Quantitative Counting of Single Fluorescent Molecules by Combined

Apr 29, 2008 - Aleeta M. Powe , Susmita Das , Mark Lowry , Bilal El-Zahab , Sayo O. Fakayode , Maxwell L. Geng , Gary A. Baker , Lin Wang , Matthew E...
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Anal. Chem. 2008, 80, 3999–4006

Quantitative Counting of Single Fluorescent Molecules by Combined Electrochemical Adsorption Accumulation and Total Internal Reflection Fluorescence Microscopy Lu Li, Xinzhe Tian,† Guizheng Zou, Zhikun Shi, Xiaoli Zhang,* and Wenrui Jin* School of Chemistry and Chemical Engineering and Key Laboratory of Colloid and Interface Chemistry, Ministry of Education, Shandong University, Jinan 250100, China We developed an ultrasensitive quantitative single-molecule imaging method for fluorescent molecules using a combination of electrochemical adsorption accumulation and total internal reflection fluorescence microscopy (TIRFM). We chose rhodamine 6G (R6G, fluorescence dye) or goat antirat IgG(H+L) (IgG(H+L)-488), a protein labeled by Alexa Fluor 488 or DNA labeled by 6-CR6G (DNA-R6G) as the model molecules. The fluorescent molecules were accumulated on a light transparent indium tin oxide (ITO) conductive microscope coverslip using electrochemical adsorption in a stirred solution. Then, images of the fluorescent molecules accumulated on the ITO coverslip sized 40 × 40 µm were acquired using an objective-type TIRFM instrument coupled with a high-sensitivity electron multiplying chargecoupled device. One hundred images of the fluorescent molecules accumulated on the coverslip were taken consecutively, one by one, by moving the coverslip with the aid of a three-dimensional positioner. Finally, we counted the number of fluorescent spots corresponding to single fluorescent molecules on the images. The linear relationships between the number of fluorescent molecules and the concentration were obtained in the range of 5 × 10-15 to 5 × 10-12 mol/L for R6G, 3 × 10-15 to 2 × 10-12 mol/L for IgG(H+L)-488, and 3 × 10-15 to 2 × 10-12 mol/L for DNA-R6G. Recently, much attention has been focused on single-molecule detection (SMD) in solutions. Fluorescence detection techniques coupled with hydrodynamically focused flows,1–15 microdroplets,16–19 capillary or microchannels,20–34 nanometer-scale pores,35 confocal * Corresponding author. E-mail: [email protected]. Fax +86-531-8856-5167. † Present address: Chemical Engineering and Pharmaceutics College, Henan University of Science and Technology, Luoyang 471003, China. (1) Zarrin, F.; Dovichi, N. J. Anal. Chem. 1985, 57, 2690–2692. (2) Nguyen, D. C.; Keller, R. A.; Jett, J. H.; Martin, J. C Anal. Chem. 1987, 59, 2158–2161. (3) Peck, K.; Stryer, L.; Glazer, A. N.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 4087–4091. (4) Shera, E. B.; Seitzinger, N. K.; Davis, L. M.; Keller, R. A.; Soper, S. A. Chem. Phys. Lett. 1990, 174, 553–557. (5) Soper, S. A.; Davis, L. M.; Shera, E. B. J. Opt. Soc. Am. B 1992, 9, 1761– 1769. (6) Castro, A.; Fairfield, F. R.; Shera, E. B. Anal. Chem. 1993, 65, 849–852. (7) Goodwin, P. M.; Johnson, M. E.; Martin, J. C.; Ambrose, W. P.; Marrone, B. L.; Jett, J. H.; Keller, R. A. Nucleic Acids Res. 1993, 21, 803–806. 10.1021/ac702534h CCC: $40.75  2008 American Chemical Society Published on Web 04/29/2008

