Ultrasensitive Electrochemical DNA Assay Based on Counting of

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Ultrasensitive Electrochemical DNA Assay Based on Counting of Single Magnetic Nanobeads by a Combination of DNA Amplification and Enzyme Amplification Xiaoli Zhang, Linlin Li, Lu Li, Jia Chen, Guizheng Zou, Zhikun Si, and Wenrui Jin* School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China An ultrasensitive electrochemical method for determination of DNA is developed based on counting of single magnetic nanobeads (MNBs) corresponding to single DNA sequences combined with a double amplification (DNA amplification and enzyme amplification). In this method, target DNA (t-DNA) is captured on a streptavidin-coated substrate via biotinylated capture DNA. Then, MNBs functionalized with first-probe DNAs (p1DNA-MNBs) are conjugated to t-DNA sequences with a ratio of 1:1. Subsequently, the p1-DNA-MNBs are released from the substrate via dehybridization. The released p1-DNA-MNBs are labeled with alkaline phosphatase (AP) using biotinylated second-probe DNAs (p2-DNAs) and streptavidin-AP conjugates. The resultant AP-p2-DNA-p1-DNA-MNBs with enzyme substrate disodium phenyl phosphate (DPP) are continuously introduced through a capillary as the microsampler and microreactor at 40 °C. AP on the AP-p2-DNA-p1DNA-MNBs converts a huge number of DPP into its product phenol, and phenol zones are produced around each moving AP-p2-DNA-p1-DNA-MNB. The phenol zones are continuously delivered to the capillary outlet and detected by a carbon fiber disk bundle electrode at 1.05 V. An elution curve with peaks is obtained. Each peak is corresponding to a phenol zone relative to single t-DNA sequence. The peaks on the elution curve are counted for quantification. The number of the peaks is proportional to the concentration of t-DNA in a range of 5.0 × 10-16 to 1.0 × 10-13 mol/L. Sensitive and selective DNA detection has become increasingly important to the investigation of the genetic basis of diseases, clinical diagnosis, and gene therapy. Although the polymerase chain reaction (PCR) is the most commonly used technique for DNA analysis, a variety of DNA detection methods relying on labeling with different probes and DNA hybridization have been developed to improve sensitivity and selectivily.1-16 In analytical chemistry, single-molecule detection (SMD) is the ultimate limit * To whom correspondence should be addressed. E-mail: [email protected]. Fax: +86-531-8856-5167. (1) Saiki, R. K.; Scharf, S.; Faloona, F.; Mullis, K. B.; Horn, G. T.; Erlich, H. A.; Arnheim, N. Science 1985, 230, 1350–1354. (2) Kopp, M. U.; De Mello, A. J.; Manz, A. Science 1998, 280, 1046–1048. (3) Weiss, S. Science 1999, 283, 1676–1683.

