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Quenching Efficiency of Quantum Dots Conjugated to Lipid Bilayers on Graphene Oxide Evaluated by Fluorescence Single Particle Tracking

Quenching Efficiency of Quantum Dots Conjugated to Lipid Bilayers on Graphene Oxide Evaluated by... applied sciences Article Quenching Efficiency of Quantum Dots Conjugated to Lipid Bilayers on Graphene Oxide Evaluated by Fluorescence Single Particle Tracking Yoshiaki Okamoto, Seiji Iwasa and Ryugo Tero * Department of Applied Chemistry and Life Science, Toyohashi University of Technology, Toyohashi 441-8580, Japan; [email protected] (Y.O.); [email protected] (S.I.) * Correspondence: [email protected] Abstract: A single particle observation of quantum dots (QDs) was performed on lipid bilayers formed on graphene oxide (GO). The long-range fluorescence quenching of GO has been applied to biosensing for various biomolecules. We demonstrated the single particle observation of a QD on supported lipid bilayers in this study, aiming to detect the quenching efficiency of lipid and protein molecules in a lipid bilayer by fluorescence single particle tacking (SPT). A single lipid bilayer or double lipid bilayers were formed on GO flakes deposited on a thermally oxidized silicon substrate by the vesicle fusion method. The QDs were conjugated on the lipid bilayers, and single particle images of the QDs were obtained under the quenching effect of GO. The quenching efficiency of a single QD was evaluated from the fluorescence intensities on the regions with and without GO. The quenching efficiency reflecting the layer numbers of the lipid bilayers was obtained. Keywords: lipid bilayer membrane; graphene oxide; fluorescence quenching; quantum dot; single particle tracking; atomic force microscopy Citation: Okamoto, Y.; Iwasa, S.; Tero, R. Quenching Efficiency of Quantum Dots Conjugated to Lipid Bilayers on Graphene Oxide Evaluated by 1. Introduction Fluorescence Single Particle Tracking. Lipid bilayers are the fundamental structure of plasma membranes and play important Appl. Sci. 2022, 12, 3733. roles as a reaction field for various membrane reactions such as the transport of material, https://doi.org/10.3390/app12083733 energy, and information into and out of cells [1–3]. Artificial lipid bilayer membranes such as the black membrane, vesicle, and supported lipid bilayer (SLB) have been used Academic Editor: Takahito Ohshiro as cell membrane model systems to understand the behaviors of lipids, peptides, and proteins in and on lipid bilayers [4–8]. The artificial lipid bilayer system at the interface Received: 20 March 2022 between a solid substrate and an aqueous solution is the SLB. The lipid bilayer exists in Accepted: 5 April 2022 the vicinity of approximately 1 nm to the substrate, and thus, has a high technical affinity Published: 7 April 2022 with functionalized surfaces and sensors [9–12]. SLBs on functional materials are valuable Publisher’s Note: MDPI stays neutral as a platform for investigating the function of membrane proteins because the lateral and with regard to jurisdictional claims in vertical distribution, and assembly of lipids and proteins, significantly affect the efficiency published maps and institutional affil- of the transportation reaction though cell membranes [2,3]. iations. Graphene oxide (GO) is a chemical derivative of graphene, which is a two-dimensional atomic sheet of sp2-carbon. GO is a single-atomic sheet comprising aromatic carbons modi- fied with oxygen functional groups such as hydroxy, epoxy, and carboxy groups [13,14]. Because of these functional groups, GO becomes hydrophilic and is available in aqueous Copyright: © 2022 by the authors. systems, whereas pristine graphene is hydrophobic. Recently, various biological appli- Licensee MDPI, Basel, Switzerland. cations of GO, utilizing its unique properties, were reported [14–22]. GO possesses a This article is an open access article unique fluorescence quenching ability that works independently of the wavelength of distributed under the terms and a donor fluorescence probe and presents a longer effective range than that of general conditions of the Creative Commons molecular accepters [23]. The quenching function of GO has been applied to biosensing Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ based on fluorescence resonance energy transfer (FRET) for DNA hybridization and protein 4.0/). binding [15,16,24,25]. In the theoretical calculation based on the Förster mechanism, the Appl. Sci. 2022, 12, 3733. https://doi.org/10.3390/app12083733 https://www.mdpi.com/journal/applsci Appl. Sci. 2022, 12, x FOR PEER REVIEW 2 of 9 Appl. Sci. 2022, 12, 3733 2 of 9 and protein binding [15,16,24,25]. In the theoretical calculation based on the Förster mech- anism, the efficiency of fluorescence quenching by the two-dimensional graphene-based materials has the dependence to the minus forth power of the distance between the donor efficiency of fluorescence quenching by the two-dimensional graphene-based materials has molecule and GO [26,27], while dye molecules assumed as a point dipole have that to the the dependence to the minus forth power of the distance between the donor molecule and minus sixth power of the distance. The former shows a longer effective range compared GO [26,27], while dye molecules assumed as a point dipole have that to the minus sixth to the latter (Figure 1). The fluorescence lifetime measurement for fluorescence-tagged power of the distance. The former shows a longer effective range compared to the latter DNA molecules at the different positions demonstrated that the quenching efficiency of (Figure 1). The fluorescence lifetime measurement for fluorescence-tagged DNA molecules GO was expressed as Equation (1) [28]: at the different positions demonstrated that the quenching efficiency of GO was expressed as Equation (1) [28]: 𝐸 = (1) E = 𝑑 (1) 1+( ) 1 + 0 where d is the distance between GO and a fluorescent probe, and d0 is the Förster distance, where d is the distance between GO and a fluorescent probe, and d is the Förster distance, at which the quenching efficiency becomes 0.5. The estimated d0 of GO is 7.5 nm [28]. It is at which the quenching efficiency becomes 0.5. The estimated d of GO is 7.5 nm [28]. It is larger than the d0 of typical dye molecules, approximately 5 nm at maximum. larger than the d of typical dye molecules, approximately 5 nm at maximum. Figure 1. Schematic comparing the distance dependence of the FRET efficiency between a dye Figure 1. Schematic comparing the distance dependence of the FRET efficiency between a dye mol- molecule and GO. The abscissa and ordinate axes represent the FRET efficiency (E) and the distance ecule and GO. The abscissa and ordinate axes represent the FRET efficiency (E) and the distance (d), (d), respectively. The effective range of the FRET to GO, whose rate of energy transfer (R ) depends eg respectively. The effective range of the FRET to GO, whose rate of energy transfer (Reg) depends on 4 6 on d , reaches longer than that to dye molecules, whose R depends on d . The typical thickness −4 eg −6 d , reaches longer than that to dye molecules, whose Reg depends on d . The typical thickness of a of a single lipid bilayer membrane is indicated. single lipid bilayer membrane is indicated. We aim to apply the fluorescence quenching of GO to detect lipids and protein We aim to apply the fluorescence quenching of GO to detect lipids and protein mol- molecules in and on lipid bilayers. A typical thickness of a lipid bilayer is approximately ecules in and on lipid bilayers. A typical thickness of a lipid bilayer is approximately 5 5 nm. Therefore, the quenching effect of GO reaches to the other side of the lipid bilayer nm. Therefore, the quenching effect of GO reaches to the other side of the lipid bilayer that is formed on GO (Figure 1). We have reported that