applied as a special bio-linker cal nanosensors. Gold nanoparticles (AuNPs) are mostly owing to the strong adsorption to carbon nanostructures used nanoparticles capable of introducing various bonds 1 3 36 Page 6 of 27 Biomedical Microdevices (2025) 27:36 such as the gold-thiol (Hua et al. 2018) or gold-streptavidin biochemical events as they usually comprise of a real part (Daneshpour et al. 2016) conjugation for covalently bond- (i.e., charge transfer resistance) and an imaginary part (i.e., ing with DNA probes. Interestingly, AuNPs in combina- capacitance). Electrochemical techniques for assessment of tion with other metal or metal oxide nanoparticles such as DNA on functionalized electrode-based sensors are mostly silver and ferroferric oxide nanoparticles (Fe O NPs) can voltammetric measurements such as cyclic voltammetry 3 4 form nanoparticle-based composites as silver coated gold (CV) (You et al. 2023; Garcia-Melo et al. 2023; Jagan- nanoparticles (Ag@AuNPs) (Pareek et al. 2023) and Fe O - nathan et al. 2023), differential pulse voltammetry (DPV) 3 4 Au core–shell nanoparticles (Rasouli et al. 2023), respec- (Meftah et al. 2023; Bo et al. 2011; Hushiarian et al. 2015; tively. The increased surface area allows more DNA probes Daneshpour et al. 2016; Rasouli et al. 2023; Liu et al. 2023; anchoring on the electrode functionalized with nanoparti- Ali et al. 2023; Aamri et al. 2023), square wave voltam- cle-based composites, compared with that of untreated elec- metry (SWV) (Duan et al. 2023; Bahri et al. 2023; Hua et trode surfaces. Owning a strong magnetic feature, ferroferric al. 2018; Wasiewska et al. 2023), and linear sweep voltam- oxide nanoparticles (Fe O NPs) can be also integrated with metry (LSV) (Pareek et al. 2023). As the typical technique 3 4 DNA probes such as magnetic nanoparticle-capture probes of impedimetric measurements, electrochemical impedance (MNP-Capture probes) (Hushiarian et al. 2015), which can spectroscopy (EIS) is another effective approach to charac - be used for target DNA enrichment. Quantum dots (QD) terizing DNA at trace amount (Pareek et al. 2023; Wan et such as SnO -QD (Richard et al. 2023), belong to a spe- al. 2023; Zhang et al. 2019; Richard et al. 2023; Han et al. cial class of nanoparticles with geometrical size less than 2023). The use of EIS recent years has received a growing 100 nm. They have been recently applied in the process of number of attentions owing to powerful properties of simple electrode fabrication to achieve a downsized device with program setting, rapid analysis, high sensitivity, and setup higher sensitivity and selectivity (Kolhe et al. 2023; Hansen miniaturization. Amperometric measurements in contrast et al. 2006). Nanowire-based structures play essential roles are less reported in the determination of DNA. This is prob- in electrochemical characterization of DNA molecules. ably due to the mechanism not suitable for low concentra- Sensing devices with polyaniline nanowires (PANIws) are tion monitoring. of particular interest, since polyaniline possesses strengths Electrodes are also the important component in electro- including straightforward fabrication, large surface area, chemical sensing systems as they play a role in electron and tunable conductivity. (Maynor et al. 2002; Wu and Bein transportation from bioactive interfaces to signal transfor- 1994; Noll et al. 1998) Zinc oxide nanowires (ZnO NWs), mation devices. For functionalized electrode-based sen- a nanomaterial with a broad band gap of 3.37 eV, recently sors, screen-printed carbon electrodes (SPCE) (Duan et al. has been applied in sensor fabrication (Cao et al. 2016; 2023; Meftah et al. 2023; Daneshpour et al. 2016; Rasouli Kong et al. 2009; Tereshchenko et al. 2016). The growth of et al. 2023; Aamri et al. 2023) or gold electrodes (Hua et al. ZnO NWs on various substrates can be easily achieved via 2018; Hushiarian et al. 2015; Richard et al. 2023; Garcia- a hydrothermal method (Zhang et al. 2019). Additionally, Melo et al. 2023; Han et al. 2023) are mostly used electrodes the positively charged surface is of non-negligible charac- for electrochemical detection of DNA. SPCEs are usually teristic upon ZnO NWs, which directly attaches negatively designed as “three route” pattern catering for the three- charged probe DNA strands to the surface through electro- electrode system, of which the sensing window is always static interaction (Zhang et al. 2019). with a concentric shape to maximumly increase the effective contacting area. Other than SPCEs, the screen-printing tech- 1.3 Electrochemical techniques and electrodes nique can be also utilized in fabrication of electrodes with gold, forming screen-printed gold electrodes (SPGE) (You Electrochemical techniques suitable for functionalized elec- et al. 2023). Polydimethylsiloxane (PDMS) films are some - trode-based sensors consist of voltammetric, amperometric times used to cover around the sensing window of SPCEs and impedimetric categories. Voltammetric measurements to form a cylindrical chamber avoiding the testing solution provide information of current variation over an assigned to spread out of the electrode edge (Fig. 2a) (Yuan et al. range of potential, where peak currents normally represent 2023). In comparison to simple gold electrodes, interdigi- oxidation or reduction level of signal indicators. Ampero- tated gold microelectrodes engraved at the surface of silicon metric measurements offer a picture of current variation chip (Si-Au IDEs) have sophisticated patterns capable of versus time, and information of tested concentrations can simultaneously detecting multiple DNA sequences (Fig. 2b) be directly indicated according to a series of current levels. (Wasiewska et al. 2023). A gold electrode coupled with a Impedimetric measurements can convert the actual micro- novel 2D material of graphitic carbon nitride (g-C N ) is 3 4 environment of electrode-solution interface into an equiva- recently reported for real-time determination of 5-hydroxy- lent circuit, of which impedance signals vividly reflect the methylcytosine (5hmC) in genomic DNA molecules. The 1 3 Biomedical Microdevices (2025) 27:36 Page 7 of 27 36 Fig. 2 Various electrodes used for electrochemical assessment of c AuE attached with g-C3N4. Image reprinted with permission from DNA: a SPCE electrode coupled with a PDMS cover forming a con- Imran et al. (2023); d LIG electrode integrated with a three-electrode fined sensing window. Image reprinted with permission from Yuan et electrochemical sensing system. Image reprinted with permission from al. (2023); b structure of Si-Au IDE along with the chip inserted in a Bahri et al. (2023) holder. Image reprinted with permission from Wasiewska et al. (2023); Fig. 3 Flexible electrodes used for electrochemical analysis of DNA: of wax-on-PET chips, along with its mass production and high flex - a preparation workflow of graphene paper electrodes. Image reprinted ibility. Image reprinted with permission from Das et al. (2023) with permission from Jagannathan et al. (2023); b fabrication process use of g-C N makes the sensor having highly selective terephthalate (PET) substrates subjected to wax patterning 3 4 “antennas” capturing target DNA sequences via hydrogen are made as an underlayer for deposition of AuNPs and gra- bonding (Fig. 2c) (Imran et al. 2023). Graphene-based elec- phene oxide (GO), yielding a multiplexed electrode for pep- trodes are another commonly used electrodes applied for tide nucleic acid (PNA) adsorption. The wax-on-PET chip electrochemical assessment of DNA. Laser-induced gra- presents good flexibility and can be massively produced phene (LIG) electrodes possess similar designs like SPCEs, from batch to batch (Fig. 3b) (Das et al. 2023). Other soft which are fabricated through a photothermal pyrolysis substrates such as indium tin oxide (ITO) (Ali et al. 2023) sourced from laser pulses (Fig. 2d) (Bahri et al. 2023). can also be made as flexible electrodes, however, ITO elec - Flexible electrodes recently have earned a great amount trodes used for electrochemical analysis of DNA are less of interest owing to their light weight, disposability, and reported because of the high price of the material. Interest- low cost. Heteroatom doped graphene electrodes coated ingly, there are also only a few reports on electrochemi- on a paper substrate have been used for rapid DNA detec- cal determination of DNA using glassy carbon electrodes tion, which the cost-effective preparation starts from corn (GCE), while GCEs are top-preferred electrodes in other cobs (Fig. 3a) (Jagannathan et al. 2023). Polyethylene 1 3 36 Page 8 of 27 Biomedical Microdevices (2025) 27:36 sensing applications (Wan et al. 2023; Bo et al. 2011; Liu a versatile nanoelectrode used for label-free monitoring et al. 2023). of lung cancer DNA biomarkers (Fig. 4b) (Richard et al. 2023). The use of AuNPs enables the electrode surface to 1.4 Negative correlation between electrochemical have a uniform coverage of DNA probes, compared with responses and target DNA concentrations the surface without AuNPs. Impedimetric responses pro- duced by the sensor were observed in an increasing behav- There are many studies in which resulting electrochemical ior as more target DNA molecules captured, reaching an –8 6 outcomes present a negative correlation with concentra- extremely wide detection range of 10 ~ 10 pM, coupled –10 tions of target DNA sequences. Such inversely proportional with an ultralow LOD of 7.4 × 10 pM. The probe-target relationship can be explained as the electrostatic resistance DNA combination presented the highest charge transfer or