Quantitative Detection of Single Molecules Using Enhancement of Dye

Oct 27, 2010 - Subsequently, a 30 base pair double-stranded oligonucleotide terminated with biotin (BT-dsDNA) is conjugated to the SA-MNBs. The result...
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Bioconjugate Chem. 2010, 21, 1987–1993

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Quantitative Detection of Single Molecules Using Enhancement of Dye/DNA Conjugate-Labeled Nanoparticles Qingwang Xue,† Dafeng Jiang,† Lei Wang,*,‡ and Wei Jiang*,† School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, P.R. China, and School of Pharmacy, Shandong University, 250012 Jinan, P.R. China. Received April 29, 2010; Revised Manuscript Received September 27, 2010

An ultrasensitive fluorescence immunoassay method for quantitative detection of single molecules is developed on the basis of counting single magnetic nanobeads (MNBs) with combined amplification of DNA and dye/DNA conjugate. Highly amplified fluorescence signal and low background signal are achieved by using mutilabel bioconjugates made by linking multiple dye/DNA conjugates to streptavidin-coated magnetic nanobeads (SAMNBs) and magnetic separation. In this method, human IgG (Ag) is captured on the silanized glass substrate surface, followed by immunoreaction with biotinylated mouse antihuman antibody (BT-Ab). Then, SA-MNBs are attached to the BT-Ab through the biotin/streptavidin interaction at a ratio of 1:1. Subsequently, a 30 base pair double-stranded oligonucleotide terminated with biotin (BT-dsDNA) is conjugated to the SA-MNBs. The resultant Ag-BT-Ab-SA-MNBs/BT-dsDNA/SYBR Green I is achieved after a fluorescent DNA probe, SYBR Green I, is added to the substrate and bound to the oligonucleotide at high ratios. Finally, epifluorescence microscopy coupled with a high-sensitivity electron multiplying charge-coupled device is employed for human IgG fluorescence imaging and detection. The number of fluorescent spots corresponding to single protein molecules on the images is counted. It is found that the number of fluorescent spots resulting from the SA-MNBs/BT-dsDNA/SYBR Green I immuotargeted on the glass slides is correlated with the concentration of human IgG target antigen in the range 3.0-50 fM.

INTRODUCTION Recently, single-molecule detection (SMD), based on the analysis of individual molecules, has attracted a great deal of attention in the area of analytical chemistry and life science (1, 2). In analytical chemistry, single-molecule detection (SMD) is the ultimate limit of detection (3). Fluorescence detection techniques combined with capillary or microchannels (4-8), confocal fluorescence microscopy (9-16), total internal reflection fluorescence microscopy (TIRFM) (17-23), and epifluorescence microscopy (EFM) (24, 25) are the most popular techniques in SMD. Of these SMD studies with fluorescence quantitative detection, all research performed the counting of single fluorescence molecules for quantitative analysis at the singlemolecule level. The SMD quantitative research technique relies on directly counting one by one the number of target molecules rather than measuring signal intensity to quantitative analysis of the concentration, which is suitable for ultralow concentrations of target biological macromolecule identification and detection. As for SMD-based quantitative research, on the assumption of target biological macromolecules being identified, 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 (3). To ensure that the target biological macromolecule can be identified, a better signal-tonoise (S/N) ratio is needed, which can improve the sensitivity. Fluorescent antibodies have been widely used in singlemolecule fluorescence immunodetection. The fluorescent labels for the labeling of antibodies, which provide exquisite sensitivity * Corresponding author. W.J.: Tel +86 531 88363888; fax +86 531 88564464; e-mail [email protected]. L.W.: Tel +86 531 88382330; e-mail [email protected]. † School of Chemistry and Chemical Engineering. ‡ School of Pharmacy.