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copy66,67 are the most popular techniques in SMD. Of these SMD studies with fluorescence detection, only a few reports are concerned with quantitative analysis. All research concerned with quantitative analysis performs the counting of single fluorescent molecules. The most important advantage of the quantitative technique that relies on counting single molecules is that the detected signal intensity is not important, which guarantees the reliability of the quantitative determination. Yeung and coworkers34 developed an electrophoretic imaging approach for the quantification of β-Actin DNA (838 bp) labeled with ∼168 YOYO-1 molecules. The YOYO-1-labeled DNA molecules are induced to migrate electrophoretically in a fused-silica square capillary with a polymer solution. There, the DNA molecules are excited by a planar laser beam and imaged with a sequence of 3000 consecutive frames using a cooled charge-coupled device (CCD) camera kept at -35 °C. All fluorescent spots corresponding to single DNA molecules are counted in the 3000 frames. A linear relation between the DNA concentration and the number of molecules counted is obtained in the range of 1 × 10-15 to 1 × 10-12 mol/L. Hanley and Harris66 report an epifluorescence microscopy for the quantification of dye molecules. They apply substrate-withdrawal quantitative deposition of dye molecules on a substrate. Then, bright spots corresponding to single molecules on the substrate are imaged with a cooled CCD detector using a laser excited epiillumination. By counting single molecules for 50 frames on average, an exponential relationship between the number of spots and the number of molecules is obtained in the range of 2.0 × 10-11 to 4.0 × 10-10 mol/L. Klenerman and co-workers55 developed a confocal fluorescence microscopy to quantify protein analytes tagged with labeled antibodies in solution. The protein (41) Lyon, W. A.; Nie, S. Anal. Chem. 1997, 69, 3400–3405. (42) Lo ¨scher, F.; Bo ¨hme, S.; Martin, J.; Seeger, S. Anal. Chem. 1998, 70, 3202– 3205. (43) Haab, B. B.; Mathies, R. A. Anal. Chem. 1999, 71, 5137–5145. (44) Prummer, M.; Hubner, C. G.; Sick, B.; Hecht, B.; Renn, A.; Wild, U. P. Anal. Chem. 2000, 72, 443–447. (45) Knemeyer, J.; Marme´, N.; Sauer, M. Anal. Chem. 2000, 72, 3717–3724. (46) Byassee, T. A.; Chan, W. C.; Nie, S. Anal. Chem. 2000, 72, 5606–5611. (47) Wabuyele, M. B.; Ford, S. M.; Stryjewski, W.; Barrow, J.; Soper, S. A. Electrophoresis 2001, 22, 3939–3948. (48) Tadakuma, H.; Yamaguchi, J.; Ishihama, Y.; Funatsu, T. Biochem. Biophys. Res. Commun. 2001, 287, 323–327. (49) Do ¨rre, K.; Stephan, J.; Lapczyna, M.; Stuke, M.; Dunkel, H.; Eigen, M. J. Biotechnol. 2001, 86, 225–236. (50) Do ¨rre, K.; Stephan, J.; Eigen, M. Single Mol. 2001, 2, 165–175. (51) Weston, K. D.; Dyck, M.; Tinnefeld, P.; Mu ¨ ller, C.; Herten, D. P.; Sauer, M. Anal. Chem. 2002, 74, 5342–5349. (52) Shelby, J. P.; Chiu, D. T. Anal. Chem. 2003, 75, 1387–1392. (53) Li, H.; Ying, L.; Green, J. J.; Balasubramanian, S.; Klenerman, D. Anal. Chem. 2003, 75, 1664–1670. (54) Prummer, M.; Sick, B.; Renn, A.; Wild, U. P. Anal. Chem. 2004, 76, 1633– 1640. (55) Li, H.; Zhou, D.; Browne, H.; Balasubramanian, S.; Klenerman, D. Anal. Chem. 2004, 76, 4446–4451. (56) Sun, B.; Chiu, D. T. Anal. Chem. 2005, 77, 2770–2776. (57) Hirschfeid, T. Appl. Opt. 1976, 15, 2965–2966. (58) Dickson, R. M.; Norris, D. J.; Tzeng, Y. L.; Moerner, W. E. Science 1996, 274, 966–969. (59) Xu, X. H.; Yeung, E. S. Science 1997, 275, 1106–1109. (60) Xu, X. H.; Yeung, E. S. Science 1998, 281, 1650–1653. (61) Fang, X.; Tan, W. Anal. Chem. 1999, 71, 3101–3105. (62) Ma, Y.; Shortreed, M. R.; Yeung, E. S. Anal. Chem. 2000, 72, 4640–4645. (63) Kang, S. H.; Yeung, E. S. Anal. Chem. 2002, 74, 6334–6339. (64) Singh-Zocchi, M.; Dixit, S.; Ivanov, V.; Zocchi, G. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7605–7610. (65) Li, H. W.; Yeung, E. S. Anal. Chem. 2005, 77, 4374–4377. (66) Hanley, D. C.; Harris, J. M. Anal. Chem. 2001, 73, 5030–5037.