steric hinderance sourced from the probe-target DNA resistance over other groups including SnO -QD-Au only, hybridization against the charge transfer of redox ion pairs. probe, non-complementary, and single base mismatch Wan et al., prepared an electrochemical DNA sensor DNA, providing evidence of good selectivity. The sensor for sensitive determination of antibiotic-resistant bacteria also exhibited outstanding electrode reproducibility in the (ARG) or resistance genes (ARGs) (Fig. 4a) (Wan et al. charge transfer measurement for both single-stranded DNA 2023). An increasing impedance against the electron trans- (ssDNA) and double-stranded DNA (dsDNA) cases, along 3−/4− fer of [Fe(CN) ] was observed as more target genes with a long-term stability of up to 21 days. Although target were introduced and hybridized with DNA probes. The sen- analytes were not always the biomarker for lung cancer in sor presented a linear behavior towards ARGs with different other studies in regard of cancer biomarker detection using concentrations ranging from 21.6 to 477.12 pM, reaching a electrochemical sensors, this work verified the technique detection limit of 21.6 pM. The strong environmental adapt- of EIS sometimes can reach an ultrasensitive detection ability makes the sensor successful in detecting the target towards DNA strands. Beyond this, the sensor displayed gene in actual sewage or river water specimens even inter- an extra function of detecting anti-p53 antibody. Neverthe- fering with ions and other pollutants. Richard et al. reported less, it worked only at the perfect dsDNA coverage. Liu Fig. 4 Schematic illustration of functionalized electrode-based sensors biomarker and anti-p53 antibody. Image reprinted with permission used for assessing DNA molecules, where electrochemical results are from Richard et al. (2023); c A special “one-pot” sensing strategy used caused by steric hinderance sourced from double-stranded structures: for detection of p53 genes. Image reprinted with permission from Liu a Impedimetric DNA sensor prepared for determination of ARGs. et al. (2023); d Gold based electrochemical genosensor applied for Image reprinted with permission from Wan et al. (2023); b EIS based detection of p53 gene mutation. Image reprinted with permission from electrochemical DNA sensor used for detection of lung cancer DNA Garcia-Melo et al. (2023) 1 3 Biomedical Microdevices (2025) 27:36 Page 9 of 27 36 et al. depicted a special “one-pot” sensing process for the al. 2023). In this case, the resistance against the charge 3−/4− detection of p53 gene. The Fe O /α-Fe O nanocomposite transfer of [Fe(CN) ] consists of two parts: one is the 3 4 2 3 6 tethered with gold nanoparticles served as a substrate for intense electrostatic repulsion between the redox couple 3−/4− immobilization of thiolated peptide nucleic acid (SH-PNA) of [Fe(CN) ] and the hybridized probe-target DNA probes and subsequent hybridization with target DNA, all complex, the other is the steric hinderance caused by the in a single centrifuge tube (Fig. 4c) (Liu et al. 2023). 6-mer- probe-target DNA hybridization. The sensor provided a lin- captohexanol (MCH) was also involved and attached to the ear response towards the concentration of target genes of nanocomposite for blocking non-specific binding sites or HPV-16 from 0.1 to 10 pM (Fig. 5b), along with a LOD pinholes. The resulting solution was finally cast onto the of 100 pM. Selectivity analysis was performed by compar- electrode surface to increase the steric hinderance against the ing the peak current value of DNA probe control, non-com- redox couple, causing reduced DPV signals. The dynamic plementary, three-base mismatch, one-base mismatch, and range regarding the p53 gene detection based on the sen- complementary DNA. Results revealed the sensor presented sor was 1 ~ 10 pM, with a LOD of 0.85 pM. The superior less difference in the peak current between the detection of selectivity of the sensor made it capable of accurately differ - complementary DNA and mismatch sequences. No repro- entiating single-, double-, and triple- base mismatched DNA ducibility and real sample testing were presented in their sequences. The good reproducibility was demonstrated by work. However, these results could be further improved by testing seven electrodes with the target DNA, yielding cur- another HPV-16 sensor made by Pareek et al. (Fig. 5c), both rent responses with a negligible relative standard deviation LSV and EIS characterizations were performed, reaching a –4 6 (RSD). Sensor stability was also studied, with the response wider dynamic range of 10 ~ 10 pM (Fig. 5d) and a LOD –4 at the twelfth day only presenting a 16.8% drop of the initial of 10 pM (Pareek et al. 2023). The selectivity showed the response. Furthermore, the target p53 gene was spiked into highest impedance against the target DNA but also presented human serum samples to assess the practicability of the sen- non-negligible responses after hybridization with non- sor. The ultraviolent (UV) absorbance method was used to complementary sequences. Two concentrations of HPV-16 provide a double verification that the sensor is reliable in were spiked into real samples in this work to evaluate the real clinical applications. Garcia-Melo et al. constructed a feasibility for practical usage, and reasonable recovery val- simple electrochemical genosensor for sensitive detection ues were obtained. The sensors fabricated by Rasouli et al. of the 175p2 mutation of the p53 gene (Fig. 4d) (Garcia- and Pareek et al., were also compared with other recently Melo et al. 2023). In comparison to the detection without reported electrochemical biosensors used for detection of doxorubicin (Dox), the LOD was lowered by one order HPV, showing a broader dynamic range and a competi- of magnitude in the presence of Dox, based on a detec- tive LOD. Han et al. reported an on-demand EIS sensor for tion range of 0.001 ~ 100 pM. Three non-complementary detection of the r60 DNA (Fig. 5e, f) (Han et al. 2023). The and the target DNA probes were tested using the sensor use of mixed SAMs containing MCH and MUA aimed to to evaluate the selectivity and only the target DNA probes form a dense and uniform layer for connection with organic presented reduced current signals after the hybridization linkers (e.g., NHS coupled with DANP). DNA probes fea- for both cases of with or without Dox. Nonetheless, results turing hairpin structures contain the cytosine bulge that can on reproducibility, stability, and real sample testing were covalently bind to the ligand of DANP. The charge transfer not discussed in the work. The proposed sensor was also resistance (R ) increased as more target r60 strands were ct 4 7 compared to electrochemical nanodevices reported in other captured, with linear response shown in a range of 10 ~ 10 studies, confirming a relatively wide detection range and a and a LOD of 10 pM. Zhang et al. developed a DNA lower LOD. The use of MB in the work of Meftah et al. nanosensor where the growth of ZnO NWs on the graph- (Meftah et al. 2023) results in greater currents measured as ite fiber surface was achieved via a hydrothermal method higher concentrations of target DNA are introduced, while (Fig. 5g) (Zhang et al. 2019). The EIS was used to assess decreased current reponses were observed when the other the concentration of target DNA ranging from 0.01 ~ 10 pM signal indicator of Dox is applied in this work. A relatively (Fig. 5h). Increased impedance signals were observed as reasonable explanation is the introduction of Dox causes the higher concentrations of target DNA were introduced. compaction of the superficial layer which alters the spatial After the demonstration with good signal reproducibility structure of double-helix complexes near the electrode sur- and stability, the selectivity of the sensor was also assessed face (Malanina et al. 2022). by testing non-complementary DNA (non-cDNA), single- Rasouli et al., created an electrochemical DNA nanosen- mismatched DNA, and cDNA, all at same concentration of sor for sensitive detection of human papillomavirus type 10 pM. The highest resistance of charge transfer (i.e., 250 3−/4− 16 (HPV-16), aiming to provide a reliable sensing platform Ω) against the redox ion pair of [Fe(CN) ] was recorded for early warning of cervical cancer (Fig. 5a) (Rasouli et as the target cDNA was added, while the hybridization with 1 3 36 Page 10 of 27 Biomedical Microdevices (2025) 27:36 Fig. 5 Functionalized electrode-based sensors used for electrochemi- NHS, and DANP for detection of target r60 DNA. Image reprinted cal detection of DNA, where redox ion pairs are usually suffered from with permission from Han et al. (2023); f Nyquist plot revealing steric hinderance and negative-negative charge repulsion: a SPCE impedance increased after Cb-HP probe sequence attachment and tar- coated with Fe O -Au core shell nanocomposites used for detection of get DNA hybridization. Image reprinted with permission from Han et 3 4 HPV-16. Image reprinted with permission from Rasouli et al. (2023); al. (2023); g Graphite fiber wrapped with ZnO NWs for monitoring of b DPV results with concentrations of HPV-16 ranging from 0.1 to target DNA sequences. Image reprinted with permission from Zhang 10 pM. Image reprinted with permission from Rasouli et al. (2023); c et al. (2019); h EIS measurements with concentrations of target DNA ITO electrode attached with GONR/Ag@AuNPs for determination of sequence ranging from 0.01 to 10 pM. Image reprinted with permis- HPV-16. Image reprinted with permission from Pareek et al. (2023); d sion from Zhang et al. (2019); i