a single lipid bilayer or double that is formed on GO (Figure 1). We have reported that a single lipid bilayer or double bilayers form on GO flakes after the SLB formation by the vesicle fusion method [29,30]. bilayers form on GO flakes after the SLB formation by the vesicle fusion method [29,30]. However, fluorescence single particle tracking (SPT) showed that dye molecules labeled to However, fluorescence single particle tracking (SPT) showed that dye molecules labeled lipids are quenched too effectively to be detected in the SLB on GO [30]. As a first step for to lipids are quenched too effectively to be detected in the SLB on GO [30]. As a first step detecting the quenching efficiency of a single molecule by the SPT, we demonstrated the for detecting the quenching efficiency of a single molecule by the SPT, we demonstrated single particle observation of the quantum dot (QD) in this study. We expected that the the single particle observation of the quantum dot (QD) in this study. We expected that fluorescence intensity of a QD is sufficiently high for the SPT even under the quenching the fluorescence intensity of a QD is sufficiently high for the SPT even under the quench- effect of GO. ing effect of GO. 2. Materials and Methods The graphene oxide was prepared through the chemical exfoliation of graphite fol- lowing the modified Hummer ’s method [31,32]. Briefly, graphite particles (Ito Graphite Co., Ltd., Kuwana, Japan) were oxidized in two steps with peroxydisulfuric acid and potas- sium permanganate in sulfuric acid, and the oxidized graphite was dispersed into pure water to prepare an aqueous suspension of single-layered GO flakes that were exfoliated from the graphite particles. Residual oxidized graphite particles and multi-layered GO Appl. Sci. 2022, 12, 3733 3 of 9 flakes were removed by centrifugation. The GO suspension was deposited by drop-casting on a piranha-cleaned thermally oxidized silicon (SiO /Si) substrate. The details of the preparation of the GO suspension and the SiO /Si substrate are described elsewhere [33]. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3- phosphothioethanol (DPPTE), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000] (PEG-DSPE) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA) and used without further purification. Chloroform solutions of DOPC, PEG-DSPE, and DPPTE were mixed in a glass vial at a molar ratio of 95:5:5  10 for SPT or (100C ):C :0 (C represents PEG-DSPE concentration, C = 5.0 or PEG PEG PEG PEG 7.5 mol%) for AFM. The PEG-DSPE was added to suppress the non-specific adsorption of the QD [30]. The mixed lipid solution was dried in a nitrogen stream followed by vacuum-drying for at least 6 h. The dried lipid film was suspended in a buffer solution (100 mM KCl, 25 mM HEPES, pH 7.4/NaOH) to obtain multilamellar vesicles. The sus- pension was extruded through 800 nm and 100 nm polycarbonate filters to prepare the unilamellar vesicles. We prepared the SLBs on the SiO /Si substrates with and without GO by the vesicle fusion method [12,34], following the protocols in the previous study [30]. The substrates were incubated in the suspension of the unilamellar vesicle at 45 C for 1 h. Excess vesicles were removed by exchanging the suspension with the buffer solution. A carboxyl-coated QD (Qdot 655 ITK™, Life Technologies, Carlsbad, CA, USA) was modified with a maleimide-hydrazide hetero-cross-linker (Quanta BioDesign Ltd., Plain City, OH, USA) and 2-(2-aminoethoxy) ethanol (AEE) using 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC), and then added to the SLB containing DPPTE [35]. AEE was co-adsorbed with the cross-linker to control the number of efficient maleimide groups on the QD surface for the covalent bond formation with DPPTE in the SLB, and to reduce multivalent conjugation of the QD with the DPPTE [30]. Single particle tracking was performed with an inverted fluorescence microscope (IX-71, Olympus, Inc., Tokyo, Japan) and a 532 nm DPSS laser. The GO/SiO /Si substrate after SLB formation was sealed in a cell made of cover glass slips with a silicone resin spacer. We adopted the diagonal illumination setup [36], which enables single molecule imaging on an opaque silicon wafer [37,38]. The single particle images of the QDs were recorded at a time resolution of 30 ms (33 frames per second) using an EM-CCD camera (iXon DU-897, Andor Technology, Ltd., Belfast, UK)). The pixel size of the SPT recording was 140.84 nm. We obtained the trajectory coordinates and fluorescence intensity of each QD from the movies using an ImageJ (NIH, http://imagej.nih.gov/ij/, (accessed on 19 March 2022)) and the Particle Tracker plug-in on the basis of the theory and protocol developed by Sbalzarini and Koumoutsakos [39]. Atomic force microscopy (AFM) topographies were obtained with a PicoPlus 5500 (Keysight Technologies, Inc., Santa Rosa, CA, USA, formerly Molecular Imaging, Corp.) us- ing a magnetically coated cantilever (Agilent Type I MAC Lever, spring constant 0.6 N/m) in the magnetic AC mode in the buffer solution at ~25 C. 3. Results and Discussion After the SLB formation on the GO/SiO /Si substrate, the position of the GO flakes was recognized in the AFM topographies as shown in Figure 2a,b. The regions with GO flakes were higher than the surrounding SiO region by ~6.5 nm or ~1.5 nm. They are assigned to double layers and a single layer of the SLB as with the previous study [29]. The typical height of a GO flake on a SiO /Si solid substrate in the AFM topographies is approximately 1.5 nm, which includes the thicknesses of GO and a water layer confined between the GO flake and the substrate [29]. Since the SiO /Si substrate is covered with a single layer of SLB, the height difference of 1.5 nm indicates a single layer of SLB existing on the GO flake, and that of 6.5 nm indicates another SLB layer, whose typical thickness is approximately 5 nm [12,29,40], stacking on the first SLB on GO. The domains of the PEG- DSPE appeared as depressed regions in Figure 2a,b as reported in the previous study [41]. The DOPC-SLB with C = 5.0 mol% and 7.0 mol% on the SiO /Si substrate without GO PEG 2 Appl. Sci. 2022, 12, x FOR PEER REVIEW 4 of 9 typical height of a GO flake on a SiO2/Si solid substrate in the AFM topographies is ap- proximately 1.5 nm, which includes the thicknesses of GO and a water layer confined be- tween the GO flake and the substrate [29]. Since the SiO2/Si substrate is covered with a single layer of SLB, the height difference of 1.5 nm indicates a single layer of SLB existing on the GO flake, and that of 6.5 nm indicates another SLB layer, whose typical thickness is approximately 5 nm [12,29,40], stacking on the first SLB on GO. The domains of the Appl. Sci. 2022, 12, 3733 4 of 9 PEG-DSPE appeared as depressed regions in Figure 2a,b as reported in the previous study [41]. The DOPC-SLB with CPEG = 5.0 mol% and 7.0 mol% on the SiO2/Si substrate without GO are shown in Figure 2c,d, respectively, for comparison. The depressed region in- creased with CPEG. The apparent AFM topography of the PEG-DSPE domains is highly are shown in Figure 2c,d, respectively, for comparison. The depressed region increased with force-dependent: in the force range of the conventional intermittent contact mode, they C . The apparent AFM topography of the PEG-DSPE domains is highly force-dependent: PEG appear lower than the surrounding SLB region where diffusing PEG-DSPE molecules ex- in the force range of the conventional intermittent contact mode, they appear lower than the ist [41,42]. It is interesting that the PEG-DSPE domains were localized on the GO flakes surrounding SLB region where diffusing PEG-DSPE molecules exist [41,42]. It is interesting (Figure 2a,b) probably due to the difference in the substrate-SLB interaction between GO that the PEG-DSPE domains were localized on the GO flakes (Figure 2a,b) probably due to and SiO2. Hydration