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of detection. Recently, much attention has been focused on SMD in solution. Laser-induced fluorescence (LIF) detection is the most popular technique in SMD.17-32 LIF detection needs expensive fluorescence detection setups, such as confocal fluorescence microscopy with avalanche photodiodes or total internal reflection fluorescence microscopy with a high-sensitivity charge-coupled device. In all SMD-based quantitative methods, counting of single (4) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757–1760. (5) He, L.; Musick, M. D.; Nicewarner, S. R.; Salinas, F. G.; Benkovic, S. J.; Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 9071–9077. (6) Nicewarner-Pena, S. R.; Freeman, R. G.; Reiss, B. D.; He, L.; Pena, D. J.; Walton, I. D.; Cromer, R.; Keating, C. D.; Natan, M. J. Science 2001, 294, 137–141. (7) Han, M.; Gao, X.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631–635. (8) Yu, C. J.; Wan, Y.; Yowanto, H.; Li, J.; Tao, C.; James, M. D.; Tao, C. L.; Blackburn, G. F.; Meade, T. J. J. Am. Chem. Soc. 2001, 123, 11155–11161. (9) Park, S.-J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 1503–1506. (10) Makrigiorgos, G. M.; Chakrabarti, S.; Zhang, Y.; Kaur, M.; Price, B. D. Nat. Biotechnol. 2002, 20, 936–939. (11) Nam, J.; Park, S.; Mirkin, C. A. J. Am. Chem. Soc. 2002, 124, 3820–3821. (12) Wang, J.; Xu, D.; Polsky, R. J. Am. Chem. Soc. 2002, 124, 4208–4209. (13) Saghatelian, A.; Guckian, K. M.; Thayer, D. A.; Ghadiri, M. R. J. Am. Chem. Soc. 2003, 125, 344–345. (14) Zhao, X.; Tapec-Dytioco, R.; Tan, W. J. Am. Chem. Soc. 2003, 125, 11474– 11475. (15) Nam, J.; Stoeva, S. I.; Mirkin, C. A. J. Am. Chem. Soc. 2004, 126, 5932– 5933. (16) Liu, C.; Li, Z.; Du, B.; Duan, X.; Wang, Y. Anal. Chem. 2006, 78, 3738– 3744. (17) Nie, S.; Chiu, D. T.; Zare, R. N. Science 1994, 266, 1018–1021. (18) Xue, Q.; Yeung, E. S. Nature 1995, 373, 681–683. (19) Craig, D. B.; Arriaga, E. A.; Wong, J. C. Y.; Lu, H.; Dovichi, N. J. J. Am. Chem. Soc. 1996, 118, 5245–5253. (20) Xu, X. H.; Yeung, E. S. Science 1998, 281, 1650–1653. (21) Fister, I. J. C.; Jacobson, S. C.; Davis, L. M.; Ramsey, J. M. Anal. Chem. 1998, 70, 431–437. (22) Fang, X.; Tan, W. Anal. Chem. 1999, 71, 3101–3105. (23) Polakowski, R.; Craig, D. B.; Skelley, A.; Dovichi, N. J. J. Am. Chem. Soc. 2000, 122, 4853–4855. (24) Lagally, E. T.; Medintz, I.; Mathies, R. A. Anal. Chem. 2001, 73, 565–570. (25) Anazawa, T.; Matsunaga, H.; Yeung, E. S. Anal. Chem. 2002, 74, 5033– 5038. (26) Singh-Zocchi, M.; Dixit, S.; Ivanov, V.; Zocchi, G. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7605–7610. (27) Zhao, X.; Tapec-Dytioco, R.; Tan, W. J. Am. Chem. Soc. 2003, 125, 11474– 11475. (28) Li, H.; Zhou, D.; Browne, H.; Balasubramanian, S.; Klenerman, D. Anal. Chem. 2004, 76, 4446–4451. (29) Li, H. W.; Yeung, E. S. Anal. Chem. 2005, 77, 4374–4377. (30) Agrawal, A.; Zhang, C.; Byassee, T.; Tripp, R. A.; Nie, S. Anal. Chem. 2006, 78, 1061–1071. (31) Lee, J.; Yeung, E. S. Anal. Chem. 2007, 79, 8083–8089. (32) Li, L.; Tian, X.; Zou, G.; Shi, Z.; Zhang, X.; Jin, W. Anal. Chem. 2008, 80, 3999–4006. 10.1021/ac802183u CCC: $40.75  2009 American Chemical Society Published on Web 02/05/2009

phosphatase conjugates (streptavidin-AP) (Figure 1D). The resulting AP-p2-DNA-p1-DNA-MNBs with disodium phenyl phosphate (DPP, enzyme substrate of AP) are continuously propelled through a capillary by microsyringe injection. AP on the moving AP-p2-DNA-p1DNA-MNBs converts a large number of DPP into its product phenol, and phenol zones are formed around each MNB. The phenol zones are continuously delivered to the capillary outlet under hydraulic flow and detected by a carbon fiber disk bundle electrode (CFDBE) (Figure 1E), obtaining an elution curve with peaks. Each peak corresponds to the product zone of AP from a single MNB, and it is assumed that a single t-DNA in Figure 1C produces a single MNB. The number of peaks on the elution curve is counted for quantification. A 27-base oligonucleotide sequence (5′-GGA TTA TTG TTA AAT ATT GAT AAG GAT-3′) associated with the anthrax lethal factor sequence is chosen as the t-DNA. DNA concentration can be quantified as low as 5.0 × 10-16 mol/L.