CRISPR-Cas 12a dependent HRCA –4 EIS measurements with concentrations of HPV-16 ranging from 10 in combination with electrochemical sensing system for detection of to 10 pM. Image reprinted with permission from Pareek et al. (2023); KRAS G12D. Image reprinted with permission from You et al. (2023) e On-demand gold-based EIS sensor functionalized with MCH, MUA, 1 3 Biomedical Microdevices (2025) 27:36 Page 11 of 27 36 single-mismatched DNA resulted in a lower resistance of electrode surface since non-target DNA sequences failed 125 Ω, and the case of the non-cDNA provided with almost to induce the trans-cleavage activity, leaving obvious cur- no resistance. The decrease of resistance is accordance with rent signals. The CRISPR-Cas 12a along with the program- the synergistic effect of steric hinderance and negatively ming CRISPR-RNA (crRNA) triggers the trans-cleavage charged repulsion. Moreover, the electrode after the probe activity once target DNA sequences hybridize with the MB DNA immobilization presented strong reusability of up to conjugated ssDNA, causing reduced peak currents. The pro- ten cycles. Compared with LODs shown in other works, a posed sensor exhibited excellent selectivity for both HPV- –3 value of 3.3 × 10 pM yielded from the sensor is considered 16 and HPV-18, while had no response towards other HPV as a highly competitive LOD, which implies that the imped- subtypes. Based on the good performance in the real urine imetric methods can offer ultrasensitive detection over other sample testing, human immunoglobulin G (IgG) and bovine electrochemical techniques particularly with redox couples serum albumin (BSA) were tested in both urinal and stan- suffered from dual hinderance. You et al., introduced the dard buffered samples to evaluate the sensor selectivity for CRISPR-Cas 12a dependent hyperbranched rolling circle non-target proteins, with negligible differences observed amplification (HRCA) into the sensing system for the elec - in current signals. Beyond a highly competitive detection trochemical detection of Kristen rat sarcoma viral onco- range of 10 ~ 500,000 pM and a LOD of 1.95 pM, the sensor gene homolog (KRAS G12D) (Fig. 5i) (You et al. 2023). also equipped with good stability and less response time, A variety of bioactive enzymes including ligase and poly- compared to other DNA detections based on CRISPR-Cas merases (e.g., phi29 and Bst 2.0) were used contributing to 12a powered electrochemical sensors. amplification of target sequences from both sense and anti- DNA probes used are mostly ssDNA strands. However, sense direction. The resulting target strands then bound to other biological sequences complementary to DNA targets the probe DNA, leading to increased spatial resistance and such as RNA are also applied for electrochemical detection more intense repulsion force against the redox couple. The of DNA. Güzel et al., developed an effective electrochemi - –6 sensor enabled a wide range of detection ranging from 10 cal sensor for detection of antibiotic susceptibility, where the –5 to 1 pM, along with a LOD of 10 pM. The good selectiv- surface of the C223AT-SPE was uniformly functionalized ity of the sensor was demonstrated by detecting the KRAS with 16S rRNA probes for recognizing M. Smegmatis DNA G12D mixed with other DNA interferents, and only the tar- targets (Fig. 7b) (Güzel et al. 2021). The backfilling solu - get owned the highest current over 300 μA. tion containing 3-mercapto-1-propanol (MCP) and tris(2- Hybridization between complementary DNA (cDNA) carboxyethyl) phosphine (TCEP), along with the probe and probe DNA (pDNA) molecules was quantified by solution was prepared for the formation of SAM. The tar- means of a LIG based electrochemical sensor (Fig. 6a) geted fragment DNA was obtained after the culture of bac- (Bahri et al. 2023). Square wave voltammogram showed a teria coupled with a series of centrifugation (Fig. 7a). Two weakened variation of peak current after series of concen- different electrochemical techniques (i.e., EIS and SWV) trations of cDNA from 0.1 to 100,000 pM were measured. were applied to achieve the detection of the M. Smegmatis Ferrocene (Fc) was used as an electrochemical tag aiming DNA target. Current values in the SWV measurement nega- to yield obvious current signals. Decreased peak currents tively varied after the introduction of DNA targets (Fig. 7d), are recorded due to Fc molecules lifted from the electrode which was in accordance with the mechanism that increased surface after the hybridization, extending the distance of electrostatic resistance or steric hinderance could debilitate electron transfer towards the electrode. A femto-level LOD the electron transfer of redox couples. While EIS results was eventually obtained using this sensing strategy. Non- presented the trend of variation in an opposite behavior complementary DNA (ncDNA) and several mismatch DNA (Fig. 7c). The impedance drastically increased after stages strands with a concentration of 100,000 pM were involved from “pre probe” and “past probe” to “past target”, imply- to evaluate the selectivity of the sensor, yielding negligible ing the positive correlation between electrochemical signals signals compared to that of the cDNA. Good reproducibility and DNA analyte concentrations. More studies revealing was investigated by the determination of cDNA at 1,000 pM this positive correlation can be found below. on three individual sensors. The sensor was also tested in tenfold diluted human serum, verifying a good adaptabil- 1.5 Positive correlation between electrochemical ity in real sample measurement. Based on a similar sens- responses and target DNA concentrations ing mechanism, Duan et al., provided an electrochemical DNA sensor integrated with a biological component of Electrochemical responses recorded have a positive corre- CRISPR-Cas 12a for highly sensitive detection of HPV- lation with target DNA concentrations, which are mainly 18 (Fig. 6b) (Duan et al. 2023). Serving as a signal indica- attributed to the use of redox tags intercalated in probe- tor, methylene blue (MB) molecules are confined near the target DNA complex. A restriction endonuclease EcoRI 1 3 36 Page 12 of 27 Biomedical Microdevices (2025) 27:36 Fig. 6 Functionalized electrode-based sensors used for electrochemi- modified with MXene Ti C and AuNPs for target DNA determination. 3 2 cal assessment of DNA, from which signal variations are sourced from Amount of MB molecules attached at electrode surface are controlled increased distance between electrochemical tags and electron transfer via target activated trans-cleavage, thus causing different electrochem - interface: a LIG based electrochemical sensor for detection of cDNA. ical signals. Image reprinted with permission from Duan et al. (2023) Image reprinted with permission from Bahri et al. (2023); b SPCE assisted bio-platform prepared by Meftah et al. was used for IL6 and TGFβ1, respectively. The selectivity towards to determine polymorphisms in both interleukin-6 (IL6) two genes was assessed by mixing with their 1-, 2- mis- and transforming growth factor β1 (TGFβ1) genes from match, and non-complementary sequences and only tar- patients suffered from ovarian cancer (Fig. 8a) (Meftah et get IL6 and TGFβ1 genes feedbacked a significant high al. 2023). MCH acting as a blocking agent was applied to response even the concentration used was 30 times lower the electrode surface to avoid any non-specific adsorption at than those of rest interferents. Moreover, the sensor was uti- active sites. MB molecules used as a signal indicator prom- lized in real samples containing TT, TG/TC, and GG/CC ised an increased peak current once they intercalated into genotypes, and achieved good results on the discrimina- the double-stranded oligonucleotide sequences (ODN). The tion in heterozygous and homozygous genes. Sensing per- sensor presented wide linear ranges of 0.1 ~ 100,000 and formances regarding the proposed sensor were compared 0.05 ~ 100 pM, along with LODs of 0.048 and 0.017 pM, with those from other reported electrochemical nanosensors 1 3 Biomedical Microdevices (2025) 27:36 Page 13 of 27 36 Fig. 7 16S rRNA probe functionalized sensors for electrochemical M. Smegmatis DNA target hybridization. Image reprinted with permis- detection of M. Smegmatis DNA target: a Preparation of M. Smegma- sion from Güzel et al. (2021); c Positively varied EIS responses after tis DNA target from bacteria culture. Image reprinted with permission a sensing process. Image reprinted with permission from Güzel et al. from Güzel et al. (2021); b Detection of M. Smegmatis DNA target (2021); d Negatively varied SWV responses after a sensing process. using C223AT-SPE, including immobilization of 16S rRNA probe and Image reprinted with permission from Güzel et al. (2021) for DNA polymorephism detection. However, target genes reaching a positive correlation between DPV responses and restriction endonuclease applied were not very consis- and concentrations of the target DNA, probably due to the tent as the current study, which were not able to provide accommodation of the AQMS in the LT-RP double-stranded convincible results in the literature comparison. Ali et al. structure. The sensor presented an extremely wide detection –9 6 developed an electrochemical DNA nanosensor for moni- range from 10 to 10 pM, along with an ultralow LOD –10 toring genes extracted from Shigella flexneri ( S. flexneri ) of 7.4 × 10 pM. The developed nanodevice can be uti- bacteria (Fig. 8b) (Ali et al. 2023). Capture probes (CP) lized for determination of other target sequences by easily were introduced