repulsion on a SiO2 surface, which is due to the hydrogen-bonded the difference in the substrate-SLB interaction between GO and SiO . Hydration repulsion water layer to the surface hydroxy groups, reduces with the decrease in the surface hy- on a SiO surface, which is due to the hydrogen-bonded water layer to the surface hydroxy drophilicity [43]. The surface of GO is partially hydrophobic because of the pristine gra- groups, reduces with the decrease in the surface hydrophilicity [43]. The surface of GO phene patches [13,14]; therefore, it induces less hydration repulsion compared to the SiO2 is partially hydrophobic because of the pristine graphene patches [13,14]; therefore, it surface. Hydrophilic polymers, including PEG, cause thermal fluctuation repulsions induces less hydration repulsion compared to the SiO surface. Hydrophilic polymers, [41,42,44]; therefore, we surmise that the PEG-DSPE preferred the less repulsive GO re- including PEG, cause thermal fluctuation repulsions [41,42,44]; therefore, we surmise that gion than the SiO2 region. the PEG-DSPE preferred the less repulsive GO region than the SiO region. Figure 2. AFM topographies of DOPC-SLB containing PEG-DSPE on the SiO2/Si substrates (a,b) Figure 2. AFM topographies of DOPC-SLB containing PEG-DSPE on the SiO /Si substrates (a,b) with and (c,d) without GO, and their cross-section profiles at the white lines. CPEG = (a,c) 5.0%, and with and (c,d) without GO, and their cross-section profiles at the white lines. C = (a,c) 5.0%, and PEG (b,d) 7.5%. Scale bar = 500 nm. (b,d) 7.5%. Scale bar = 500 nm. The QD was conjugated to the SLB with CPEG = 5.0 mol% on the GO/SiO2/Si substrate, The QD was conjugated to the SLB with C = 5.0 mol% on the GO/SiO /Si substrate, PEG 2 to perform the SPT. Each QD appeared as bright spots in the SPT movie, as shown in to perform the SPT. Each QD appeared as bright spots in the SPT movie, as shown in Video S1 of the Supplementary Materials and its snapshot in Figure 3a. Mobile bright Video S1 of the Supplementary Materials and its snapshot in Figure 3a. Mobile bright spots spots (Figure 3a, indicated by a white arrow) were QDs conjugated with one or a few (Figure 3a, indicated by a white arrow) were QDs conjugated with one or a few DPPTE DPPTE molecules in the SLB, while immobile ones (Figure 3b, indicated by black arrows) molecules in the SLB, while immobile ones (Figure 3b, indicated by black arrows) were were QDs with an excessively multivalent conjugation or those nonspecifically adsorbed QDs with an excessively multivalent conjugation or those nonspecifically adsorbed on on the SLB. The shape of the GO flakes was recognized because of the fluorescence from the SLB. The shape of the GO flakes was recognized because of the fluorescence from GO, GO, but its intensity was sufficiently lower than that from QDs. QDs conjugated on the but its intensity was sufficiently lower than that from QDs. QDs conjugated on the SLB SLB surface were visible in the GO region even under the effect of the fluorescence surface were visible in the GO region even under the effect of the fluorescence quenching quenching by GO. The fluorescence intensity of the QDs was not homogeneous as seen in by GO. The fluorescence intensity of the QDs was not homogeneous as seen in Figure 3a. It may be because of the heterogeneous conjugation states of the QDs that possibly affect the fluorescence intensity by itself and also the effects of dissolved oxygen in the buffer solution. Oxygen induces various effects on the fluorescence of QDs including quenching, brightening, and blinking [45]. Appl. Sci. 2022, 12, x FOR PEER REVIEW 5 of 9 Figure 3a. It may be because of the heterogeneous conjugation states of the QDs that pos- sibly affect the fluorescence intensity by itself and also the effects of dissolved oxygen in the buffer solution. Oxygen induces various effects on the fluorescence of QDs including quenching, brightening, and blinking [45]. Figure 3b shows the trajectory of a mobile QD that diffused from the GO region (de- picted in blue in Figure 3b) to the SiO2 region without GO (depicted in red in Figure 3b). The fluorescence intensity of this QD during the diffusion in Figure 3b was obtained from the SPT movie (Video S1) and plotted as the time trace in Figure 3c. The fluoresce from Appl. Sci. 2022, 12, 3733 5 of 9 the QD was attenuated in the GO region (depicted in blue in Figure 3c) compared to the SiO2 region (depicted in red in Figure 3c). −7 Figure 3. (a) A single particle image of QDs conjugated on DOPC-SLB including DPPTE (5 × 10 Figure 3. (a) A single particle image of QDs conjugated on DOPC-SLB including DPPTE mol%) and PEG-DSPE (CPEG = 5.0 mol%) on the GO/SiO2/Si substrate. Representative mobile and (5  10 mol%) and PEG-DSPE (C = 5.0 mol%) on the GO/SiO /Si substrate. Representa- PEG 2 immobile QDs are indicated with the white arrow and black arrows, respectively. Scale bar = 5 μm. tive mobile and immobile QDs are indicated with the white arrow and black arrows, respectively. (b) The diffusion trajectory of the QD indicated by the white arrow in (a). Blue and red parts depict Scale bar = 5 m. (b) The diffusion trajectory of the QD indicated by the white arrow in (a). Blue the trajectory in the GO region, and the SiO2 region without GO, respectively. (c) Time trace of the and red parts depict the trajectory in the GO region, and the SiO region without GO, respectively. fluorescence intensity of the QD during the diffusion in (b). The intensities obtained in the GO re- (c) Time trace of the fluorescence intensity of the QD during the diffusion in (b). The intensities gion and the SiO2 region without GO are depicted in blue and red, respectively. The histogram of the obtained fluorescenc in the e GO intens region ity and in each the SiO regiorn egion is illustrate without d GO on ar the e depicted right. The inav blue erag and e intensi red, respectively ty of each . reg The iohistogram n is indicated of the with fluor the escence dotted li intensity ne. in each region is illustrated on the right. The average intensity of each region is indicated with the dotted line. We evaluated the fluorescence quenching efficiency (E) from the fluorescence inten- Figure 3b shows the trajectory of a mobile QD that diffused from the GO region sities of the QD at the GO and SiO2 regions, where the QD was under and free from the (depicted in blue in Figure 3b) to the SiO region without GO (depicted in red in Figure 3b). quenching effect of GO, respectively, by2 Equation (2): The fluorescence intensity of this QD during the diffusion in Figure 3b was obtained from GO the SPT movie (Video S1) and plotted as the time trace in Figure 3c. The fluoresce from the 𝐸 = 1− (2) SiO2 QD was attenuated in the GO region (depicted in blue in Figure 3c) compared to the SiO region (depicted in red in Figure 3c). where IGO and ISiO2 are the fluorescence intensity at the GO and SiO2 regions, respectively. We evaluated the fluorescence quenching efficiency (E) from the fluorescence inten- Using the average fluorescence intensities in Figure 3c for IGO and ISiO2, we obtained E = sities of the QD at the GO and SiO regions, where the QD was under and free from the 0.29 for the QD in Figure 3. Note that 2 IGO and ISiO2 of a single QD are needed to calculate E quenching effect of GO, respectively, by Equation (2): because each QD has different brightness as mentioned above. We calculated E of five other QDs at different positions from their fluorescence intensities at the GO and SiO2 GO regions (Figure S1 in the Supplementary Material). The values of IGO, ISiO2, and E of six E = 1 (2) SiO2 QDs in total are summarized in Table 1. QDs #1 (Figure 3c) and #2–#4 (Figure S1a–c) showed E in the range between 0.1–0.3, whereas E of QDs #5 and #6 (Figure S1d,e) were where I and I are the fluorescence intensity at the GO and SiO regions, respectively. GO