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target is labeled with red- and blue-excited antibodies. Two or more antibodies can bind to the same or different antigens on one target molecule. Coincident bursts of fluorescence are detected for the target molecules labeled with both red- and blueexcited antibodies, which are diffused into the probe volume. By directly counting the single coincidence events corresponding to the single protein target molecules, the protein can be quantified molecule by molecule. A linear range of 5 × 10-14 to 5 × 10-11 mol/L for protein G holds between the coincidence event and the concentration. Nie and co-workers67 report the use of colorcoded nanoparticles and dual-color fluorescence coincidence for the real-time detection of single biomolecules in a microfluidic channel by epifluorescence microscopy. With the use of green and red nanoparticles to simultaneously recognize two binding sites on one set of target molecules, single molecules of genes and proteins can be detected. A linear concentration dependence on the single molecule counting is obtained in the range of 5 × 10-15 to 5 × 10-12 mol/L. TIRFM is also an excellent technique for SMD in solution.57–65 Fang and Tan61 report a quantitative approach based on TIRFM that uses single molecule counting. An optic fiber is used to produce an evanescent wave on its surface. The fluorescent signals emitted by fluorophores excited in the evanescence field are collected at a microscope objective and detected by an intensified CCD. They apply the system to detect rhodamine 6G (R6G) and DNA molecules labeled by R6G at the single-molecule level. The fluorescent molecules entering the evanescence field are imaged. Then, the bright spots corresponding to the fluorescent molecules in each image are counted. A linear relationship between the average number of bright spots and the concentration is in the range of 2.5 × 10-9 to 1.7 × 10-8 mol/L. Recently, Porter and co-workers report an approach to single-molecule spectroelectrochemistry.68 In a 5.0 × 10-11 mol/L YOYO-I-labeled λ-DNA solution, individual λ-DNA molecules electrochemically adsorbed on a carbon-based optically transparent electrode are probed by TIRFM. We also develop TIRFM equipped with an electron multiplying CCD (EMCCD) for SMD of goat anti-rat IgG(H+L) labeled by Alexa Fluor 488 (IgG(H+L)-488).69 The number of fluorescent spots in the image is a linear function of the IgG(H+L)488 concentration in the range of 5.4 × 10-11 mol/L to 8.1 × 10-10 mol/L. Yeung’s group uses TIRFM to quantify human papilloma virus-16 DNA-labeled with Alexa Fluor 532 dye. The limit of detection (LOD) is 10-14 mol/L.70 For conventional TIRFM, the probe volume is very smalls on the order of tens to several hundreds of pixels, depending on the magnification of the objective and the image area acquired by the CCD camera. In solutions with very low concentrations, the number of molecules in an acquired image is very low, if there are any at all. This explains the high LOD. To capture single molecules by TIRFM, enough images should be acquired. However, this is difficult to achieve for small molecules in an aqueous solution due to the large diffusion rate and the short residence time in the evanescent field. The best way to solve these problems is to immobilize the molecules of interest onto a substrate and then perform TIRFM detection. In this work, we use an electrochemical method to immobilize and accumulate fluorescence molecules on a light transparent indium tin oxide (ITO) conductive microscope coverslip. Then, 100 images of the