(26) and guarantee the reliability of quantitative determination, should be bright and stable enough in single-molecule fluorescence detection. Usually, a fluorescent dye molecule is coupled to the target molecules to generate a detectable signal. However, two major difficulties limit the sensitivity when these fluorophores are used. The first difficulty is the relatively low fluorescence signal intensity. Because one antibody can only be labeled with one or a few fluorophores, the fluorescence signal is too weak to be detected when the target concentration is low. The second difficulty is the poor photostability of many fluorophores. Most organic dyes suffer serious photobleaching. To improve the sensitivity of single-molecule fluorescence immunodetection by using highly fluorescent and photostable fluorescence labels, quantum dots (QDs) have been developed and explored as novel signal labels for single-molecule fluorescence immunodetection (23, 26-29). Although QDs have many unique prominent properties, there are many problems involved in using QDs as fluorescence labels, such as the stability of multifunctionalized QDs in different reaction systems, the toxity of heavy metal ions, and the blinking and physical adsorption (27, 30). Thus, efforts to develop novel fluorescence labels to reduce the background signal from nonspecific adsorption of labels and increase fluorescence signal intensity have gained considerable momentum in singlemolecule fluorescence detection. Along with the efficient magnetic particle amplification strategy for sensitive detection of biomolecules (31, 32), a magnetic particle amplification strategy could be also applied to single-molecule immunodetection. The magnetic particle acts as a carrier and a bridge when functionalized magnetic particles are used as signal labels. On one hand, the significant signal intensity could be gained from the application of amplification strategy. On the other hand, the background signals from nonspecific adsorption of labels could be reduced because of the application of magnetic particles. Recently, Jin’s group (3)

10.1021/bc100212w  2010 American Chemical Society Published on Web 10/27/2010

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Figure 1. Schematic representation of the process of immunoassay labeling format by using the conjugate SA-MNBs/BT-dsDNA/SYBR Green I label.

developed an electrochemical immunoassay strategy for quantitative detection of single molecules based on counting the magnetic nanobeads released from a substrate with the combination of DNA amplification and enzyme amplification. If SAMNB does not need to be dissociated, further sensitivity improvement may be obtained by directly counting the number of functionalized magnetic nanobeads attached to single antibodies for quantitative detection of single molecules in a fluorescence image. In this work, we present an ultrasensitive fluorescence immunoassay method for quantitative detection of single molecules based on counting of single magnetic nanobeads with combined amplification of DNA and DNA/SYBR Green I conjugate. The principle of this method is shown in Figure 1. In this method, we employed a streptavidin-coated magnetic nanobead as a carrier for the conjugation of multiple fluorescent BT-dsDNA/SYBR Green I conjugates. It was attached to a biotin-modified mouse antihuman antibody through the highspecificity, high-affinity biotin-streptavidin interaction at a ratio of 1:1. Human IgG captured on the silanized glass substrate surface was detected by its immunoreaction with biotinylated mouse antihuman antibody. EFM with EMCCD was employed for human IgG fluorescence imaging and detection. In this case, only one MNB is bound to one single protein. Each fluorescent spot corresponds to one SA-MNB/BT-dsDNA/SYBR Green I immuotargeted on the glass slides. Finally, the number of fluorescent spots corresponding to single protein molecules on the images was counted. It is found that the number of fluorescent spots resulting from the SA-MNBs/BT-dsDNA/ SYBR Green I immuotargeted on the glass slides could be applied to the quantitative detection of human IgG target antigen.

EXPERIMENTAL SECTION Materials and Instrumentation. Human immunoglobulin G (IgG), monoclonal antihuman IgG-Biotin conjugate (Clone HP6017), 3-glycidyloxypropyltrimethoxysilane, and bovine serum albumin were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Strand A, biotin-5′-TTT TTT TTT GCG GGT AAC GTC AAT ATT AAC TTT ACT CCC-3′, and strand B, 5′GGG AGT AAA GTT AAT ATT GAC GTT ACC CGC-3′, were obtained from the Genscript Corporation (Nanjing, China).