forming a uniform coverage for immobili- changing the CP, LT, and RP, revealing a strong applica- zation of linear targets (LT). Report probes (RP) were also tion versatility. The largest current response was produced involved in hybridizing with the LT. The anthraquinone- by the hybridization with LT, compared with those yielded 2-sulfonic acid monohydrate sodium salt (AQMS) played from mismatched and non-complementary LT sequences. In as a role of signal indicator and was applied in the detection, detection of pathogenic bacteria, S. flexneri series exhibited 1 3 36 Page 14 of 27 Biomedical Microdevices (2025) 27:36 Fig. 8 Schematic illustration of electrochemical sensors used for (2023); c Multiplex silicon supported DNA nanosensor for simultane- assessing DNA molecules, where electrochemical results are caused ous detection of stx1 and stx2 genes. Image reprinted with permis- by electron transfer promotion in presence of signal indicators inter- sion from Wasiewska et al. (2023); d Paper-based electrochemical calated in double-stranded structures: a EcoRI assisted electrochemi- sensor with nanostructures of N-doped graphene used for nucleobase cal bio-platform prepared for determination of polymorphisms in IL6 detection of A and G from calf thymus DNA. Image reprinted with and TGFβ1 genes. Image reprinted with permission from Meftah et permission from Jagannathan et al. (2023); e f-CB/SPE based electro- al. (2023); b Flexible ITO based nanosensor used for monitoring of chemical sensor used for determination of A and G released from DNA S. flexneri genes. Image reprinted with permission from Ali et al. hydrolysis. Image reprinted with permission from Aamri et al. (2023) –7 –1 higher current signals over other categories. These demon- 10 to 10 pM, for stx1 and stx2 genes respectively, caus- strate high selectivity of the sensor for both DNA and bac- ing increase of square wave voltammetry (SWV) responses –7 teria detection. Moreover, the eminent stability makes the and an ultralow LOD of 10 pM. The nanosensor was also sensor still provide measurable signals even after very long- tested after simultaneously introducing two types of probe term storage (e.g., up to 8 weeks). The reusability was also sequences complementary to target genes, showing strong tested by breaking the DNA duplex and rebinding with the discrimination ability particularly with presence of the MB. LT several times, and results showed that not much appar- In comparison to previously reported DNA sensors used ent signal decreasing was recorded within 5 ~ 6 regeneration for detection of genes from the STEC, the sensor presented cycles. The target S. flexneri DNA added real food samples the lowest LOD. However, target genes compared were not were eventually analyzed, with recovery values ranging always stx1 and stx2. Jagannathan et al., developed a type from 90 ~ 103%, evidencing good practicability of the sen- of paper-based electrodes functionalized with nanostruc- sor. Wasiewska et al., prepared a multiplex silicon supported tures of N-, P-, and S- doped graphene and used to detect DNA nanoelectrode for simultaneous determination of stx1 calf thymus DNA (Jagannathan et al. 2023). The cathodic and stx2 genes from the Shiga toxin-producing E. coli cyclic voltammetry was applied throughout the detec- (STEC) (Fig. 8c) (Wasiewska et al. 2023). The robust func- tion towards guanine (G) and adenine (A). Results yielded tional layer with very good conductivity was achieved by from the N-doped graphene electrode present characteriz- electrodepositing AuNPs and the chitosan-gold nanocom- able current signals for both G and A, compared with other posite (Cht-Au) on the gold interdigitated electrode (Au- heteroatom doped (e.g., P-, S- doped) or untreated ones IDE), providing biocompatible circumstance for attachment that can only detect G with less discrimination (Fig. 8d). of probe DNA sequences (Wasiewska et al. 2022). MB mol- The sensor eventually presented a concentration range of 5 6 6 ecules increasingly intercalated into the probe-target DNA 4 × 10 ~ 4 × 10 pM, and a LOD of 1.5 × 10 pM for detection –7 –2 5 6 duplex as concentrations detected from 10 to 10 pM and of G, and a concentration range of 4.4 × 10 ~ 4.4 × 10 pM, 1 3 Biomedical Microdevices (2025) 27:36 Page 15 of 27 36 and a LOD of 2.38 × 10 pM for detection of A. Aamri et apparently improved. Additionally, there are many reports al., performed a similar work of determining both G and on determination of G and A, or thymine (T), based on vari- A released from DNA hydrolysis based on a f-CB assisted ous electrochemical sensors, yielding more broad ranges of electrochemical sensor (Fig. 8e) (Aamri et al. 2023). Glass determination along with several order-of-magnitude lower microbeads were used for immobilization of probe DNA LODs, compared with the current two works, indicating strands or probe-target DNA duplexes. The reaction was further development upon the sensor is needed to improve allowed to be carried out in a tube, from which the resulting sensing performances for detection of DNA bases. solution was drop-casted onto the f-CB layered electrode In the work of Bo et al. (Bo et al. 2011), the surface of surface, followed by a DPV measurement to evaluate the oxidized graphene coupled with PANIws was elaborately amount of released G or A molecules. The proposed DNA fabricated to provide an adequate, biocompatible, and highly nanosensor exhibited good selectivity towards target strands, conductive room for immobilization of probe sequences since electrochemical responses of the target are remarkably (Fig. 9a). A series of linear DPV signals were observed after higher than those of the probe only or non-complementary a wide dynamic range of 2.12 ~ 2.12 × 10 pM was tested, sequences. The repeatability was also assessed by testing where reduction peaks became stronger with the incre- three equally prepared sensors incubated with the target of ment of target DNA concentration (Fig. 9b). Daunomycin microRNA-21 at different concentrations, yielding rela - was used as the electroactive signal indicator, of which the tively consistent results with a RSD of 5.6%. In comparison reduction was achieved via transfer of electrons after the to the work of Jagannathan et al., the sensor presented in formation of probe-target double helix structure. The pro- this study enables a slightly wider detection range towards posed nanosensor demonstrated that the electrode surface both G and A, although LOD values obtained have not been modified with probe-target DNA duplexes presented the Fig. 9 a GCE modified with GO and PANIws used for detection of ful hybridization. Image reprinted with permission from Hushiarian target DNA molecules. Image reprinted with permission from Bo et et al. (2015); f DPV responses in relation to different concentrations al. (2011); b DPV responses in relation to different concentrations of of methylated target strands ranging from 0.01 to 5,000 pM. Image target DNA from g to a representing a range of 2.12 ~ 2.12 × 10 pM. reprinted with permission from Daneshpour et al. (2016); g DPV Image reprinted with permission from Bo et al. (2011); c AuNWE responses in relation to different concentrations of target probes rang - covered with MCH and thiolated DNA probes for electrochemical ing from 3,130 to 10. pM (a to g). Image reprinted with permission analysis of DNA. Image reprinted with permission from Hua et al. from Hushiarian et al. (2015); h SPCE covered with PT and anti-5mC (2018); d T4 DNA ligase involved DNA sensing mechanism, along antibody used for immobilization of methylated target strands. Nano- with presence of MNP for target sequence enrichment. Image reprinted composites of Fe3O4/TMC/Au/streptavidin hybridized with biotin- with permission from Hushiarian et al. (2015); e Gold-based biosensor labeled probe sequences forming a “sandwich” sensing strategy after coupled with PEDOT-PSS, AuNPs, and DPA used for sensitive detec- they recognize methylated target strands. Image reprinted with permis- tion of genomic DNA, an image with more details revealing success- sion from Daneshpour et al. (2016) 1 3 36 Page 16 of 27 Biomedical Microdevices (2025) 27:36 highest current response in the presence of daunomycin, only (Fig. 9d). Ru(dppz), another redox complex, was compared to cases of base mismatched sequences and non- applied in this work to produce sensitive signals after they complementary DNA. Other than the low detection limit of inserted into double-stranded structures. A positive cor- 0.325 pM, the reproducibility and repeatability were also relation between DPV signals and target DNA concentra- studied on seven sensors, with a small RSD of 1.15%. The tions was observed (Fig. 9g), yielding a LOD of 0.054 pM. storability was evaluated by placing the electrode in 0.1 M This work employs magnetic nanoparticles and DNA ligase phosphate buffered saline (PBS), pH 7.3, at 4 °C for over for separation of target sequences from non-specific sub - seven days, and current signals with a slight decrease were stances, which presents great significance in the detection observed. The proposed sensor owns a wider linear range of genomic DNA with various types, although the value coupled with a lower LOD in comparison to values shown in of detection limit is not as low as that shown in their pre- other reports. Hua et al., allowed MB molecules to undergo viously reported paper (Dutse et al. 2014). A “sandwich” a reduction into methylene white (LB) on a single gold sensing strategy for the electrochemical assessment of RAS nanowire electrode (AuNWE), serving as indicator of