Figure 1. Schematic representation of the process of ECD of DNA: (A) p1-DNA-MNBs are fabricated; (B) streptavidin-coated substrate is modified with biotin-c-DNA, followed by t-DNA being hybridized with c-DNA; (C) p1-DNA-MNBs are brought to the substrate and then released from the substrate; (D) the p1-DNA-MNBs are hybridized with biotin-p2-DNAs and then labeled with AP; (E) resulting AP-p2-DNA-p1-DNA-MNBs with DPP are propelled through a capillary; phenol zones are formed around each MNB and electrochemically detected at the capillary outlet.

fluorescent molecules is used to quantify analytes of interest, which guarantees the reliability of the quantitative determination because reproducibility of detected signal intensity is not important. Usually, the reproducibility of detected signal intensity is a problem in electrochemical detection (ECD) methods due to chemical passivation and/or fouling of the electrode surface. If the strategy of quantitative SMD is used to ECD of DNA with a conventional device, the sensitivity will be increased and the reproducibility of the method will be improved. In this paper, we report an ultrasensitive electrochemical method for DNA quantification based on counting of magnetic nanobeads (MNBs) combining with a double amplification (DNA amplification and enzyme amplification). The principle of this method is shown in Figure 1. First, streptavidin-coated MNBs (streptavidin-MNBs) are functionalized with biotinylated first-probe DNA (biotin-p1-DNA), fabricating p1DNA-MNBs (Figure 1A). Then, the streptavidin-coated substrate is modified with biotinylated capture DNA (biotin-c-DNA), followed by capturing target DNA (t-DNA) on the substrate (Figure 1B). Subsequently, p1-DNA-MNBs are conjugated onto the substrate by utilizing a hybridization reaction between p1-DNA and t-DNA (Figure 1C). In this case, only one MNB is bound to one t-DNA sequence. Then, the p1-DNA-MNBs are released from the substrate by dehybridization and then hybridized with a large number of biotinylated second-probe DNA (biotin-p2-DNA), followed by labeling them with streptavidin-alkaline

EXPERIMENTAL SECTION Chemicals and Materials. Streptavidin-MNBs (350 nm diameter, 1.343 g/mL, aqueous suspension containing 0.1% bovine serum albumin (BSA), 0.05% Tween-20, and 10 mmol/L EDTA with a concentration of 3.324 × 1011 beads/mL) from Bangs Laboratories Inc. (Fishers, IN), streptavidin-coated quantum dots (SA-QDs) with a maximum emission at 655 nm from Invitrogen (Eugene, OR), streptavidin-AP solution (3 mg/2 mL) from Zymed Laboratories (San Diego, CA), biotinconjugated AP (biotin-AP) solution (1 mg/mL) from Rockland (Philadelphia, PA), Tris (>99.8%) from Amresco Inc. (Solon, OH) as well as streptavidin HC coated plates (S6940, 96 wells) and Tween-20 from Sigma (St. Louis, MO) were used in the work. Other chemicals (analytical grade) were obtained from standard reagent suppliers. The physiological buffer saline (PBS) consisted of 0.15 mol/L NaCl, 7.6 × 10-3 mol/L NaH2PO4, and 2.4 × 10-3 mol/L Na2HPO4 (pH 7.4). TE buffer consisted of 0.010 mol/L Tris-HCl and 0.001 mol/L Na2EDTA (pH 8.0). TTL buffer consisted of 0.100 mol/L Tris-HCl (pH 8.0), 0.1% Tween-20, and 1 mol/L LiCl. TT buffer consisted of 0.250 mol/L Tris-HCl (pH 8.0) and 0.1% Tween-20. TTE buffer consisted of 0.250 mol/L Tris-HCl (pH 8.0), 0.1% Tween-20, and 0.020 mol/L Na2EDTA (pH 8.0). Synthetic biotinylated 32-mer single-stranded c-DNA (5′-TAA CAA TAA TCC-T20-3′-biotin), biotinylated 35-mer singlestranded p1-DNA (biotin-5′-T20-ATC CTT ATC AAT ATT-3′), biotinylated 16-mer single-stranded p2-DNA (biotin-5′-T AAT ATT GAT AAG GAT-3′), 16-mer single-stranded FAM-labeled p2-DNA (FAM-5′-T AAT ATT GAT AAG GAT-3′), 27-mer single-stranded t-DNA (5′-GGA TTA TTG TTA AAT ATT GAT AAG GAT-3′), single-stranded noncomplementary DNAs (5′TAG CCT CCA GAC TCA CAA ATA CTT ATG-3′, 5′-CTC CAA ATG TAG GAG CTA TCG TT-3′, 5′-AAG CCA TGA AGC GGC TTA TGA TTC TTA CCG CCC ACT-3′, 5′-GCG AGG ATT TGA CGA AAG CGC ACC TTA AAG-3′, and 5′-GGC AAG CCG ATA ACG GGA TTA-3′), and 27-mer single-stranded single base mismatched DNA (1b-m-DNA, 5′-GGA TAA TTG TTA AAT ATT GAT AAG GAT-3′) were purchased from SBS Genetech Co., Ltd. (Beijing, China). The 1.00 × 10-4 mol/L stock solutions for each DNA were prepared by centrifuging for 1 min at 1000 rpm and dissolving in an appropriate volume of TE buffer and stored at -20 °C. The dilute DNA solutions were Analytical Chemistry, Vol. 81, No. 5, March 1, 2009