Streptavidin-MNBs (350 nm diameter, 1.343 g mL-1, aqueous suspension containing 0.1% bovine serum albumin (BSA), 0.05% Tween-20, and 10 µM EDTA at a concentration of 3.324 × 1011 beads mL-1) from Bangs Laboratories Inc. (Fishers, IN), Tris (>99.8%) from Amresco Inc. (Solon, OH), and Tween-20 from Sigma (St. Louis, MO) were used in the work. Other chemicals (analytical grade) were obtained from standard reagent suppliers. Microscope cover glasses (22 × 22 mm2) were purchased from Cole-Parmer (Illinois, USA). The physiological buffer saline (PBS) consisted of 0.15 M NaCl, 7.6 mM NaH2PO4, and 2.4 mM Na2HPO4 (pH 7.4). PBS-T buffer consisted of 0.15 M NaCl, 7.6 mM Na2HPO4, 2.4 mM NaH2PO4, and 0.05% Tween-20 (pH 7.4). TE buffer consisted of 10 mM Tris-HCl and 1.0 mM Na2EDTA (pH 8.0). TTL buffer consisted of 0.10 M Tris-HCl (pH 8.0), 0.1% Tween-20, and 1.0 M LiCl. TT buffer consisted of 0.25 M Tris-HCl (pH 8.0) and 0.1% Tween-20. TTE buffer consisted of 0.25 M Tris-HCl (pH 8.0), 0.1% Tween-20, and 20 mM Na2EDTA (pH 8.0). A phosphate buffer (pH 7.0) containing 1% BSA was used as a buffer for blocking. We used an inverted microscope (model IX81, Olympus, Tokyo, Japan) equipped with a high-numerical-aperture 40× objective lens (PlanApo, Olympus, Tokyo, Japan), a mercury lamp (OSRAM, HBO, 103w/2, Germany), a mirror unit consisting of a 470-490 nm excitation filter (BP470-490), a 505 nm dichromatic mirror (DM 505), a 510-550 nm emission filter (IF510-550), and a 16 bit thermoelectrically cooled EMCCD (Cascade 512B, Tucson, AZ, USA). The EMCCD was used for collecting the fluorescent images. Imaging acquisition and data analysis were performed using the MetaMorph software (Universal Imaging, Downingtown, PA, USA). DNA Hybridization. A 30 base pair biotinylated doublestranded oligonucleotide (BT-dsDNA) was obtained by hybridizing two complementary single-stranded oligonucleotides. The two oligonucleotides were mixed in 2 × SSC buffer, denatured at 95 °C for 5 min (33), annealed at 72 °C for 10 min, and naturally cooled down to 48 °C for 6 h. Substrate Preparation and Antigen Coating. The epoxyfunctionalized glass surfaces were prepared according to the modification described in the literature (27). The freshly prepared substrate surface was coated with 100 µL human IgG

Quantitative Detection of Single Molecules

solutions of various concentrations (50, 30, 10, 8.0, 5.0, 3.0 fM). The substrate was immediately placed in a sealed Petri dish at 37 °C for 5 h. After that, the substrate was washed three times with PBS-T washing buffer to remove unbound human IgG and impurities. Blocking. We used a phosphate buffer containing 1% BSA as a blocking buffer. To the substrate of antigen coating was added 100 µL of blocking, and then, the substrate was incubated for 3 h. After that, the substrate was washed three times with PBS-T washing buffer. Immunoreaction. Antihuman IgG-Biotin solution was 40fold diluted with a phosphate buffer (pH 7.4) before being used, and 100 µL of the diluted solution of various concentrations (2, 1.2, 0.4, 0.32, 0.2, 0.12 pM) was then added to the substrate. The biotinylated monoclonal antihuman IgG (BT-Ab) molecule was bound to the human IgG through the immunoreaction. After that, the substrate was deposited hermetically for 2 h at 37 °C. Finally, nonspecifically absorbed antibodies were washed by PBS-T washing buffer. Binding of SA-MNBs to BT-Ab. Five microliters of streptavidin-MNB suspension (3.324 × 1011 beads/mL) was washed five times with 400 µL of TTL buffer to remove surfactants. Then, 50 µL of TTL buffer was added. Subsequently, streptavidin-MNB was conjugated to the biotinylated monoclonal antihuman IgG molecules by adding 16 µL of streptavidin-MNB suspension (0.52 × 109 beads) and incubating for 2 h at 37 °C. After the incubation, the redundant MNB were separated magnetically from the substrate using a neodymiumboron (Nd-B) magnet that produced an inhomogeneous magnetic field on the top of the sample cell for 5 min. Then, the substrate of the cell was gently rinsed eight times with 200 µL of PBS-T by drawing these solutions out along the wall of the cell. Finally, nonspecifically absorbed SA-MNBs were removed with magnetic separation. Modification of SA-MNBs with BT-DNA. 100 µL of 0.14 µM BT-DNA solution was added to the substrate, and then the substrate was incubated hermetically for 2 h at 37 °C. Doublestranded oligonucleotides terminated with biotin (BT-dsDNA) were conjugated to the streptavidin-MNBs. Finally, the unreacted biotin-dsDNA was washed away with 400 µL PBS-T washing buffer. Then, the substrate was washed twice with 400 µL of TT buffer and twice with 400 µL of TTE buffer. SYBR Green I Adding and Fluorescence Image Detection. 100 µL 2.0 µM of SYBR Green I was added to the substrate and incubated for 10 min. SYBR Green I was bound to the oligonucleotide at high ratios by the specific binding interaction with the oligonucleotide. The resultant Ag-BT-AbSA-MNBs/BT-dsDNA/SYBR Green I was formed. All images were taken using an Olympus IX81 microscope (40× objective) equipped with a 16 bit thermoelectrically cooled EMCCD. The fluorescence images were obtained by the excitation light within the wavelength 470-490 nm of the mercury lamp and exposure time of 100 ms. Thirty images on the coverslip substrate were acquired one by one by moving the coverslip. Finally, the bright spots corresponding to single protein molecules on all the images were counted.