fluorescent molecules accumulated on the coverslip are taken consecutively, one by one, by moving the coverslip with the aid of a three-dimensional (3D) positioner. Finally, we count the bright spots corresponding to single fluorescent molecules on all the images. Since R6G is commonly used for the investigation of SMD methods,16,17,22,26,61,66 it is chosen as the small molecule model. Additionally, an SMD method for biomacromolecule IgG(H+L)488 and DNA labeled by the 6-CR6Ga derivative of R6G (DNAR6G) is also investigated in this study. The LOD of the method reaches 10-15 mol/L. EXPERIMENTAL SECTION Chemicals. R6G purchased from Sigma-Aldrich (Germany) was dissolved in methanol and then diluted with water. IgG(H+L)488 (2 mg/mL, MW ) 148 000, Molecular Probes, Eugene, OR) was dissolved in phosphate-buffered saline consisting of 0.15 mol/L NaCl, 7.6 × 10-3 mol/L Na2HPO4, and 2.4 × 10-3 mol/L NaH2PO4 (pH ) 7.4) and then diluted with 0.01 mol/L acetate buffer (pH ) 5) or 0.01 mol/L phosphate buffer (pH ) 7.5) or 0.01 mol/L borate buffer (pH ) 8.5). Single-stranded DNA with a size of 16 bases was supplied and labeled with 6-CR6G by Jima Co., Shanghai, China. The sequence of the DNA is TTATAACTATTCCTAT. The DNA labeled with 6-CR6G (DNA-R6G) was prepared in water. Before use, DNA-R6G was diluted with a 0.01 mol/L borate buffer (pH ) 8.5). Other chemicals (analytical grade) were obtained from standard reagent suppliers. All solutions were prepared with trebly distilled water that was previously photobleached under an ultraviolet lamp for 24 h and passed through a 0.22 µm filter. Before use, the ITO coverslips were irradiated for 24 h by ultraviolet light. Apparatus. A CHI 802A electrochemical analyzer (CH Instruments, Austin, TX) was used to perform the electrochemical adsorption accumulation and voltammetric measurements of R6G adsorbed on an ITO coverslip with a thickness of 170 µm for the glass substrate and a thickness of ∼150 nm for the ITO (The Institute of Optics and Electronics of Shandong University). Electrochemical experiments were carried out with a threeelectrode system that consisted of an ITO conductive coverslip as the working electrode, an Ag/AgCl electrode as the reference electrode, and a Pt wire as the auxiliary electrode. The ITO coverslip was positioned horizontally and parallel (within an angle of less than 5 degrees) to the rotating magnetic bar. The experimental setup is shown in Figure 1A. An inverted microscope (Model IX81, Olympus, Tokyo, Japan) was equipped with a high-numerical-aperture 60 × (1.45 NA) TIRFM oil-immersion objective (PlanApo TIRFM, Olympus, Tokyo, Japan), a fluorescence microscope control unit (IX2-UCB, Olympus, Tokyo, Japan), a multiline Ar ion laser or a solid laser with an output power of 10 mW for 488 nm (Melles Griot, Carlsbad, CA), a laser incidence angle adjustment knob, a mirror unit consisting of a 470-490 nm excitation filter (BP470-490), a 505 nm dichromatic mirror (DM 505), a >510 nm emission filter (67) Agrawal, A.; Zhang, C.; Byassee, T.; Tripp, R. A.; Nie, S. Anal. Chem. 2006, 78, 1061–1071. (68) Donner, S.; Li, H.; Yeung, E. S.; Porter, M. D. Anal. Chem. 2006, 78, 2816– 2822. (69) Wang, L.; Xu, G.; Shi, Z.; Jiang, W.; Jin, W. Anal. Chim. Acta 2007, 590, 104–109. (70) Lee, J.; Li, J.; Yeung, E. S. Anal. Chem. 2007, 79, 8083–8089.

Figure 1. Schematic diagrams of instrumental setup for (A) electrochemical adsorption accumulation and (B) single-molecule imaging using TIRFM.