SiO2 2 nearly zero. A negative E value was numerically obtained for QD #6 because the variation Using the average fluorescence intensities in Figure 3c for I and I , we obtained GO SiO2 in the fluorescence intensity was larger than the difference between IGO and ISiO2. E = 0.29 for the QD in Figure 3. Note that I and I of a single QD are needed to GO SiO2 calculate E because each QD has different brightness as mentioned above. We calculated E of five other QDs at different positions from their fluorescence intensities at the GO and SiO regions (Figure S1 in the Supplementary Material). The values of I , I , and E of 2 GO SiO2 six QDs in total are summarized in Table 1. QDs #1 (Figure 3c) and #2–#4 (Figure S1a–c) showed E in the range between 0.1–0.3, whereas E of QDs #5 and #6 (Figure S1d,e) were nearly zero. A negative E value was numerically obtained for QD #6 because the variation in the fluorescence intensity was larger than the difference between I and I . GO SiO2 The difference in E among the QDs is attributed to the difference in the distance between the GO flake and the QD. The relationship between E and the distance from GO (d) based on Equation (1) is plotted in Figure 4a, and the values of E of QDs #1–#5 (Table 1) are indicated. The estimated d of QDs #1–4 were 9.4, 10.9, 10.1, and 11.9 nm, respectively, and d = 17.4 nm was obtained for QD #5. These values are included in Table 1. We did not calculate d for QD #6 that had a negative value of E. Appl. Sci. 2022, 12, x FOR PEER REVIEW 6 of 9 Table 1. Quenching efficiency (E) and distance from GO (d) of QDs. Number IGO (a.u.) ISiO2 (a.u.) E d (nm) 1 14.4 20.2 0.29 9.4 2 14.5 17.8 0.19 10.9 3 22.5 29.4 0.23 10.1 4 15.1 17.5 0.13 11.9 5 20.2 20.9 0.03 17.4 6 13.3 12.9 −0.03 - a b Figure 3c. Figure S1a–e, respectively. The difference in E among the QDs is attributed to the difference in the distance be- tween the GO flake and the QD. The relationship between E and the distance from GO (d) based on Equation (1) is plotted in Figure 4a, and the values of E of QDs #1–#5 (Table 1) Appl. Sci. 2022, 12, 3733 are indicated. The estimated d of QDs #1–4 were 9.4, 10.9, 10.1, and 11.9 nm, respectively 6 of 9 , and d = 17.4 nm was obtained for QD #5. These values are included in Table 1. We did not calculate d for QD #6 that had a negative value of E. The QDs used in this study have a cylindrical shape, with a longer axis of 10 nm and Table 1. Quenching efficiency (E) and distance from GO (d) of QDs. a diameter of 5 nm, approximately [46]. Considering the size of the QD and the thickness Number I (a.u.) I (a.u.) E d (nm) GO SiO2 of a single lipid bilayer (~5 nm), d = ~10 nm obtained for QDs #1–#4 is reasonable as a distance 1from the GO to 14.4 the center of a QD 20.2 existing on a single 0.29 lipid bilayer (Figur 9.4 e 4b, 2 14.5 17.8 0.19 10.9 the left image). As shown in the AFM topographies in Figure 2a, double lipid bilayers also 3 22.5 29.4 0.23 10.1 stacked on GO. The center of the QD on the double SLB positions at d = ~15 nm (Figure 4 15.1 17.5 0.13 11.9 4b, the right image), where the QD is rarely affected by quenching of GO as shown in 20.2 20.9 0.03 17.4 Figure 4a. QDs #5 and #6, whose E was nearly zero, existed on the double lipid bilayers 6 13.3 12.9 0.03 - on GO. The difference in the quenching efficiency was attributed to the layer numbers of a b Figure 3c. Figure S1a–e, respectively. the lipid bilayers. Figure 4. (a) Relationship between the quenching efficiency (E) and the distance from GO (d) based Figure 4. (a) Relationship between the quenching efficiency (E) and the distance from GO (d) based on Equation (1). The values of E of QDs #1–#5 (Table 1) are indicated with dotted lines. (b) Schematics on Equation (1). The values of E of QDs #1–#5 (Table 1) are indicated with dotted lines. (b) Schemat- representing d of a QD on a single lipid bilayer (left) and on double lipid bilayers (right). ics representing d of a QD on a single lipid bilayer (left) and on double lipid bilayers (right). The QDs used in this study have a cylindrical shape, with a longer axis of 10 nm and a QDs are used as donor fluorophores for various FRET-based biosensing [47–50]. In diameter of 5 nm, approximately [46]. Considering the size of the QD and the thickness of a the FRET theory, the energy of a molecule in the excited state is transferred by the dipole– single lipid bilayer (~5 nm), d = ~10 nm obtained for QDs #1–#4 is reasonable as a distance dipole interaction from a donor to an acceptor. Despite QDs being semi-conductor parti- from the GO to the center of a QD existing on a single lipid bilayer (Figure 4b, the left cles, the energy of excited QDs also transfers to the acceptor molecule. The efficiency of image). As shown in the AFM topographies in Figure 2a, double lipid bilayers also stacked the energy transfer depends on the minus six power of the distance between the QD center on GO. The center of the QD on the double SLB positions at d = ~15 nm (Figure 4b, the right image), where the QD is rarely affected by quenching of GO as shown in Figure 4a. QDs #5 and #6, whose E was nearly zero, existed on the double lipid bilayers on GO. The difference in the quenching efficiency was attributed to the layer numbers of the lipid bilayers. QDs are used as donor fluorophores for various FRET-based biosensing [47–50]. In the FRET theory, the energy of a molecule in the excited state is transferred by the dipole– dipole interaction from a donor to an acceptor. Despite QDs being semi-conductor particles, the energy of excited QDs also transfers to the acceptor molecule. The efficiency of the energy transfer depends on the minus six power of the distance between the QD center and the acceptor, similarly to the case of a dye molecule as a donor [51,52]. The distance dependence of Equation (1) is derived for the FRET between a dye molecule and GO [28,53], but is also valid for the FRET between a QD and GO [54,55]. 4. Conclusions Single particle observation of a QD was performed on SLBs that were formed on GO flakes on a SiO /Si substrate. A single bilayer membrane or double bilayer membranes existed on the GO flakes. SPT measurement of the QD was achieved under the effect of GO quenching, and the fluorescence intensity of the single QD was obtained during the Appl. Sci. 2022, 12, 3733 7 of 9 lateral diffusion in the regions with and without GO to evaluate the quenching efficiency. The distance between the QD and GO that was estimated from the quenching efficiency distributed ~5 nm and ~15 nm, reflecting the layer numbers of the lipid bilayers on the GO flakes. The results of this study demonstrated the evaluation of the vertical positions of a single molecule in lipid bilayers via SPT by applying the fluorescence quenching of GO. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/app12083733/s1, Figure S1: Time course of the fluorescence intensity of quantum dots; Video S1: Single particle tracking movie. Author Contributions: Conceptualization, Y.O., S.I. and R.T.; methodology, Y.O. and R.T.; validation, Y.O., S.I. and R.T.; investigation, Y.O.; formal analysis, Y.O.; writing—original draft preparation, Y.O.; writing—review and editing, S.I. and R.T.; visualization, Y.O.; supervision, S.I. and R.T.; project administration, R.T.; funding acquisition, R.T. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by JSPS KAKENHI Grant Nos. JP20H02690 and JP20K21125, and the Nagai Foundation for Science and Technology, Japan. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We acknowledge support from the Cooperation Research Project of the Re- search Institute of Electrical Communication (RIEC), Tohoku University, and the Electronics-Inspired Interdisciplinary Research Institute (EIIRIS) Project of Toyohashi University of Technology. Conflicts of Interest: The authors declare no conflict of interest. References 1. Rothman, J.E. The Principle of Membrane Fusion in the Cell (Nobel Lecture). Angew. Chem. Int. Ed. 2014, 53, 12676–12694. [CrossRef] [PubMed] 2. Tillman, T.S.; Cascio, M. Effects of membrane lipids on ion channel structure and function. Cell Biochem. 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Quenching Efficiency of Quantum Dots Conjugated to Lipid Bilayers on Graphene Oxide Evaluated by Fluorescence Single Particle Tracking