SWV association domain family (RASSF) 1 A tumor suppressor signals for electrochemical analysis of DNA (Fig. 9c). (Hua DNA methylation was reported by Daneshpour et al., where et al. 2018) Probe DNA sequences were fixed on the sensor the electrode was functionalized with polythiophene (PT) surface via the gold-thiol bonding, followed by the hybrid- and the anti-5-methylcytosin (anti-5mC) monoclonal anti- ization with target DNA strands to enable MB molecules body (Fig. 9h) (Daneshpour et al. 2016). Fe O nanospheres 3 4 to intercalate into the probe-target DNA duplex through the coated with N-trimethyl chitosan (TMC) are preferable for intense electrostatic effect (Dai et al. 2012; Yau et al. 2003). attachment of AuNPs, forming Fe O /TMC/Au nanocom- 3 4 However, the mismatched hybridization led to the failure posites to anchor streptavidin molecules. Biotin-labeled of MB intercalation, thus providing no reduction signal. probe sequences subsequently grew at the surface of the The nanosensor presented a linear response over concentra- nanosphere owing to the strong affinity between streptavi - –3 4 tions ranging from 10 ~ 10 pM, yielding a detection limit din and biotin. Methylated target DNA capable of being cap- –4 of 4.8 × 10 pM. The LOD was three orders of magnitude tured by the anti-5mC antibody bound to probe sequences lower than that presented in the work of Bo et al. (Bo et dragging the Fe O /TMC/Au nanocomposite nearby the 3 4 al. 2011), two orders of magnitude lower than that obtained electrode surface, forming a “sandwich” configuration for from the GCE modified with AuNPs, reduced graphene electron transfer of AuNPs (Fig. 9h). The DPV characteriza- oxide (rGO), and Adriamycin (Zhang and Jiang 2012), as tion evidenced that the genosensor had an extremely wide well as lower than results in other MB-assisted electro- concentration range of methylated DNA ranging from 0.01 –4 chemical sensors. An even lower LOD of 1.1 × 10 pM to 5,000 pM. The reduction peak current varied in a posi- was produced by Wang et al. (Wang et al. 2014), through a tive correlation with the concentration of target sequences simple GCE covered with electropolymerized Eriochrome (Fig. 9f). A reasonable explanation is the more methylated Black T (pEBT). Nevertheless, the detection range was target DNA strands are introduced, the more Fe O /TMC/ 3 4 –3 4 very narrow compared with this work (i.e., 10 ~ 10 pM). Au nanocomposites land at the electrode surface, caus- In addition, the sensor stability was also tested, however, ing reduction peak current signals with higher intensity as current signals recorded were with non-negligible decreases increased amount of gold ions are reduced into the elemental only after 6 days. A gold-based biosensor modified with gold. An estimated LOD of 0.002 pM was presented in their poly(3,4-ethyllenedioxythiophen)-poly(styrenesulfonate) work and was proved lower than LODs yielded from other (PEDOT-PSS) and AuNPs were constructed for detection relative studies. The specificity of the sensor was evaluated of the genomic DNA in white rot fungus, Ganoderma bon- by analyzing signals of methylated sequences, unmethylated ienese (Fig. 9e) (Hushiarian et al. 2015). Initially, MNP- sequences, and sheared genomic DNA, at concentrations of Capture probes bound to the complementary template and 500 and 1,000 pM, and only the methylated sequence pro- intended to link with the target DNA sequence in the pres- vided measurable responses. The excellent signal reproduc- ence of T4 DNA ligase, yielding extended DNA strands ibility and repeatable electrode fabrication make the work (35-mer). The 35-mer DNA fragment then dissociated from highly useful in many practical applications. The proposed the template after a heating treatment and was isolated from sensor was tested in real plasma solutions containing five other undesired DNA sequences under an applied magnetic different concentrations of the methylated DNA, reaching a field, followed by hybridization with DNA reporter probes good recovery range with a relatively small RSD. Addition- that were immobilized on the electrode surface by means ally, the sensor also displayed acceptable long-term stability of a self-assembled DPA monolayer (Fig. 9d). While the with a slight decrease of peak reduction current obtained use of non-complementary template resulted in the failure from electrodes already stored for 1 to 8 weeks at 4 °C. of probe-target hybridization, leaving MNP-Capture probes 1 3 Biomedical Microdevices (2025) 27:36 Page 17 of 27 36 1.6 Electrochemical assessment of DNA using membrane. Blockages against ions are created while bio- nanopore-based sensors logical molecules (e.g., DNA) pass through the pore, which are recorded via a real-time signal transformation setup. Nanopores, another class of prevalently advocated sensing Current responses with different magnitudes, shapes, and devices, have been widely developed for high-resolution dwell times, revealing information of tested sequences, are assessment of DNA molecules. In a scheme of nanopore- deemed as evaluation criteria for DNA sequencing (Deamer based sensing, ssDNA molecules thread through a nano- et al. 2016). An excellent overview reported by Crnkovic ́ dimensional channel under an applied potential, causing et al., mainly focuses on commonly used porin-based nano- blockage of ions and thus yielding current signals varied pores, outlining information about geometric dimensions with time. By virtue of rapid data recording, excellent regarding proteinaceous nanochannels (Crnković et al. precision, and cost-effectiveness, nanopores have been 2021). Among them, α-hemolysin (α-HL) and Mycobacte- employed in characterization of various biological ana- rium smegmatis porin A (MspA) are commonly reported lytes such as ssDNA or double-stranded DNA (dsDNA) porin-based nanopores, which can be massively produced sequences, aptamers (i.e., short-length of ssDNA or RNA), through a mature process of Escherichia coli ( E. coli) peptides, proteins, and DNA-peptide conjugates (Ven- induced protein expression. Although it is complicated and katesan et al. 2009). DNA translocation events are usually time-consuming, this method ensures porin-based nano- measured through ionic current when a DNA sequence pores, prepared from batch to batch, are with an identical threading through a nanopore. Other measuring protocols dimension at a molecular level. Up to now, porin-based such as tunnelling current is also used in nanopore-based nanopores have been developed into various mutated forms assessments of DNA, although there is no successful report catering for high-resolution DNA and protein sequencing. in identifying DNA bases using tunneling current (Ivanov DNA oligonucleotides with different sequences were et al. 2011). Nanopores can be largely classified into two successfully discriminated through the α-HL nanopore with subtypes namely biological nanopores and solid-state nano- a probe DNA sequence anchored at the Cys mutation site pores. Biological nanopores can be made through a vari- (Fig. 10a) (Howorka et al. 2001). Target sequences hybrid- ety of materials including toxins (Sarangi and Basu 2018), izing with the probe caused physical hindrance against ion porins (Wang et al. 2021), pore-forming proteins (Parker flows, yielding signals with a lowered current level. They and Feil 2005), and substrate-specific channels such as DNA were subsequently torn apart from its complementary domains (Langecker et al. 2012). However, the diameter of sequence due to the electric field force and threaded through channels within most of them is too large to detect DNA. the β-barrel region of the engineered α-HL nanopore, caus- Some of the porins where the size of constriction zone can ing unique downward spike-shaped signals. Nevertheless, be down to 1 ~ 2 nm are considered as terrific nanopore can - mismatched sequences (e.g., single-base altered DNA) didates for DNA sequencing. Therefore, this review mainly directly passed through the pore without any obstruction focuses on the porin-based nanopores rather than other owing to the absence of base-to-base hybridization, present- biological counterparts. Also, breakthroughs particularly ing downward spikes merely (Fig. 10b). This strategy is for ssDNA sequencing at individual-nucleobase resolution useful in recognizing target DNA sequences in hodgepodge facilitate porin-based nanopores to be developed into more DNA samples but may not be very suitable for continu- promising nanodevices suitable for high-throughput bio- ous DNA sequencing as lack of a component serving as a logical analyses or medical diagnoses. As another crucial ‘brake’ for rapid DNA translocation. Moreover, the protein nanopore subtype, solid-state nanopores are discussed at the mutation involved in this work makes the nanopore fabrica- later stage of the review. tion more complicated and increases time cost. Facing the challenge of high translocation speed of DNA, some enzy- 1.7 Porin-based nanopores for DNA sequencing matic components specific to DNA strands such as DNA polymerases or helicases were introduced to slow down the Porin-based nanopores, also named transmembrane pro- translocation event. Nanopore sensing integrated with DNA teins, are the initial elaboration of scientific groups aiming polymerases, as so-called sequencing by synthesis (SBS), for continuous DNA sequencing. The double-layer phos- has been regarded as one of routes to DNA sequencing at pholipid membrane prefers to cover onto a chip surface a single-molecule level. Tagged nucleotides are assembled containing an artificial aperture