RESULTS AND DISCUSSION Binding one MNB to One IgG onto the Substrate. Human IgG was captured on the epoxy-functionalized glass surfaces as the substrate by the covalent cross-linking reaction. The basic assumption in this method is that one protein brings one MNB onto the substrate. This assumption can be satisfied when the distance between two adjacent proteins captured on the substrate is larger than that between two adjacent MNBs. Thus, the concentration of protein should be low enough. It can be roughly calculated that 0.52 × 109 MNBs with a diameter of 350 nm

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Figure 2. EFM fluorescence image of single QD-labeled human IgG on the substrate using 50 fM human IgG.

can completely occupy the substrate within an area of 50 mm2. This means that the amount of protein should be lower than 86 fM. In our experiments, 100 µL of human IgG solutions with concentrations lower than 50 fM was used. Thus, if all proteins are captured on the substrate, the average distance between two proteins is longer than the diameter of the MNB. Although there are many streptavidin binding sites on each MNB, it is not possible that one MNB is bound to more than two human IgG molecules because the space between two adjacent human IgG molecules is big enough. In order to prove this assumption, we measured the average distance between two adjacent proteins using EFM that can take a single-molecule image. To do so, the antigen captured on the substrate was labeled with QDs using biotin-antibody and SA-QDs. The fluorescence image for 50 fM target protein is shown in Figure 2. In the image, each bright spot corresponds to a target protein. It is obvious that the interspot distance is greater than 4 µm, which is much bigger than the diameter between two adjacent MNBs. Thus, we can conclude that one target protein should bring one MNB onto the substrate when the concentration of target protein is lower than 50 fM. This conclusion was demonstrated by a control experiment, in which the target protein solution was replaced by a buffer. In this case, only 10-11 bright spots were observed in 25 images, i.e., < 0.5 spot appeared in an image on average, which was in accordance with the result mentioned in Jin’s literature (3). Reducing the Background Signal with Magnetic Separation. In this experiment, it has been roughly calculated that 0.52 × 109 MNBs with a diameter of 350 nm could completely occupy the substrate within an area of 50 mm2. 100 µL of human IgG solutions with concentrations lower than 50 fM was used. This means that the amount of protein is lower than the number of MNB. Through the irreversible interaction between streptavidin and biotin, the 350-nm-diameter SA-MNB was conjugated to BT-Ab. Along with reducing the amount of protein, the MNB was far more than the antigen. The BT-dsDNA/SYBR Green I conjugate that was subsequently added to the substrate was easily bound to the redundant MNB on the substrate, leading to the background effect. To address this problem, we employed magnetic separation to remove the unbound MNB on the substrate. With the same experimental conditions, the fluorescence intensity images of the substrate of negative experiments for 50 fM target proteins without and with magnetic separation are shown in Figure 3, which indicated that the redundant MNB could be removed from the substrate with magnetic separation. Multilabel Amplification. The high available surface-tovolume ratio of the streptavidin magnetic bead provides more

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Figure 3. Fluorescent intensity subframe image of negative experiment for 50 fM target proteins in solution. Conditions: (A) without magnetic separation, (B) with magnetic separation. Scale bar, 30 pixel.