(IF510), and a 16 bit thermoelectrically cooled EMCCD (Cascade 512B, Roper Scientific, Tuscon, AZ) to accomplish the TIRFM measurements. A CHI 900 scanning electrochemical microscope (SECM) with a 3D positioner and controller (CH Instruments, Austin, TX) was employed to move the ITO conductive coverslip with adsorbed fluorescent molecules in the TIRFM. The experimental setup is shown in Figure 1B. Electrochemical Adsorption Accumulation of Fluorescent Molecules on ITO Conductive Coverslip. The ITO conductive coverslip acting as the working electrode was cut to 1.0 × 0.5 cm and cleaned by ultrasonication in household cleaning liquid (detergent) for 5 min. After the cleaning liquid was removed by rinsing with tap water, the ITO coverslip was sequentially cleaned by ultrasonication with acetone, alcohol, distilled water, and trebly distilled water for 15 min and then dried under a pure nitrogen flow. The coiled end of a 0.5 mm diameter, ∼8 cm long orthogonal copper lead was glued to one end of the ITO coverslip with silver epoxy. The ensemble was cured for 30 min at 150 °C in an oven. Epoxy resin was applied to the junction between the ITO coverslip and the copper lead in order to isolate and protect the electrical junction. The ITO working electrode, the Ag/AgCl reference electrode, and the Pt auxiliary electrode were inserted into the electrochemical cell containing R6G and 0.2 mol/L KCl or IgG(H+L)-488 and 0.01 mol/L borate buffer (pH 8.5) or DNAR6G and 0.01 mol/L borate buffer (pH 8.5). The ITO coverslip was parallel to the rotating magnet bar in the electrochemical cell. In order to electrochemically accumulate these molecules on the ITO conductive coverslip, the solution was stirred for an accumulation time (ta) and the ITO conductive coverslip was held at an accumulation potential (Ea) of -0.30 V for R6G or 0.70 V for Analytical Chemistry, Vol. 80, No. 11, June 1, 2008

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IgG(H+L)-488 or 0.80 V for DNA-R6G. After a rest period (tr) of 30 s, the ITO coverslip with the fluorescent molecules was taken out for single-molecule imaging by TIRFM. All potentials were measured against the Ag/AgCl electrode. Single-Molecule Imaging by TIRFM. After the ITO conductive coverslip with the accumulated molecules was washed three times by immersing it into trebly distilled water, it was dried in a nitrogen flow at room temperature. In the TIRFM experiments that followed, the incident angle of the laser beam (488 nm) was first adjusted carefully to produce total internal reflection of the laser beam from the 60× (1.45 NA) TIRFM oil-immersion objective lens of the inverted microscope. This was accomplished by turning the laser incidence angle adjustment knob. Then, the ITO coverslip with the accumulated molecules was placed in a frame of the same size as the ITO coverslip. The frame was fixed on the stand of the 3D positioner of the SECM (Figure 1B). The accumulated molecules were excited by the 488 nm line of the laser through the TIRFM oil-immersion objective. The fluorescence emitted by the accumulated molecules was collected by the same objective lens, and the fluorescent image was acquired by the EMCCD, which was controlled by the MetaMorph software (Universal Imaging, Downingtown, PA). One-hundred images on the ITO coverslip were acquired one by one by moving the coverslip with a step length of 140 µm, using the 3D positioner controlled by the SECM. The data on the images were analyzed by the MetaMorph software. RESULTS AND DISCUSSION Electrochemical Adsorption Accumulation of R6G on the ITO Coverslip. The R6G molecule has been used as a model molecule in SMD.16,17,22,26,61,66 Therefore, R6G was chosen as the model small molecule in this study. Voltammetry is a useful technique for the investigation of the adsorption characteristics of electroactive species on an electrode. The profile of the peakshaped voltammogram for the electroactive species adsorbed, as well as the relationship between the peak current on the voltammograms and the potential scan rate, is different from those of the species in the solution. Since R6G could be electrochemically reduced on gold electrodes in 0.2 mol/L KCl,71 voltammetry was used to judge whether R6G can be adsorbed on the ITO conductive coverslip. To avoid interference from the reduction of oxygen from the solution, the R6G solution was deaerated for 8 min using ultrapure nitrogen before electrochemical adsorption accumulation. Then, electrochemical adsorption accumulation was performed and the voltammogram of the R6G adsorbed on the ITO coverslip was recorded by scanning the potential in the negative direction in 5.0 × 10-8 mol/L R6G. For such a low concentration, the reduction current of R6G can not be detected. Curve 1 in Figure 2A shows the voltammogram of the adsorbed R6G that was accumulated during 150 s at an accumulation potential of -0.30 V in a quiescent solution containing 5.0 × 10-8 mol/L R6G. The reduced peak on the voltammogram is very low. The reduction signal of the adsorbed R6G could be enhanced by stirring the solution to increase mass transport during accumulation. Curves 2 and 3 in Figure 2A show the voltammograms of the adsorbed R6G that was accumulated for an accumulation time (ta) of 120 s in a stirred solution containing 5.0 × 10-8 mol/L R6G (71) Yu, J.; Zhou, T. J. Electroanal. Chem. 2001, 504, 89–95.