normally with a diameter into a DNA strand in the presence of DNA polymerases, of several micrometers. Driven by the electrophoretic force while tags indicative of different nucleobases dissociates (i.e., cathode in the cis- compartment and anode in the from nucleotides and are sequentially move through the trans- compartment), transmembrane proteins then move nanopore, leading to base-corresponding current signals towards the membrane and ultimately embed within the (Fig. 10c). In Kumar’s work, detectable tags containing a 1 3 36 Page 18 of 27 Biomedical Microdevices (2025) 27:36 Fig. 10 a α-HL nanopore with a DNA oligonucleotide anchored at polymerase immobilized α-HL nanopore, along with different current mutated site of Cys 17. Image reprinted with permission from Howorka blockages revealing four nucleobases. Image reprinted with permis- et al. (2001); b Probe oligo-1 hybridized with complementary oligo- sion from Kumar et al. (2012); d Statistical study on tags passing 2G sequences or one-base mutated oligo-2C, leading to different through nanopore to achieve discrimination of bases. Image reprinted threading signals. Image reprinted with permission from Howorka et with permission from Kumar et al. (2012) al. (2001); c Schematic illustration of tags threading through a DNA small molecule of coumarin coupled to polyethylene glycol A sensing ensemble of MspA nanopore integrated with (PEG) in different lengths including PEG , PEG , PEG , phi29 DNA polymerase (DNAP) was reported to suc- 16 20 24 and PEG , were prepared reflecting nucleobases of thymine cessfully realize a clear and continuous decoding of DNA (T), guanine (G), cytosine (C), and adenine (A), respectively sequences (Laszlo et al. 2014). Target analytes were elabo- (Kumar et al. 2012). Current drops with various magnitudes rately designed into a phi29 DNAP attached short length of caused by blockage of tags were statistically recorded, ssDNA linking to the target double stranded DNA (dsDNA) revealing information of four nucleobases (Fig. 10d). The molecule coupled with a second adaptor for sequence proposed method verifies an indirect DNA sequencing rely - rereading (Fig. 11a). Complementary sequences attach to ing on base-related tags. However, the preparation of tags the lipid bilayer due to presence of cholesterol ends, aim- lengthens the experimental procedure before the nanopore ing for the analyte enrichment. The ssDNA, under the force sensing. According to results, signals with respect to bases of electric field, passed through the MspA pore first, drag - of T, G, and C present a close magnitude except the base of ging the phi29 DNAP and dsDNA towards the pore as well. A, which to some extent, may increase errors in base identi- The phi29 DNAP was then blocked at the cis- entrance of fication. A possible interpretation is use of PEG chains with the MspA pore owing to the oversized proteinaceous con- similar lengths (i.e., PEG , PEG , and PEG ) for indicat- formation, conducted polymerization at a pace of single 16 20 24 ing bases of T, G, and C, while a relatively longer chain of nucleobase to reduce the speed of DNA translocation, PEG represents the base of A. It is more worth noting that yielding continuous current signals varied with clear stairs current blockages on bases shown in this study are not rig- (Fig. 11b). Comparisons in relation to ionic current read- orously consistent with outcomes presented in other works outs between the sequence measured and reference values (Stranges et al. 2016; Fuller et al. 2016), as it may be due predicted by the de Brujin quadromer map were evalu- to different chemical structures or lengths of tags that cause ated (Fig. 11b), along with the analysis of false alignment spatial blockages in different forms. based on 91 reads of phi X 174 genomic DNA aiming to 1 3 Biomedical Microdevices (2025) 27:36 Page 19 of 27 36 Fig. 11 a phi29 DNA polymerase assisted DNA sequencing based on rithm and alignment between measured current blockages and pre- MspA nanopore and continuous recording of target DNA sequences. dicted values; d Analysis regarding false alignments based on 91 reads b Prediction on current signals of previously unevaluated sequence of phi X 174 genomic DNA. Image reprinted with permission from using de Brujin quadromer map, along with the comparison with phi Laszlo et al. (2014) X 174 consensus; c Data processing by means of a level-finding algo - enhance the reliability of nanopore sequencing (Fig. 11c,d). Next development for the work was to apply a reference 1.8 Solid-state nanopores for DNA sequencing sequence to calibrate amplitude of ion current signals for the de novo DNA sequencing. Also, some locations within Solid-state nanopores for DNA sequencing can be made a piece of current signal recording were in the lack of tran- using either “top-down” or “bottom-up” methods. “Top- sient pore-threading information probably due to the phi29 down” methods such as focused ion beam (FIB) and focused DNAP movement or mathematical errors in calculation of electron beam (FEB) allow high-energy particles (e.g., gal- the level-defining algorithm (Laszlo et al. 2014). The use lium (Miles et al. 2013) and helium (Marshall et al. 2012) of DNA helicases was depicted as a future attempt to sig- ions, or electrons (Chen et al. 2017)) to penetrate the surface nificantly improve DNA sequencing because of the monoto - of thin film, creating in-plane defects with various geom - nous moving nature of helicases upon DNA strands (Laszlo etries. System temperature, beam duration, or intensity are et al. 2014). key parameters in the process of beam-induced nanopore fabrication. Altering them or one of them can yield solid- state nanopores with a very small diameter. Interestingly, electron beams acting at 2D material surfaces for a longer 1 3 36 Page 20 of 27 Biomedical Microdevices (2025) 27:36 duration also result in smaller nanopores. (Li et al. 2001) current blockages observed are not caused by a single base Reactive ion etching (RIE) applies chemically to reactive or amino acid. Nanopore contraction techniques also belong plasma (e.g., O plasma) to engrave treated surfaces, leav- to “Bottom-up” methods. Among them, metal oxide deposi- ing cavities especially for thick wafers (Bai et al. 2014), tion induced nanopore contractions are realized by introduc- which is deemed as another effective method for nanopore ing an ultrathin layer of metal oxides such as Al O , TiO , 2 3 2 creation. “Bottom-up” methods, sometimes called self- and HfO through the atomic layer deposition (Chen and Liu assembly methods to nanopore fabrication, often enable 2019). However, this technique often leads to an increase of nanopore-containing films to be constructed based on mate - membrane thickness, causing length nanopore channels that rial building blocks. In contrast with traditional nanopores are not suitable for high-resolution DNA reading. in silicon nitride substrate, there are also substantial stud- DNA sequencing based on solid-state nanopores usually ies focusing on production of hybrid nanopores. They are faces the challenge of drilling nanopores with a diameter at a class of special solid-state nanopores that are always round 1 nm. The first solid-state nanopore was successfully functionalized with bio-components for overcoming the produced within a thin layer of Si N membrane containing 3 4 drawbacks of bare solid-state nanopores including less bio- a bowl-shaped depression on the other side, via the ion-beam compatibility and difficulty of active site creation (Yusko sculpting (Fig. 12a) (Li et al. 2001). The blockage against et al. 2011; Bell et al. 2012). Yusko, et al., created hybrid ion current was verified when dsDNA threaded through the nanopores of which the surface of silicon nitride nanopore Si N nanopore with a diameter of 5 nm (Fig. 12b). This 3 4 was coated with lipid membrane. Compared with untreated work opens a new path for characterization of DNA mol- silicon nitride nanopores, the lipid-coated one can allow an ecules based on inorganic nanodevices. Nanopores with individual protein molecule translocating through the chan- further reduced diameters (i.e., 0.8 ~ 2 nm) were fabricated nel without clogging (Yusko et al. 2011). Hybrid nanopores by drilling the thin layer of SiNx (thickness of 5 ~ 8 nm) have versatile functions, however, the fabrication process coated on the Si/SiO2 chip (Fig. 12c), using as nanosensors may be time-consuming with a higher cost. Moreover, the for assessing DNA molecules (Fig. 12d). (Venta et al. 2013) length of the channel is not radically changed as most of The constriction domain of the SiNx nanopore featuring a them are still limited by the thick solid substrate, leading to mean diameter of 1.2 nm and a length of 1.7 nm is highly Fig. 12 Solid-state nanopores used for DNA sequencing: a Schematic among dA , dC , and dT . Image reprinted with permission from (30) (30) (30) illustration of a Si3N4 nanopore via argon ion-beam sculpting. Image Venta et al. (2013); f Schematic diagram of DNA sequencing on a gra- reprinted with permission from Li et al. (2001); b dsDNA translocation phene nanopore. Image at right-hand side reveals a piece of graphene event recorded. Image reprinted with permission from Li et al. (2001); layer transferred onto a silicon nitride chip containing a 5 μm aper- c SiN nanopore fabricated on a large aperture-containing Si/SiO ture. Image reprinted with permission from Schneider et al. (2010); x 2 substrate. Image reprinted with permission from Venta et al. (2013); g Translocation signals upon drilled graphene nanopore, along with d ssDNA threading