binding sites for analysis. We attached multiple BT-DNA/SYBR Green I conjugates to the streptavidin magnetic bead surfaces to increase fluorescence intensity and to improve the sensitivity. The biotinylated double-stranded oligonucleotide (BT-dsDNA) was obtained by hybridizing two complementary single-stranded oligonucleotides. The BT-dsDNA has a sequence with 9 thymine nucleotides at the biotin end acting as a spacer to reduce the steric hindrance between BT-dsDNA and the surface-confined streptavidin of MNB interaction. Through the irreversible interaction between streptavidin and biotin (Ka ) 1015/M-1) with rapid binding kinetics and strong affinity, biotin-dsDNA combines with the 350 nm diameter streptavidin-MNB and produces the resultant Ag-BT-Ab-SA-MNB/BT-dsDNA. In this method, the primary amplification factor of the final detection fluorescence intensity is correlated with the number of BTdsDNA on one MNB. The average number biotin binding sites on the surface (m) of one streptavidin-MNB was calculated using the equation reported in the manufacture’s instructions of MagPrep streptavidin beads (Bangs Laboratories Inc., Fishers, IN). With the binding capacity (g) of the streptavidin-coated magnetic bead, the molecular weight (M) of biotin-FITC and the number (C) of magnetic beads per milligram, m could be estimated to be 2.25 × 104. The average biotin binding sites on the surface of one streptavidin-MNB is ∼30 000 based on the data provided in the manufacturer’s instructions. In order to ensure all binding sites on the surface of one streptavidin-MNB conjugated with biotin-dsDNA, the amount of biotin-dsDNA used for reaction here is 10 times over the biotin binding sites. The amount of BT-dsDNA per unit weight of magnetic beads was determined by measuring the concentration of DNA solution added and washed down after magnetic separation, which was determined by the absorbance at 260 nm using ultraviolet spectrophotometry. According to the difference at the two concentrations, we worked out the number of BT-dsDNA per magnetic bead to be ∼3000, which was in accordance with the data mentioned in Jin’s report (3), implying an amplification factor of ∼3000 for DNA amplification. Although the measured value was different from that offered by the manufacturer’s instructions, the large amplification factor was achieved. Magnetic beads of diameter 350 nm provided a very high number of labels on the surface and gave better sensitivities and detection limits. Assembly of Fluorescent DNA Nanotags. In the systems reported here, DNA nanostructures serve as a template for assembling the intercalating dyes into fluorescent arrays. High densities of fluorophores are assembled in a very small region

Figure 4. Dependence of maximum fluorescence intensity on ratio of intercalating SY groups per DNA base pair. Conditions: 13.9 nM dsDNA.

of base pairs in the DNA helix, yet the DNA template keeps them far enough away from each other to prevent selfquenching. A common feature of organic dyes is that they tend to aggregate in aqueous solution, which usually leads to selfquenching of fluorescence. Steric constraints impede multiple dyes from binding at the same intercalation site. As illustrated in Figure 4, the fluorescence increased monotonically when the fluorescence probe was titrated into the BT-DNA solution at increasing dye-to-base pair ratios (dbpr), the fluorescence saturated at approximately 0.25 dye/base pair. The quenching observed for SYBR Green I at ratios in excess of 0.25 is likely the result of alternative, nonintercalative binding that can occur at relatively high loading. Nevertheless, working at a ratio of 0.25 or lower yields highly fluorescent DNA-templated dye arrays. Fluorescence Imaging Analysis. For the optimal fluorescence image, the study was optimized at a variety of imaging conditions. First, the effects of exposure time and intensifier gain were investigated, and the optimized setting values were 100 ms and 2000, respectively. In this case, the optimal fluorescence image was obtained. Second, in the experiment, the fluorescence intensity must be uniform. The intensity of the excitation light on the area illuminated by the mercury lamp through the objective was not uniform inside an area of 201 × 201 µm2 (512 × 512 pixels), as shown in Figure 5A. The light

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Figure 5. (A) Blank image and (B) distribution of light intensity along the central line of the bright region indicated in A in the x direction. Conditions: laser power of 103 W/2 mW; excitation wavelength 470-490 nm; emission wavelength of 510 nm; exposure time of 100 ms.