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Figure 2. (A) Voltammograms of the adsorbed R6G at v ) 20 mV/s after subtracting the blank. Curves: (1) Accumulation for 150 s in a quiescent solution; Ea ) -0.30 V; (2) accumulation for 120 s in a stirred solution, rest period (tr) ) 30 s, no potential application; (3) accumulation for 120 s in a stirred solution, tr ) 30 s, Ea ) -0.30 V. (B) Relationship between the peak current (ip) and the adsorption accumulation time (ta) in a stirred solution with tr, ) 30 s, Ea ) -0.30 V, v ) 20 mV/s. (C) Relationship between ip and v, with ta ) 120 s, tr ) 30 s, Ea ) -0.30 V. The ITO coverslip area is 1.0 × 0.5 cm with 0.2 mol/L KCl and 5.0 × 10-8 mol/L R6G.

with no potential applied and an accumulation potential (Ea) of -0.30 V after a rest period (tr) of 30 s. A reduction peak with a peak potential of -0.72 V appeared on the voltammograms. The peak current (ip) on the voltammogram recorded at an accumulation potential of -0.30 V was higher than that obtained in the case of no potential. This indicates that the physisorption was weak and the adsorption of R6G depended on the potential applied at the ITO coverslip. ip increased with prolonged ta before 140 s. After 140 s, ip remained nearly constant (Figure 2B), implying that the adsorption equilibrium was reached. These results suggest that the peak resulted from the reduction of the R6G adsorbed on the ITO coverslip and not from the R6G in the solution. The adsorption characteristics of the peak could also be demonstrated by the relationship between ip and the scan rate (v). ip increased linearly with increasing v, and the straight line passed through the origin (Figure 2C), which confirmed the occurrence of an interfacial reaction of an adsorbed reactant; i.e., R6G could also be electrochemically adsorbed and reduced at ITO electrodes in 0.20 mol/L KCl. In the subsequent TIRFM with

electrochemical adsorption accumulation, 120 s for ta, 30 s for tr, and -0.30 V for Ea were used. Single-Molecule Imaging of Molecules Accumulated on the ITO Coverslip Using TIRFM. In our investigation the background was one of the key factors for single-molecule imaging. To eliminate the background from water and ITO coverslips, several steps were adopted. Before use, trebly distilled water and ITO coverslips were irradiated for 24 h by ultraviolet light to quench the fluorescence from impurity. The solution was prepared with trebly distilled water and filtered through a 0.22 µm filter twice. To obtain homogeneous distribution of molecules on the ITO coverslip during electrochemical adsorption accumulation, the ITO coverslip as the working electrode was placed horizontally and parallel (within an angle 510 nm; exposure time ) 100 ms.