through the SiN nanopore. Image reprinted with DNA translocation events with different spatial conformation and cor - permission from Venta et al. (2013); e DNA homopolymers translo- responding statistical analyses. Image reprinted with permission from cate through prepared SiN nanopore and comparison of current levels Schneider et al. (2010) 1 3 Biomedical Microdevices (2025) 27:36 Page 21 of 27 36 comparable to those in commonly used porin-based nano- the capture radius (R ) of the MoS pore (Fig. 13a). The use c 2 pores. DNA homopolymers that contain 30 bases, includ- of BmimPF6 can effectively slow down the DNA translo - ing poly (dA), poly(dC), and poly(dT), were tested using cation since the highly viscous ionic liquids provide DNA the nanopore, followed by a statistical analysis on numer- strands with intense Stokes dragging force and keep them ous threading signals to achieve the base discrimination as an entwined configuration before passing through the (Fig. 12e). However, the presented open-pore baseline is nanopore (Feng et al. 2015). Two classes of DNA analytes thick probably due to intrinsic large background noise of including DNA homopolymers (30-mers) and deoxynucleo- solid-state nanopores. Moreover, only the signal of poly(dA) tides (monophosphates) were tested using the MoS nano- is differentiable from other homopolymers according to the pore, yielding downward spikes with different amplitudes to magnitude of current blockage as there is no apparent differ - identify the four nucleobases (Fig. 13b, d). The dwell time, ence observed between the signal of poly(dC) and poly(dT). another significant nature in nanopore sensing, was also Nanodevice fabrication based on two-dimensional (2D) assessed using deoxynucleotides tested, from which results materials has been becoming a hot topic since Novoselov yielded (e.g., current drop versus dwell time) were plotted and Geim first obtained a clean graphene sheet peeled off in both merged and separate ways (Fig. 13d). Separated from a bulky graphite using the ‘scotch tape’ method in plotting contains points with different densities revealing 2004 (Novoselov et al. 2004). Garaj et al., then claimed gra- the reappearing frequency of current blockages related to phene can be applied as a trans-electrode membrane with each deoxynucleotide. This work is a successful example a thickness below 1 nm (Garaj et al. 2010). By drilling a of the use of thin film nanopores to determine biological nanopore (diameter of 22 nm) within a single layer of gra- molecules. However, there are still several issues required phene on a Si/SiO chip containing a 5 μm aperture, Schnei- to be addressed before reaching the destination of continu- der et al., aimed to provide proof of concept the possibility ous DNA sequencing at high resolution: 1) nanopores with of DNA sequencing could be realized on nanopores within reduced diameters of 1 ~ 1.2 nm are more suitable for high atomically thick membranes (Fig. 12f) (Schneider et al. resolution DNA reading, while the size of the nanopore fab- 2010). Up to 1,222 events of DNA translocation presented ricated within the MoS membrane shown in this work is as conductance signals were statistically recorded revealing 5 nm; 2) there is lack of attempts on successive reading of a different behaviors, involving the open-pore (i.e., blank), strand with random base sequence, although the ionic liquid non-, partial-, and entire- folding (Fig. 12g). Same as the of BmimPF6 is applied to remarkably reduce the transloca- work of Venta et al., there was no effort on the reduction tion speed of DNA at the current work; 3) the current-drop of nanopore noise, resulting in a thick open-pore baseline values, either for 30-mer homopolymers (e.g., A30, T30, (Fig. 12g). Merchant et al. reported a graphene single layer C30, and G30) or for deoxynucleotides (e.g., dAMP, dTMP, supported on a tandem SiN/SiO /Si composite, along with a dCMP, and dGMP), are discriminated depending on count- nanopore drilled in the graphene plane for DNA transloca- ing peaks. However, from a view of comparison, signals tion (Merchant et al. 2010). Diameters of nanopores created in relation to these two types of biomolecules are not very were about 5 ~ 10 nm, they were suitable for DNA translo- consistent, which are hard to realize reasonable identifica - cation while maybe not very appropriate for reading DNA tion on current blockages reflecting each base particularly sequences. However, these works are still regarded as a pio- in random base sequencing (Fig. 13c, d). neering attempt in solid-state nanopore based DNA sequenc- With the development of computer science, an increas- ing. For future development, enzymatic components such as ing number of study groups start to use computer-aided DNA polymerase or helicase can be introduced to overcome simulation or mathematical modeling to current signals and the challenge in reading sequences under an extremely high potential interactions yielded when DNA sequences thread translocation speed. through solid-state nanopores, especially for ones created Aleksandra Radenovic et al. reported progress on solid- within 2D thin films. Wells et al. theoretically studied ssDNA state nanopore sensing regarding discrimination of four molecules passing through a graphene nanopore based on types of nucleotides via a nanopore manufactured in a all-atom molecular dynamics and atomic-resolution Brown- MoS membrane (Fig. 13a) (Feng et al. 2015). The detec- ian dynamics (Wells et al. 2012). Likewise, Zhang et al., tion pool was separated by the MoS membrane, having the used molecular dynamic simulations to investigate DNA ‘cis’ compartment loaded with viscous room-temperature strands threading through the nanopore created within a ionic liquid (RTILs) of 1-butyl-3-methylimidazolium hexa- monolayer of hexagonal boron nitride (h-BN) for the single- fluorophosphate (BmimPF ), while the ‘trans’ compart- base identification (Zhang and Wang 2016). Moreover, all- ment loaded with KCl aqueous solution. Electrophoretic atom steered molecular dynamics (SMD) simulations can and electroosmotic forces offered a synergistic ‘push-like’ provide information about effects of nanopore geometries effect upon DNA molecules once they were trapped within within a graphene film on DNA sequencing. (Zhang et al. 1 3 36 Page 22 of 27 Biomedical Microdevices (2025) 27:36 Fig. 13 DNA sequencing based on a single layer of MoS : a Detection tion events of DNA homopolymers; c Statistical evaluation upon cur- pool separated by a MoS membrane containing a nanopore, where rent blockages of A30, T30, C30 and G30; d Statistical analysis upon cis- area is loaded with an ionic liquid of BmimPF and trans- area dAMP, dTMP, dCMP, and dGMP translocation events, of which data is loaded with KCl aqueous solution, along with a TEM image of revealing current drops versus dwell times are presented in both inte- 5 nm nanopore created within the monolayer of MoS ; b Transloca- grated and individual ways 2014). Characterizations on intrinsic properties of a novel 2 Conclusions and prospects layer with atomic thickness such as graphdiyne (GDY) and status of four types of nucleobases threading through its As the review presents, functionalized electrode-based sen- in-plane nanopore can be achieved by the first-principles sors and nanopores are two primary nanodevices used for density functional theory (Kumawat and Pathak 2021). Fur- electrochemical assessment of DNA molecules. Functional- thermore, this method can be also applied to evaluate dif- ized electrode-based sensors are often equipped with func- ferent spatial conformations of adenine (A), cytosine (C), tional layers highly effective to adsorption of DNA probe guanine (G), thymine (T) and uracil (U), when they pass sequences for sensitive detection of target DNA, providing through a graphene nanopore, as well as their effects on outcomes of electrochemical responses varied with analyte ssDNA or RNA sequencing (Nelson et al. 2010). Nonethe- concentrations. Voltammetric (e.g., DPV) and impedimetric less, the transformation from computational analyses to (e.g., EIS) measurements are mostly used for trace amount practical applications (e.g., high-resolution DNA sequenc- detection of DNA molecules, while other electrochemi- ing) for solid-state nanopores is still the common aspiration cal techniques like CV, SWV, and LSV are also frequently of scientific researchers all over the world, although the reported. Detection results reveal both positive and negative detection of DNA molecules via theoretical studies have correlation between concentrations of target DNA sequences realized in-depth development. and electrochemical signals owing to use of different sensing 3−/4− mechanisms. Redox couples (e.g., [Fe(CN) ] ) against a synergistic resistive effect made of the probe-target steric hinderance and negative charge repulsion usually lead to a typical negative correlation, while electroactive indicators 1 3 Biomedical Microdevices (2025) 27:36 Page 23 of 27 36 (e.g., MB) intercalating into structural intervals formed after in current signals being hard to differentiate and analyze. the probe-target hybridization promote the electron transfer, For example, the challenge of high background noise or low resulting in a positive correlation. SNR mainly sourced from large total capacitance of entire Although functionalized electrode-based sensors in com- nanopore-containing chips or in-plane lattice vibration of bination with electrochemical techniques often achieve out- solid materials appears almost in all types of solid-state standing performances on