Okamoto, Yoshiaki; Iwasa, Seiji; Tero, Ryugo
Applied Sciences , Volume 12 (8) – Apr 7, 2022
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applied sciences Article Quenching Efficiency of Quantum Dots Conjugated to Lipid Bilayers on Graphene Oxide Evaluated by Fluorescence Single Particle Tracking Yoshiaki Okamoto, Seiji Iwasa and Ryugo Tero * Department of Applied Chemistry and Life Science, Toyohashi University of Technology, Toyohashi 441-8580, Japan; [email protected] (Y.O.); [email protected] (S.I.) * Correspondence: [email protected] Abstract: A single particle observation of quantum dots (QDs) was performed on lipid bilayers formed on graphene oxide (GO). The long-range fluorescence quenching of GO has been applied to biosensing for various biomolecules. We demonstrated the single particle observation of a QD on supported lipid bilayers in this study, aiming to detect the quenching efficiency of lipid and protein molecules in a lipid bilayer by fluorescence single particle tacking (SPT). A single lipid bilayer or double lipid bilayers were formed on GO flakes deposited on a thermally oxidized silicon substrate by the vesicle fusion method. The QDs were conjugated on the lipid bilayers, and single particle images of the QDs were obtained under the quenching effect of GO. The quenching efficiency of a single QD was evaluated from the fluorescence intensities on the regions with and without GO. The quenching efficiency reflecting the layer numbers of the lipid bilayers was obtained. Keywords: lipid bilayer membrane; graphene oxide; fluorescence quenching; quantum dot; single particle tracking; atomic force microscopy Citation: Okamoto, Y.; Iwasa, S.; Tero, R. Quenching Efficiency of Quantum Dots Conjugated to Lipid Bilayers on Graphene Oxide Evaluated by 1. Introduction Fluorescence Single Particle Tracking. Lipid bilayers are the fundamental structure of plasma membranes and play important Appl. Sci. 2022, 12, 3733. roles as a reaction field for various membrane reactions such as the transport of material, https://doi.org/10.3390/app12083733 energy, and information into and out of cells [1–3]. Artificial lipid bilayer membranes such as the black membrane, vesicle, and supported lipid bilayer (SLB) have been used Academic Editor: Takahito Ohshiro as cell membrane model systems to understand the behaviors of lipids, peptides, and proteins in and on lipid bilayers [4–8]. The artificial lipid bilayer system at the interface Received: 20 March 2022 between a solid substrate and an aqueous solution is the SLB. The lipid bilayer exists in Accepted: 5 April 2022 the vicinity of approximately 1 nm to the substrate, and thus, has a high technical affinity Published: 7 April 2022 with functionalized surfaces and sensors [9–12]. SLBs on functional materials are valuable Publisher’s Note: MDPI stays neutral as a platform for investigating the function of membrane proteins because the lateral and with regard to jurisdictional claims in vertical distribution, and assembly of lipids and proteins, significantly affect the efficiency published maps and institutional affil- of the transportation reaction though cell membranes [2,3]. iations. Graphene oxide (GO) is a chemical derivative of graphene, which is a two-dimensional atomic sheet of sp2-carbon. GO is a single-atomic sheet comprising aromatic carbons modi- fied with oxygen functional groups such as hydroxy, epoxy, and carboxy groups [13,14]. Because of these functional groups, GO becomes hydrophilic and is available in aqueous Copyright: © 2022 by the authors. systems, whereas pristine graphene is hydrophobic. Recently, various biological appli- Licensee MDPI, Basel, Switzerland. cations of GO, utilizing its unique properties, were reported [14–22]. GO possesses a This article is an open access article unique fluorescence quenching ability that works independently of the wavelength of distributed under the terms and a donor fluorescence probe and presents a longer effective range than that of general conditions of the Creative Commons molecular accepters [23]. The quenching function of GO has been applied to biosensing Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ based on fluorescence resonance energy transfer (FRET) for DNA hybridization and protein 4.0/). binding [15,16,24,25]. In the theoretical calculation based on the Förster mechanism, the Appl. Sci. 2022, 12, 3733. https://doi.org/10.3390/app12083733 https://www.mdpi.com/journal/applsci Appl. Sci. 2022, 12, x FOR PEER REVIEW 2 of 9 Appl. Sci. 2022, 12, 3733 2 of 9 and protein binding [15,16,24,25]. In the theoretical calculation based on the Förster mech- anism, the efficiency of fluorescence quenching by the two-dimensional graphene-based materials has the dependence to the minus forth power of the distance between the donor efficiency of fluorescence quenching by the two-dimensional graphene-based materials has molecule and GO [26,27], while dye molecules assumed as a point dipole have that to the the dependence to the minus forth power of the distance between the donor molecule and minus sixth power of the distance. The former shows a longer effective range compared GO [26,27], while dye molecules assumed as a point dipole have that to the minus sixth to the latter (Figure 1). The fluorescence lifetime measurement for fluorescence-tagged power of the distance. The former shows a longer effective range compared to the latter DNA molecules at the different positions demonstrated that the quenching efficiency of (Figure 1). The fluorescence lifetime measurement for fluorescence-tagged DNA molecules GO was expressed as Equation (1) [28]: at the different positions demonstrated that the quenching efficiency of GO was expressed as Equation (1) [28]: 𝐸 = (1) E = 𝑑 (1) 1+( ) 1 + 0 where d is the distance between GO and a fluorescent probe, and d0 is the Förster distance, where d is the distance between GO and a fluorescent probe, and d is the Förster distance, at which the quenching efficiency becomes 0.5. The estimated d0 of GO is 7.5 nm [28]. It is at which the quenching efficiency becomes 0.5. The estimated d of GO is 7.5 nm [28]. It is larger than the d0 of typical dye molecules, approximately 5 nm at maximum. larger than the d of typical dye molecules, approximately 5 nm at maximum. Figure 1. Schematic comparing the distance dependence of the FRET efficiency between a dye Figure 1. Schematic comparing the distance dependence of the FRET efficiency between a dye mol- molecule and GO. The abscissa and ordinate axes represent the FRET efficiency (E) and the distance ecule and GO. The abscissa and ordinate axes represent the FRET efficiency (E) and the distance (d), (d), respectively. The effective range of the FRET to GO, whose rate of energy transfer (R ) depends eg respectively. The effective range of the FRET to GO, whose rate of energy transfer (Reg) depends on 4 6 on d , reaches longer than that to dye molecules, whose R depends on d . The typical thickness −4 eg −6 d , reaches longer than that to dye molecules, whose Reg depends on d . The typical thickness of a of a single lipid bilayer membrane is indicated. single lipid bilayer membrane is indicated. We aim to apply the fluorescence quenching of GO to detect lipids and protein We aim to apply the fluorescence quenching of GO to detect lipids and protein mol- molecules in and on lipid bilayers. A typical thickness of a lipid bilayer is approximately ecules in and on lipid bilayers. A typical thickness of a lipid