across the electrode/solution interface, which determines the surface charge of the electrodes.72–75 Due to electrostatic interaction, the adsorption of proteins from aqueous solutions with different pH levels on charged surfaces will be different. Proteins in solution with pH levels different from the isoelectric point (pI) will have either positive or negative net charges. When the protein is in a solution with a pH value higher than its pI, the protein will present a net negative charge. When the solution pH is lower than its pI, the protein will have a net positive charge. In this work, the adsorption of protein was studied at different protein charges by varying the pH of the solution and different electrode charges by changing the externally applied potential. Like R6G, the number of single fluorescent molecules accumulated in a stirred solution is much greater than that accumulated in a quiescent solution. Figure 4 shows the fluorescent subframe images of adsorbed IgG(H+L)-488 molecules accumulated in a quiescent solution and in a stirred solution. To increase mass transport, the subsequent accumulation experiments were carried out in a stirred solution. Figure 5 shows the adsorption profiles for IgG(H+L)-488 with different potentials applied and with different pH levels. It was (72) Bos, M. A.; Shervani, Z.; Anusiem, A. C. I.; Giesbers, M. N. W.; Kleijn, J. M. Colloids Surf., B: Biointerfaces 1994, 3, 91–100. (73) Moulton, S. E.; Barisci, J. N.; Bath, A. S., R.; Wallace, G. G. J. Colloid Interface Sci. 2003, 261, 312–319. (74) Ying, P.; Viana, A. S.; Abrantes, L. M.; Jin, G. J. Colloid Interface Sci. 2004, 279, 95–99. (75) Moulton, S. E.; Barisci, J. N.; Bath, A.; Stella, R.; Wallace, G. G. J. Langmuir 2005, 21, 316–322.

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Figure 4. Fluorescent subframe images of single IgG(H+L)-488 molecules accumulated on the ITO coverslip (A) for 150 s in a quiescent solution and (B) for 300 s with tr ) 30 s in a stirred solution. Conditions: 0.01 mol/L borate buffer (pH 8.5) containing 1.0 × 10-12 mol/L IgG(H+L)-488; Ea ) 0.70 V.

Figure 5. Relationship between the number of single IgG(H+L)488 molecules accumulated on the ITO coverslip for 300 s with tr ) 30 s in a stirred solution and the applied potential at different pH levels of (1) pH ) 5.0, (2) pH ) 7.5, and (3) pH ) 8.5. Conditions: 0.01 mol/L borate buffer (pH 8.5) containing 1.0 × 10-12 mol/L IgG(H+L)488; Ea ) 0.70 V.

observed that applying a potential to the electrode and varying the pH had apparent effects on the adsorption of this protein. Although the exact pI of the IgG(H+L)-488 used for this work is unknown, it is considered to be between pH 5.0 and 8.0.76 In the case of pH 5.0, the amount of adsorbed IgG(H+L)-488 was minimal and almost a constant in the potential range of 0 to 0.2 V. Both positive and negative potential applied outside the potential region promoted IgG(H+L)-488 adsorption. When no external potential was applied, the amount of adsorbed IgG(H+L)-488 was very low (Figure 6). When positive charge of the surface increased by applying a positive potential to the ITO coverslip, the electrostatic interaction between the ITO coverslip and the IgG(H+L)488 molecules was enhanced, resulting in an increase of adsorption of IgG(H+L)-488. The more positive the applied potential was, the more adsorbed IgG(H+L)-488 molecules there were. Additionally, when the potentials were more positive than 0 V, more IgG(H+L)-488 was adsorbed at pH 8.5 than at pH 7.5. This is because the net negative charge on the protein molecules was higher at pH 8.5 than at pH 7.5. To enhance the adsorption of IgG(H+L)-488 on the ITO coverslip, 0.70 V for the accumulation potential and a buffer of pH 8.5 were used in this work. Curve 1 in Figure 7 shows the adsorption profile of IgG(H+L)-488 as a function of time, at an adsorption potential of 0.70 V and in a solution of pH 8.5. The number of IgG(H+L)-488 adsorbed on the ITO coverslip increased with prolonged ta. In this work, DNA-R6G with a sequence of TTATAACTATTCCTAT was chosen as the model DNA. Single-molecule imaging (76) Hidalgo-Alvarez, R.; Galisteo-Gonzalez, F. Heterog. Chem. Rev. 1995, 2, 249–268.

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Figure 6. Fluorescent subframe images of single (A and B) IgG(H+L)-488 and (C and D) DNA-R6G molecules accumulated on the ITO coverslips for 300 s with tr ) 30 s in a stirred solution. Conditions: (A and C) without and (B and D) with potential application; (B) Ea, 0.70 V and (D) Ea, 0.80 V; (A and B) 0.01 mol/L borate buffer (pH 8.5) containing 1.0 × 10-12 mol/L IgG(H+L)-488 and (C and D) 0.01 mol/L borate buffer (pH 8.5) containing 1.0 × 10-12 mol/L DNAR6G.