determination of DNA such as nanopores. A probably feasible method worth performing wide dynamic range, ultralow LOD, high selectivity, good is introducing an extra dielectric layer since successive lay- reproducibility, long storability, and reliable real sample ers can mimic tandem capacitance connection left a reduced detection, majority of them merely exhibit a single function total capacitance. Fragasso et al., offered a nice review on of concentration quantification. Multiplexed electrodes that the comparison of current noise in biological and solid- can simultaneously detect two or even more DNA analytes state nanopores (Fragasso et al. 2020). Other strategies and versatile sensors capable of screening not only DNA for lowering the background noise of nanopores are pre- strands but also macromolecules (e.g., proteins) are prob- sented in their work. Moreover, unlike porin-based nano- ably in large demand in the future. Also, flexible electrodes pores that always can couple with DNA-related enzymes based on inexpensive soft substrates and electrodes inte- to slower the transverse speed of DNA, there is extremely grated with novel 2D materials (e.g., MXenes) have potent hard to anchor such enzymatic components precisely nearby advantages of low cost, mass production, large surface area solid-state nanopores. Nanopores created within functional and light weight, which are competitive alternations that group-containing materials (e.g., graphene oxide) or metal replace conventional hard electrodes (e.g., GCE) in near organic frames (MOFs) may somehow address this issue someday. since functional groups or active metal cores exhibit strong Nanopores are a class of special electrochemical nanode- affinity towards enzymatic biomolecules via either covalent vices that are able to decipher bioinformation relying or non-covalent adsorption. Furthermore, the dimension of on nucleobase-dependent current blockages when DNA solid-state nanopores obtained from current pore-making sequences thread through the nanopore driven by an elec- techniques are unable to achieve an atomic-grade accuracy trical field force applied. Up to date, high-resolution DNA like porin-based nanopores due to the sub-nano-level atom sequencing is only realized on porin-based nanopores. α-HL arrangement of solid materials. This is currently considered and MspA nanopores as well as their mutated variants are as one of the most difficult technical bottlenecks. Creating mostly used transmembrane channels in comparison to other a pore-diameter of about or sub 1 nm within a single 2D porins. Continuous reading of a DNA strand with clear “stair- membrane is possibly accessible, but creating solid-state like” current signals can be obtained from these porin-based nanopores with configurational identity at atomic level is nanopores coupled with enzymatic translocation speed con- extremely hard based on commonly used pore-drilling trollers (e.g., DNA polymerases or helicases). However, in techniques. This drawback can be overcome through a actual studies, double-layered phospholipid membranes are computational algorithm or an alignment with consensus highly influenced by temperature, humidity, and solution sequences to rectify current-blocking levels of nucleobases osmatic pressure. Extremely thick phospholipid membrane from batch-to-batch measurements. However, the work of often leads to failure of porin embedding, while ultrathin signal collection and data analysis would be substantially phospholipid membrane easily causes break-up or multiple increased. Following the trend of nanopore studies, porin- porin embedding under a certain voltage. Polymeric mem- based nanopores still dominate the currently laboratory and branes recently have been attracting a great amount of atten- commercialized DNA sequencing technologies. Besides, tion owing to strengths of good stability, high durability, and there are increasing number of studies on peptide or organic membrane-forming efficiency, which may be employed as small molecules sequencing based on porin-based nano- the next generation membrane for porin-based nanopores. pores. Unlike negatively charged DNA molecules, electron Several studies have made great effort to recognize DNA carriage of tested peptides becomes more complicated. homopolymers or deoxynucleotides through solid-state Some scientific groups made great achievements on design - nanopores, other studies also have achieved evaluation ing different mutation sites near the constriction area of pro - on structural variations or motion behaviors of individual tein nanopores aiming to increase the signal resolution by nucleobases as they pass through 2D membrane-based nano- introducing the electroosmotic flow (EOF) (Yu et al. 2023; pores using mathematical modeling and computer-assisted Martin-Baniandres et al. 2023; Sauciuc et al. 2024; Liu et al. machine learning, there is hitherto no report of a milestone 2022). Porin-based nanopores in the future would be with on continuous sequencing of DNA at single-base resolution more various modifications catering for different purposes, making use of solid-state nanopores. This is largely attrib- while solid-state nanopores are still on the way to address uted to inherent issues upon solid-state nanopores that result 1 3 36 Page 24 of 27 Biomedical Microdevices (2025) 27:36 technical bottlenecks before they are deemed as a counter- area for high-throughput DNA sequencing, thus reducing part of porin-based nanopores someday. the cost of sample preparation and testing. According to In a view of comparison, functionalized electrode-based recent advances and future perspectives, next generations of sensors are more suitable for quantitative analysis of DNA either functionalized electrode-based sensors or nanopores than nanopores. While nanopores exhibit more strengths require to be developed into more effective, cheaper and than functionalized electrode-based sensors when they are smarter forms to meet growingly higher demands of human used to decipher information of bases in a DNA strand. beings. Functionalized electrode-based sensors have advantages of Acknowledgements We would like to thank the Shuimu Tsinghua cost effectiveness, easy to use, less-time responses, setup Scholar Program (2023SM195) and EPSRC International Doctoral miniaturization, and onsite application. However, most of Scholars—IDS grant NCZ1080-EP/W524694/1-2323187 for the sup- them are only with a single function of target quantifica - port of this work. tion reflecting concentration results after recognized with Author contribution L.W. wrote the main manuscript. W.Z. reviewed target-specific DNA probes. Nanopores instead can dis - and revised the the manuscript. criminate multiple targets via base-dependent current lev- els. However, the target quantification based on nanopore Data availability No datasets were generated or analysed during the technologies is somehow less effective than functionalized current study. electrode-based sensors since “spike-like” signals obtained from the nonenzymatic pore-threading are unable to accu- Declarations rately reveal the amount of DNA strands passing through a nanopore within a fixed duration. Moreover, the constric - Competing interests The authors declare no competing interests. tion area of porin-based nanopores only allow biomolecules Open Access This article is licensed under a Creative Commons with cross-sectional dimensions about or less than 1 nm to Attribution 4.0 International License, which permits use, sharing, pass through, which may be more friendly to “chain-like” adaptation, distribution and reproduction in any medium or format, molecules without secondary or even more complicated as long as you give appropriate credit to the original author(s) and the structures. source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this Functionalized electrode-based sensors integrated with article are included in the article’s Creative Commons licence, unless microfluidic systems achieving multiplex target monitoring indicated otherwise in a credit line to the material. If material is not may be in high demand in the future (Ghorbanpoor et al. included in the article’s Creative Commons licence and your intended 2022). Equipped with increasingly higher sensitivity, this use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright type of sensor may be very valuable in early diagnosis of holder. To view a copy of this licence, visit h t t p : //c r e a t i v e c o m m o n s. o nucleic acid-related diseases and determination of toxins r g / l i c e n s es / b y / 4 . 0 / . in water or soil specimens from natural environments. To further reduce the fabrication cost, soft substrates may be preferred in future fabrication of functionalized electrode- References based sensors though a massively produceable printing process. Moreover, majority of soft substrates own proper- M.R. Ali, M.S. Bacchu, S. Das, S. Akter, M.M. Rahman, M.A.S. 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Biomedical Microdevices – Springer Journals

Published: Jul 12, 2025

Keywords: Electrochemical; DNA; Nanosensors

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Recent progress in electrochemical assessment of DNA based on nanostructured sensors

Recent progress in electrochemical assessment of DNA based on nanostructured sensors

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Recent progress in electrochemical assessment of DNA based on nanostructured sensors

Recent progress in electrochemical assessment of DNA based on nanostructured sensors

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