bilayer is approximately 5 5 nm. Therefore, the quenching effect of GO reaches to the other side of the lipid bilayer nm. Therefore, the quenching effect of GO reaches to the other side of the lipid bilayer that is formed on GO (Figure 1). We have reported that a single lipid bilayer or double that is formed on GO (Figure 1). We have reported that a single lipid bilayer or double bilayers form on GO flakes after the SLB formation by the vesicle fusion method [29,30]. bilayers form on GO flakes after the SLB formation by the vesicle fusion method [29,30]. However, fluorescence single particle tracking (SPT) showed that dye molecules labeled to However, fluorescence single particle tracking (SPT) showed that dye molecules labeled lipids are quenched too effectively to be detected in the SLB on GO [30]. As a first step for to lipids are quenched too effectively to be detected in the SLB on GO [30]. As a first step detecting the quenching efficiency of a single molecule by the SPT, we demonstrated the for detecting the quenching efficiency of a single molecule by the SPT, we demonstrated single particle observation of the quantum dot (QD) in this study. We expected that the the single particle observation of the quantum dot (QD) in this study. We expected that fluorescence intensity of a QD is sufficiently high for the SPT even under the quenching the fluorescence intensity of a QD is sufficiently high for the SPT even under the quench- effect of GO. ing effect of GO. 2. Materials and Methods The graphene oxide was prepared through the chemical exfoliation of graphite fol- lowing the modified Hummer ’s method [31,32]. Briefly, graphite particles (Ito Graphite Co., Ltd., Kuwana, Japan) were oxidized in two steps with peroxydisulfuric acid and potas- sium permanganate in sulfuric acid, and the oxidized graphite was dispersed into pure water to prepare an aqueous suspension of single-layered GO flakes that were exfoliated from the graphite particles. Residual oxidized graphite particles and multi-layered GO Appl. Sci. 2022, 12, 3733 3 of 9 flakes were removed by centrifugation. The GO suspension was deposited by drop-casting on a piranha-cleaned thermally oxidized silicon (SiO /Si) substrate. The details of the preparation of the GO suspension and the SiO /Si substrate are described elsewhere [33]. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3- phosphothioethanol (DPPTE), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000] (PEG-DSPE) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA) and used without further purification. Chloroform solutions of DOPC, PEG-DSPE, and DPPTE were mixed in a glass vial at a molar ratio of 95:5:5  10 for SPT or (100C ):C :0 (C represents PEG-DSPE concentration, C = 5.0 or PEG PEG PEG PEG 7.5 mol%) for AFM. The PEG-DSPE was added to suppress the non-specific adsorption of the QD [30]. The mixed lipid solution was dried in a nitrogen stream followed by vacuum-drying for at least 6 h. The dried lipid film was suspended in a buffer solution (100 mM KCl, 25 mM HEPES, pH 7.4/NaOH) to obtain multilamellar vesicles. The sus- pension was extruded through 800 nm and 100 nm polycarbonate filters to prepare the unilamellar vesicles. We prepared the SLBs on the SiO /Si substrates with and without GO by the vesicle fusion method [12,34], following the protocols in the previous study [30]. The substrates were incubated in the suspension of the unilamellar vesicle at 45 C for 1 h. Excess vesicles were removed by exchanging the suspension with the buffer solution. A carboxyl-coated QD (Qdot 655 ITK™, Life Technologies, Carlsbad, CA, USA) was modified with a maleimide-hydrazide hetero-cross-linker (Quanta BioDesign Ltd., Plain City, OH, USA) and 2-(2-aminoethoxy) ethanol (AEE) using 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC), and then added to the SLB containing DPPTE [35]. AEE was co-adsorbed with the cross-linker to control the number of efficient maleimide groups on the QD surface for the covalent bond formation with DPPTE in the SLB, and to reduce multivalent conjugation of the QD with the DPPTE [30]. Single particle tracking was performed with an inverted fluorescence microscope (IX-71, Olympus, Inc., Tokyo, Japan) and a 532 nm DPSS laser. The GO/SiO /Si substrate after SLB formation was sealed in a cell made of cover glass slips with a silicone resin spacer. We adopted the diagonal illumination setup [36], which enables single molecule imaging on an opaque silicon wafer [37,38]. The single particle images of the QDs were recorded at a time resolution of 30 ms (33 frames per second) using an EM-CCD camera (iXon DU-897, Andor Technology, Ltd., Belfast, UK)). The pixel size of the SPT recording was 140.84 nm. We obtained the trajectory coordinates and fluorescence intensity of each QD from the movies using an ImageJ (NIH, http://imagej.nih.gov/ij/, (accessed on 19 March 2022)) and the Particle Tracker plug-in on the basis of the theory and protocol developed by Sbalzarini and Koumoutsakos [39]. Atomic force microscopy (AFM) topographies were obtained with a PicoPlus 5500 (Keysight Technologies, Inc., Santa Rosa, CA, USA, formerly Molecular Imaging, Corp.) us- ing a magnetically coated cantilever (Agilent Type I MAC Lever, spring constant 0.6 N/m) in the magnetic AC mode in the buffer solution at ~25 C. 3. Results and Discussion After the SLB formation on the GO/SiO /Si substrate, the position of the GO flakes was recognized in the AFM topographies as shown in Figure 2a,b. The regions with GO flakes were higher than the surrounding SiO region by ~6.5 nm or ~1.5 nm. They are assigned to double layers and a single layer of the SLB as with the previous study [29]. The typical height of a GO flake on a SiO /Si solid substrate in the AFM topographies is approximately 1.5 nm, which includes the thicknesses of GO and a water layer confined between the GO flake and the substrate [29]. Since the SiO /Si substrate is covered with a single layer of SLB, the height difference of 1.5 nm indicates a single layer of SLB existing on the GO flake, and that of 6.5 nm indicates another SLB layer, whose typical thickness is approximately 5 nm [12,29,40], stacking on the first SLB on GO. The domains of the PEG- DSPE appeared as depressed regions in Figure 2a,b as reported in the previous study [41]. The DOPC-SLB with C = 5.0 mol% and 7.0 mol% on the SiO /Si substrate without GO PEG 2 Appl. Sci. 2022, 12, x FOR PEER REVIEW 4 of 9 typical height of a GO flake on a SiO2/Si solid substrate in the AFM topographies is ap- proximately 1.5 nm, which includes the thicknesses of GO and a water layer confined be- tween the GO flake and the substrate [29]. Since the SiO2/Si substrate is covered with a single layer of SLB, the height difference of 1.5 nm indicates a single layer of SLB existing on the GO flake, and that of 6.5 nm indicates another SLB layer, whose typical thickness is approximately 5 nm [12,29,40], stacking on the first SLB on GO. The domains of the Appl. Sci. 2022, 12, 3733 4 of 9 PEG-DSPE appeared as depressed regions in Figure 2a,b as reported in the previous study [41]. The DOPC-SLB with CPEG = 5.0 mol% and 7.0 mol% on the SiO2/Si substrate without GO are shown in Figure 2c,d, respectively, for comparison. The depressed region in- creased with CPEG. The apparent AFM topography of the PEG-DSPE domains is highly are shown in Figure 2c,d, respectively, for comparison. The depressed region increased with force-dependent: in the force range of the conventional intermittent contact mode, they C . The apparent AFM topography of the PEG-DSPE domains is highly force-dependent: PEG appear lower than the surrounding SLB region where diffusing PEG-DSPE molecules ex- in the force range of the conventional intermittent contact mode, they appear lower than the ist [41,42]. It is interesting that the PEG-DSPE domains were localized on the GO flakes surrounding SLB region