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obtained by a serial dilution with TE buffer. Before experiments, the dilute solutions were heated for 5 min at 90 °C and then cooled for 10 min in an ice bath. The preparation of the DNA solutions was performed in a clean bench. To prevent the contamination from the possible repeat sampling, the commercial MNB suspension and the stock DNA solutions were divided into several small packs in disinfected plastic vessels. All aqueous solutions were prepared with doubly distilled water. Several steps described in our previous work33 were taken to minimize contamination from the outside environment. Apparatus. A CHI 802 electrochemical analyzer (CH Instruments, Austin, TX) was used to perform ECD. A syringe pump (model TCI-II, Beijing Silugao High-Techdevelop Co., Ltd., Beijing, China) coupled with a 100 µL syringe (Shanghai Gaoge Co., Ltd., Shanghai, China) was used for sample injection. The total internal reflection fluorescence microscopy (TIRFM) system described in our previous work32 was used to accomplish the TIRFM measurements of single quantum dot (QD)-labeled t-DNA sequences. Functionalization of MNBs with p1-DNA. Before functionalization, 10 µL of streptavidin-MNB suspension (3.32 × 1011 beads/mL) was washed five times with 400 µL of TTL buffer, to remove surfactants. Then, 10 µL of TTL buffer was added. An amount of 1 µL of the MNB suspension was diluted with 50 µL of TTL buffer. Subsequently, biotin-p1-DNA was conjugated to the streptavidin-MNBs by adding 10 µL of biotin-p1-DNA solution (2.5 × 10-5 mol/L) and incubating for 30 min at room temperature. The unreacted biotin-p1-DNA was washed away with 400 µL of 0.15 mol/L NaOH, obtaining p1-DNA-MNB suspension. The p1-DNA-MNBs were washed twice with 400 µL of TT buffer and twice with 400 µL of TTE buffer, respectively. Then, 400 µL of TTE buffer was added. In order to remove unbound biotin-p1-DNA on the surface of the streptavidin-MNBs, the MNB suspension was incubated for 10 min at 80 °C. After the MNBs were magnetically separated, 10 µL of TTE buffer was added for subsequent experiments. All washing steps in this work were performed under a magnetic field. Modification of Substrate with c-DNA and Capture of t-DNA onto the Substrate. The wells of the streptavidin-coated plates with high capacity were used in this work as the reactors. After the well was washed twice with 200 µL of PBS, biotin-cDNA was conjugated to the substrate of the well by adding 10 µL of biotin-c-DNA solution (5.0 × 10-5 mol/L) and 50 µL of PBS into the well and incubating for 2 h at room temperature. The solution was then removed. In order to remove the unconjugated biotin-c-DNA on the substrate, the well was washed five times with 200 µL of PBS and three times with 200 µL of 1.0 mol/L NaCl, respectively. Subsequently, 10 µL of t-DNA solution and 50 µL of 1.0 mol/L NaCl were added into the well. The well was placed in a constant-humidity chamber, and the solution was incubated for 4 h at 25 °C to capture t-DNA onto the substrate. Then, the solution was removed and the substrate was washed five times with 200 µL of PBS. Binding of p1-DNA-MNBs to the t-DNA Sequences on Substrate. An amount of 20 µL of the p1-DNA-MNB suspension (3.32 × 1010 beads/mL) obtained above and 50 µL of 1.0 mol/L (33) Sun, X.; Jin, W. Anal. Chem. 2003, 75, 6050–6055.