intensity dispersed from the center. The distribution of the fluorescence intensity corresponding to the central line in the χ direction of the light spot was given in Figure 5B. It was obvious that the light intensity dispersed from the center. In order to solve this problem, we chose a 150 × 150 pixel subregion at the center of the light spot for measuring the fluorescence intensity image. The intensity deviation inside an area of 59 × 59 µm2 (150 × 150 pixels) at the center of the light spot was less than 5%. The threshold for image acquirement was chosen at a value of 3 times the standard deviation of the background noise for 10 blank images. For each concentration, 30 (three images of each location) images were obtained from location to location on the substrate surface, which was performed by moving the XY sample stage, as shown in Figure 6. The typical fluorescence subframe images of SA-MNB/BTdsDNA/SYBR Green I conjugate at different concentrations of human IgG in the range from 3.0 to 50 fM were shown in Figure 6. At the higher concentration, it was noted that the fluorescence intensities of some bright spots were 2-3 times higher than the mean intensity of a bright spot corresponding to a single MNB, which was obtained in lower concentrations (30 fM) that exceeded a 6 × 6 pixel area was assumed to come from two nearest-neighbor MNBs. When the concentrations exceeded 30 fM, the distance of two neighbor MNBs was small. The bright spots of two nearestneighbor MNBs aggregated to one brighter spot due to diffraction of the fluorescence image. Figure 7 showed the linear relationship between the number of beads and human IgG concentration in the range 3.0-50 fM. The linear regression equation for human IgG in the range 3.0-50 fM was determined to be y ) 13.135 + 7.684 × 1015x (R ) 0.996). The present

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Figure 8. Comparison of fluorescence immunoassay for human-IgG (black column), mouse IgG (red column), BSA (blue column), and negative control (green column) on a silanized glass subframe using the SA-MNBs/BT-dsDNA/SYBR Green I conjugate label. Conditions: Each data point represents an average of 3 measurements (each error bar indicates the standard deviation); 10 images each measurement.

approach improved the sensitivity by 4 orders of magnitude compared to the IgG(H+L)-Alexa Fluor 488 labeling for adsorption equilibrium strategy (23) and by 1.5 orders of magnitude approximately compared to the biotinylated monoclonal antihuman IgG molecules immobilized on the glass substrate surface by the streptavidin-coated quantum dot labeling strategy (27, 30). A major factor in increased sensitivity is likely to be that highly amplified fluorescence signal and low background signal are achieved by using mutilabel bioconjugates made by linking multiple dye/DNA conjugates to streptavidincoated magnetic nanobeads (SA-MNBs) and magnetic separation. The improved photostability is likely to be the number of dsDNA/SYBR Green I bound per bead in the labeling development step. By comparison, the SA-MNB/dsDNA/SYBR Green I conjugate approach reported in this paper uses all the common reagents such as biotin, oligonucleotides, streptavidin-coated magnetic nanobead, and fluorescent DNA probes. To evaluate the specificity of the assay for human IgG, an experiment was carried out in which the surface-immobilized protein was replaced with either mouse IgG or BSA, while keeping the protein concentration the same as that for human IgG. The subsequent assay procedure was also the same as for human IgG. As illustrated in Figure 8, at a protein concentration of 8.0 fM, 30 images were obtained from location to location on the substrate surface. The fluorescence spots of either mouse IgG or BSA sample are very low by comparison with that of human IgG. The result indicates that the immunoassay is highly specific for human IgG.

CONCLUSION In this paper, we reported an ultrasensitive fluorescence immunoassay for quantitative detection of single protein molecules based on counting of single magnetic nanobeads corresponding to single proteins with combined amplification of DNA and BT-DNA/SYBR Green I conjugate. As described above, the method offers several significant advantages. First, our strategy is to use a magnetic nanobead as a bridge carrier to attach multiple BT-DNA/SYBR Green I conjugates to an antibody at a single site, that is to say, many multiple BT-DNA/ SYBR Green I conjugates are attached to an antigen to generate a strong detectable signal. Second, such a low background is attributed to a blocking step and extensive washing steps with

Quantitative Detection of Single Molecules

magnetic separation. Third, the BT-DNA/SYBR Green I conjugate is not pre-synthesized, but rather formed in situ as part of the immunoassay. A fluorescent DNA probe was bound to the oligonucleotide at high ratio. The binding stoichiometry is preferably high to ensure the high ratio, and the quantum yield of SYBR Green I is also high. The unbound probe does not emit fluorescence, and measurements can be performed without a wash step. Fourth, the multiple labeling approach uses readily available reagents and MNBs that are commercially available.

ACKNOWLEDGMENT This project was supported by the National Natural Science Foundation of China (Grant Nos. 20775043, 20875056) and the Natural Science Foundation of Shandong Province in China (Grant No. Z2008B05). The authors would also like to acknowledge funding from the Graduate Independent Innovation Foundation of Shandong University.

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