where diffusing PEG-DSPE molecules exist [41,42]. It is interesting (Figure 2a,b) probably due to the difference in the substrate-SLB interaction between GO that the PEG-DSPE domains were localized on the GO flakes (Figure 2a,b) probably due to and SiO2. Hydration repulsion on a SiO2 surface, which is due to the hydrogen-bonded the difference in the substrate-SLB interaction between GO and SiO . Hydration repulsion water layer to the surface hydroxy groups, reduces with the decrease in the surface hy- on a SiO surface, which is due to the hydrogen-bonded water layer to the surface hydroxy drophilicity [43]. The surface of GO is partially hydrophobic because of the pristine gra- groups, reduces with the decrease in the surface hydrophilicity [43]. The surface of GO phene patches [13,14]; therefore, it induces less hydration repulsion compared to the SiO2 is partially hydrophobic because of the pristine graphene patches [13,14]; therefore, it surface. Hydrophilic polymers, including PEG, cause thermal fluctuation repulsions induces less hydration repulsion compared to the SiO surface. Hydrophilic polymers, [41,42,44]; therefore, we surmise that the PEG-DSPE preferred the less repulsive GO re- including PEG, cause thermal fluctuation repulsions [41,42,44]; therefore, we surmise that gion than the SiO2 region. the PEG-DSPE preferred the less repulsive GO region than the SiO region. Figure 2. AFM topographies of DOPC-SLB containing PEG-DSPE on the SiO2/Si substrates (a,b) Figure 2. AFM topographies of DOPC-SLB containing PEG-DSPE on the SiO /Si substrates (a,b) with and (c,d) without GO, and their cross-section profiles at the white lines. CPEG = (a,c) 5.0%, and with and (c,d) without GO, and their cross-section profiles at the white lines. C = (a,c) 5.0%, and PEG (b,d) 7.5%. Scale bar = 500 nm. (b,d) 7.5%. Scale bar = 500 nm. The QD was conjugated to the SLB with CPEG = 5.0 mol% on the GO/SiO2/Si substrate, The QD was conjugated to the SLB with C = 5.0 mol% on the GO/SiO /Si substrate, PEG 2 to perform the SPT. Each QD appeared as bright spots in the SPT movie, as shown in to perform the SPT. Each QD appeared as bright spots in the SPT movie, as shown in Video S1 of the Supplementary Materials and its snapshot in Figure 3a. Mobile bright Video S1 of the Supplementary Materials and its snapshot in Figure 3a. Mobile bright spots spots (Figure 3a, indicated by a white arrow) were QDs conjugated with one or a few (Figure 3a, indicated by a white arrow) were QDs conjugated with one or a few DPPTE DPPTE molecules in the SLB, while immobile ones (Figure 3b, indicated by black arrows) molecules in the SLB, while immobile ones (Figure 3b, indicated by black arrows) were were QDs with an excessively multivalent conjugation or those nonspecifically adsorbed QDs with an excessively multivalent conjugation or those nonspecifically adsorbed on on the SLB. The shape of the GO flakes was recognized because of the fluorescence from the SLB. The shape of the GO flakes was recognized because of the fluorescence from GO, GO, but its intensity was sufficiently lower than that from QDs. QDs conjugated on the but its intensity was sufficiently lower than that from QDs. QDs conjugated on the SLB SLB surface were visible in the GO region even under the effect of the fluorescence surface were visible in the GO region even under the effect of the fluorescence quenching quenching by GO. The fluorescence intensity of the QDs was not homogeneous as seen in by GO. The fluorescence intensity of the QDs was not homogeneous as seen in Figure 3a. It may be because of the heterogeneous conjugation states of the QDs that possibly affect the fluorescence intensity by itself and also the effects of dissolved oxygen in the buffer solution. Oxygen induces various effects on the fluorescence of QDs including quenching, brightening, and blinking [45]. Appl. Sci. 2022, 12, x FOR PEER REVIEW 5 of 9 Figure 3a. It may be because of the heterogeneous conjugation states of the QDs that pos- sibly affect the fluorescence intensity by itself and also the effects of dissolved oxygen in the buffer solution. Oxygen induces various effects on the fluorescence of QDs including quenching, brightening, and blinking [45]. Figure 3b shows the trajectory of a mobile QD that diffused from the GO region (de- picted in blue in Figure 3b) to the SiO2 region without GO (depicted in red in Figure 3b). The fluorescence intensity of this QD during the diffusion in Figure 3b was obtained from the SPT movie (Video S1) and plotted as the time trace in Figure 3c. The fluoresce from Appl. Sci. 2022, 12, 3733 5 of 9 the QD was attenuated in the GO region (depicted in blue in Figure 3c) compared to the SiO2 region (depicted in red in Figure 3c). −7 Figure 3. (a) A single particle image of QDs conjugated on DOPC-SLB including DPPTE (5 × 10 Figure 3. (a) A single particle image of QDs conjugated on DOPC-SLB including DPPTE mol%) and PEG-DSPE (CPEG = 5.0 mol%) on the GO/SiO2/Si substrate. Representative mobile and (5  10 mol%) and PEG-DSPE (C = 5.0 mol%) on the GO/SiO /Si substrate. Representa- PEG 2 immobile QDs are indicated with the white arrow and black arrows, respectively. Scale bar = 5 μm. tive mobile and immobile QDs are indicated with the white arrow and black arrows, respectively. (b) The diffusion trajectory of the QD indicated by the white arrow in (a). Blue and red parts depict Scale bar = 5 m. (b) The diffusion trajectory of the QD indicated by the white arrow in (a). Blue the trajectory in the GO region, and the SiO2 region without GO, respectively. (c) Time trace of the and red parts depict the trajectory in the GO region, and the SiO region without GO, respectively. fluorescence intensity of the QD during the diffusion in (b). The intensities obtained in the GO re- (c) Time trace of the fluorescence intensity of the QD during the diffusion in (b). The intensities gion and the SiO2 region without GO are depicted in blue and red, respectively. The histogram of the obtained fluorescenc in the e GO intens region ity and in each the SiO regiorn egion is illustrate without d GO on ar the e depicted right. The inav blue erag and e intensi red, respectively ty of each . reg The iohistogram n is indicated of the with fluor the escence dotted li intensity ne. in each region is illustrated on the right. The average intensity of each region is indicated with the dotted line. We evaluated the fluorescence quenching efficiency (E) from the fluorescence inten- Figure 3b shows the trajectory of a mobile QD that diffused from the GO region sities of the QD at the GO and SiO2 regions, where the QD was under and free from the (depicted in blue in Figure 3b) to the SiO region without GO (depicted in red in Figure 3b). quenching effect of GO, respectively, by2 Equation (2): The fluorescence intensity of this QD during the diffusion in Figure 3b was obtained from GO the SPT movie (Video S1) and plotted as the time trace in Figure 3c. The fluoresce from the 𝐸 = 1− (2) SiO2 QD was attenuated in the GO region (depicted in blue in Figure 3c) compared to the SiO region (depicted in red in Figure 3c). where IGO and ISiO2 are the fluorescence intensity at the GO and SiO2 regions, respectively. We evaluated the fluorescence quenching efficiency (E) from the fluorescence inten- Using the average fluorescence intensities in Figure 3c for IGO and ISiO2, we obtained E = sities of the QD at the GO and SiO regions, where the QD was under and free from the 0.29 for the QD in Figure 3. Note that 2 IGO and ISiO2 of a single QD are needed to calculate E quenching effect of GO, respectively, by Equation (2): because each QD has different brightness as mentioned above. We calculated E of five other QDs at different positions from their fluorescence intensities at the GO and SiO2 GO regions (Figure S1 in the Supplementary Material). The values of IGO, ISiO2, and E of six E = 1 (2) SiO2 