Figure 7. Relationship between the number of (1) single IgG(H+L)488 and (2) single DNA-R6G molecules accumulated on the ITO coverslip with tr ) 30 s in a stirred solution and the adsorption accumulation time. Conditions: (1) 0.01 mol/L borate buffer (pH 8.5) containing 1.0 × 10-12 mol/L IgG(H+L)-488, Ea, 0.70 V; and (2) 0.01 mol/L borate buffer (pH 8.5) containing 1.0 × 10-12 mol/L DNA-R6G, Ea, 0.80 V.

was also used to evaluate the electrochemical adsorption accumulation of DNA-R6G. In basic solution, DNA has a negative charge.77 The adsorption of DNA-R6G on ITO coverslips depended on the applied potentials. When no external potential was applied, very few DNA-R6G molecules were adsorbed (Figure 6). After adsorption accumulation in a stirred solution for 300 s, the relationship between the number of bright spots, corresponding to single DNA-R6G molecules, and the externally applied potential is shown in Figure 8. The number of DNA-R6G molecules adsorbed increased with increasing positive potential due to electrostatic attraction between the ITO coverslip and the DNAR6G molecules. To enhance the adsorption of DNA-R6G on the ITO coverslip, an accumulation potential of 0.80 V and a buffer of pH 8.5 were used in this work. Curve 2 in Figure 7 shows the adsorption profile of DNA-R6G as a function of time at an (77) Ganachaud, F.; Elaı¨ssari, A.; Pichot, C.; Laayoun, A.; Cros, P. Langmuir 1997, 13, 701–707.

Figure 8. Relationship between the number of single DNA-R6G molecules accumulated on the ITO coverslip for 300 s with tr ) 30 s in a stirred solution and the applied potential at pH 8.5. Conditions: 0.01 mol/L borate buffer (pH 8.5) containing 1.0 × 10-12 mol/L DNAR6G.

adsorption potential of 0.80 V and in a solution of pH 8.5. The number of DNA-R6G molecules adsorbed on the ITO electrode also increased with prolonged ta. Quantification of Fluorescent Compounds based on Electrochemical Adsorption Accumulation and Single-Molecule Counting Using TIRFM. In our experiments mentioned above, when a potential was applied to the ITO coverslip for a certain time in a stirred solution, molecules of interest were accumulated on the surface of the ITO coverslip by electrochemical adsorption. After electrochemical adsorption accumulation, the fluorescence images of these molecules accumulated on the ITO coverslip were taken by TIRFM. To capture more single molecules on an ITO coverslip, 100 consecutive images were taken. In concentrations of less than 4.0 × 10-14 mol/L for R6G, 1.0 × 10-14 mol/L for

IgG(H+L)-488, and 1.0 × 10-14 mol/L for DNA-R6G, only parts of the 100 subframe images had bright spots. All bright spots occupied one pixel on the subframe images acquired. The higher the concentration, the more subframe images there were where each bright spot occupied one pixel. For R6G and DNA-R6G, the mean fluorescence intensity of all bright spots on these subframes was the same as that of the bright spots obtained by dropping the diluted standard solutions on an ITO coverslip, indicating that the bright spots were from accumulated single fluorescence molecules. The subframe images for R6G, IgG(H+L)-488, and DNA-R6G at different concentrations (one set for each concentration level) are shown in the Supporting Information. The typical subframe images of R6G, IgG(H+L)-488, and DNAR6G for different concentrations in the range from 5.0 × 10-13 to 5.0 × 10-12 mol/L for R6G, 1.0 × 10-13 to 2.0 × 10-12 mol/L for IgG(H+L)-488, and 1.0 × 10-13 to 2.0 × 10-12 mol/L for DNAR6G are shown in Figure 9. Three sets of subframe images for R6G, IgG(H+L)-488, and DNA-R6G at different concentrations are shown in the Supporting Information. It was noted that the fluorescence intensity of some bright spots was 2 or more times higher than the mean intensity of a bright spot corresponding to a single fluorescent molecule, which was obtained in lower concentrations (