QDs in total are summarized in Table 1. QDs #1 (Figure 3c) and #2–#4 (Figure S1a–c) showed E in the range between 0.1–0.3, whereas E of QDs #5 and #6 (Figure S1d,e) were where I and I are the fluorescence intensity at the GO and SiO regions, respectively. GO SiO2 2 nearly zero. A negative E value was numerically obtained for QD #6 because the variation Using the average fluorescence intensities in Figure 3c for I and I , we obtained GO SiO2 in the fluorescence intensity was larger than the difference between IGO and ISiO2. E = 0.29 for the QD in Figure 3. Note that I and I of a single QD are needed to GO SiO2 calculate E because each QD has different brightness as mentioned above. We calculated E of five other QDs at different positions from their fluorescence intensities at the GO and SiO regions (Figure S1 in the Supplementary Material). The values of I , I , and E of 2 GO SiO2 six QDs in total are summarized in Table 1. QDs #1 (Figure 3c) and #2–#4 (Figure S1a–c) showed E in the range between 0.1–0.3, whereas E of QDs #5 and #6 (Figure S1d,e) were nearly zero. A negative E value was numerically obtained for QD #6 because the variation in the fluorescence intensity was larger than the difference between I and I . GO SiO2 The difference in E among the QDs is attributed to the difference in the distance between the GO flake and the QD. The relationship between E and the distance from GO (d) based on Equation (1) is plotted in Figure 4a, and the values of E of QDs #1–#5 (Table 1) are indicated. The estimated d of QDs #1–4 were 9.4, 10.9, 10.1, and 11.9 nm, respectively, and d = 17.4 nm was obtained for QD #5. These values are included in Table 1. We did not calculate d for QD #6 that had a negative value of E. Appl. Sci. 2022, 12, x FOR PEER REVIEW 6 of 9 Table 1. Quenching efficiency (E) and distance from GO (d) of QDs. Number IGO (a.u.) ISiO2 (a.u.) E d (nm) 1 14.4 20.2 0.29 9.4 2 14.5 17.8 0.19 10.9 3 22.5 29.4 0.23 10.1 4 15.1 17.5 0.13 11.9 5 20.2 20.9 0.03 17.4 6 13.3 12.9 −0.03 - a b Figure 3c. Figure S1a–e, respectively. The difference in E among the QDs is attributed to the difference in the distance be- tween the GO flake and the QD. The relationship between E and the distance from GO (d) based on Equation (1) is plotted in Figure 4a, and the values of E of QDs #1–#5 (Table 1) Appl. Sci. 2022, 12, 3733 are indicated. The estimated d of QDs #1–4 were 9.4, 10.9, 10.1, and 11.9 nm, respectively 6 of 9 , and d = 17.4 nm was obtained for QD #5. These values are included in Table 1. We did not calculate d for QD #6 that had a negative value of E. The QDs used in this study have a cylindrical shape, with a longer axis of 10 nm and Table 1. Quenching efficiency (E) and distance from GO (d) of QDs. a diameter of 5 nm, approximately [46]. Considering the size of the QD and the thickness Number I (a.u.) I (a.u.) E d (nm) GO SiO2 of a single lipid bilayer (~5 nm), d = ~10 nm obtained for QDs #1–#4 is reasonable as a distance 1from the GO to 14.4 the center of a QD 20.2 existing on a single 0.29 lipid bilayer (Figur 9.4 e 4b, 2 14.5 17.8 0.19 10.9 the left image). As shown in the AFM topographies in Figure 2a, double lipid bilayers also 3 22.5 29.4 0.23 10.1 stacked on GO. The center of the QD on the double SLB positions at d = ~15 nm (Figure 4 15.1 17.5 0.13 11.9 4b, the right image), where the QD is rarely affected by quenching of GO as shown in 20.2 20.9 0.03 17.4 Figure 4a. QDs #5 and #6, whose E was nearly zero, existed on the double lipid bilayers 6 13.3 12.9 0.03 - on GO. The difference in the quenching efficiency was attributed to the layer numbers of a b Figure 3c. Figure S1a–e, respectively. the lipid bilayers. Figure 4. (a) Relationship between the quenching efficiency (E) and the distance from GO (d) based Figure 4. (a) Relationship between the quenching efficiency (E) and the distance from GO (d) based on Equation (1). The values of E of QDs #1–#5 (Table 1) are indicated with dotted lines. (b) Schematics on Equation (1). The values of E of QDs #1–#5 (Table 1) are indicated with dotted lines. (b) Schemat- representing d of a QD on a single lipid bilayer (left) and on double lipid bilayers (right). ics representing d of a QD on a single lipid bilayer (left) and on double lipid bilayers (right). The QDs used in this study have a cylindrical shape, with a longer axis of 10 nm and a QDs are used as donor fluorophores for various FRET-based biosensing [47–50]. In diameter of 5 nm, approximately [46]. Considering the size of the QD and the thickness of a the FRET theory, the energy of a molecule in the excited state is transferred by the dipole– single lipid bilayer (~5 nm), d = ~10 nm obtained for QDs #1–#4 is reasonable as a distance dipole interaction from a donor to an acceptor. Despite QDs being semi-conductor parti- from the GO to the center of a QD existing on a single lipid bilayer (Figure 4b, the left cles, the energy of excited QDs also transfers to the acceptor molecule. The efficiency of image). As shown in the AFM topographies in Figure 2a, double lipid bilayers also stacked the energy transfer depends on the minus six power of the distance between the QD center on GO. The center of the QD on the double SLB positions at d = ~15 nm (Figure 4b, the right image), where the QD is rarely affected by quenching of GO as shown in Figure 4a. QDs #5 and #6, whose E was nearly zero, existed on the double lipid bilayers on GO. The difference in the quenching efficiency was attributed to the layer numbers of the lipid bilayers. QDs are used as donor fluorophores for various FRET-based biosensing [47–50]. In the FRET theory, the energy of a molecule in the excited state is transferred by the dipole– dipole interaction from a donor to an acceptor. Despite QDs being semi-conductor particles, the energy of excited QDs also transfers to the acceptor molecule. The efficiency of the energy transfer depends on the minus six power of the distance between the QD center and the acceptor, similarly to the case of a dye molecule as a donor [51,52]. The distance dependence of Equation (1) is derived for the FRET between a dye molecule and GO [28,53], but is also valid for the FRET between a QD and GO [54,55]. 4. Conclusions Single particle observation of a QD was performed on SLBs that were formed on GO flakes on a SiO /Si substrate. A single bilayer membrane or double bilayer membranes existed on the GO flakes. SPT measurement of the QD was achieved under the effect of GO quenching, and the fluorescence intensity of the single QD was obtained during the Appl. Sci. 2022, 12, 3733 7 of 9 lateral diffusion in the regions with and without GO to evaluate the quenching efficiency. The distance between the QD and GO that was estimated from the quenching efficiency distributed ~5 nm and ~15 nm, reflecting the layer numbers of the lipid bilayers on the GO flakes. The results of this study demonstrated the evaluation of the vertical positions of a single molecule in lipid bilayers via SPT by applying the fluorescence quenching of GO. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/app12083733/s1, Figure S1: Time course of the fluorescence intensity of quantum dots; Video S1: Single particle tracking movie. Author Contributions: Conceptualization, Y.O., S.I. and R.T.; methodology, Y.O. and R.T.; validation, Y.O., S.I. and R.T.; investigation, Y.O.; formal analysis, Y.O.; writing—original draft preparation, Y.O.; writing—review and editing, S.I. and R.T.; visualization, Y.O.; supervision, S.I. and R.T.; project administration, R.T.; funding acquisition, R.T. 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Journal

Applied Sciences – Multidisciplinary Digital Publishing Institute

Published: Apr 7, 2022

Keywords: lipid bilayer membrane; graphene oxide; fluorescence quenching; quantum dot; single particle tracking; atomic force microscopy

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