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NaCl were added into the well with the substrate containing captured t-DNA. The well was placed in a constant-humidity chamber, and the solution was incubated for 4 h to bind p1DNA-MNBs to t-DNA sequences on the substrate. The solution was then removed, and the substrate of the well was gently rinsed eight times with 200 µL of PBS by drawing these solutions out along the wall of the well. Release of p1-DNA-MNBs from Substrate and Labeling AP onto the Released p1-DNA-MNBs. To release p1DNA-MNBs from the substrate through dehybridization, 200 µL of 50% (w/w) urea solution was added to the well with MNBs on the substrate. After 10 min, the urea solution containing released p1-DNA-MNBs was transferred to a vessel. The p1-DNA-MNBs were magnetically separated, followed by washing the p1DNA-MNBs five times with 400 µL of 1.0 mol/L NaCl. Subsequently, 10 µL of 2.5 × 10-5 mol/L biotin-p2-DNA and 50 µL of 1.0 mol/L NaCl were added to the vessel, and the suspension was incubated for 4 h at 25 °C, followed by washing the resulting biotin-p2-DNA-p1-DNA-MNBs five times with 400 µL of PBS. Then, 50 µL of PBS was added. To label AP to biotin-p2-DNA-p1-DNA-MNBs, 30 µL of 1.0 × 10-5 mol/L streptavidin-AP was added and the suspension was incubated for 30 min at 25 °C, followed by washing the resultant AP-p2DNA-p1-DNA-MNBs three times with 400 µL of PBS at room temperature and three times with 400 µL of 0.010 mol/L borate buffer (pH 9.8) at 0 °C. Finally, 10 µL of 0.010 mol/L borate buffer (pH 9.8) was added to the vessel for subsequent ECD. Preparation of Hybrids of p1-DNA-MNBs and FAMLabeled p2-DNA (FAM-p2-DNA), as well as Quantification of FAM-p2-DNA Released from the Hybrids Using Fluorescence Detection. In order to optimize the hybridization time between p1-DNA-MNBs and other DNAs, p1-DNA-MNBs was hybridized with FAM-p2-DNA. An amount of 10 µL of 3.32 × 1010 beads/mL p1-DNA-MNB was incubated for 2-6 h with 10 µL of 2.5 × 10-5 mol/L FAM-p2-DNA and 50 µL of 1.0 mol/L NaCl at 25 °C in the dark, followed by washing the resulting FAM-p2DNA-p1-DNA-MNBs five times with 400 µL of 1.0 mol/L NaCl to remove the unhybridized FAM-p2-DNA. Then, 50 µL of 1.0 mol/L NaCl was added. The fluorescence intensity of 20 µL of the FAM-p2-DNA-p1-DNA-MNB suspension was measured. In order to determine the amount of p1-DNA conjugated on MNBs, the FAM-p2-DNA-p1-DNA-MNBs obtained after 4 h of hybridization was dehybridized by adding 200 µL of 50% (w/ w) urea solution and incubating for 10 min at 25 °C in the dark. The p1-DNA-MNBs were magnetically removed. The fluorescence intensity of 20 µL of the solution containing FAM-p2-DNA released from the FAM-p2-DNA-p1-DNA-MNBs was measured, and the concentration of FAM-p2-DNA in the solution was determined using the calibration curve of FAM-p2-DNA, which was obtained by measuring the fluorescence intensity of the standard solutions of FAM-p2-DNA. Acquiring Fluorescence Images of Single Quantum Dot Labeled t-DNA Sequences Attached on the Substrate by TIRFM. A well consisted of a streptavidin-coated substrate, which was obtained by removing the plastic wall of the commercial streptavidin-coated well, and four thin glass slides glued to the substrate with epoxy served as the reactor. After t-DNA was captured on the substrate via c-DNA mentioned above, the t-DNA

was hybridized with biotin-p1-DNA by incubating the hybrids on the substrate for 4 h with 10 µL of 5.0 × 10-6 mol/L biotin-p1DNA and 50 µL of 1.0 mol/L NaCl at room temperature, followed by washing the well three times with 200 µL of 1.0 mol/L NaCl and three times with PBS, respectively, to remove the unhybridized biotin-p1-DNA. Then, the biotin-p1-DNA-tDNA-c-DNA was labeled with QD by adding 10 µL of 1.0 × 10-8 mol/L SA-QD and 50 µL of PBS into the well and incubating for 2 h at room temperature. The unreacted SAQDs was removed by washing six times with PBS, obtaining QD-labeled t-DNA on the substrate. The glued glass well walls were removed, and the substrate with the QD-labeled t-DNA was covered with a microscope coverslip. The coverslip with the substrate was placed on the stage of the inverted microscope of the TIRFM system to acquire the fluorescence images of single QD-labeled t-DNA sequences on the substrate according to the same procedure as in ref 32. Acquiring Fluorescence Images of FAM-p2-DNA-p1DNA-MNBs Attached on the Substrate by Epifluorescence Microscopy. After binding p1-DNA-MNBs to the t-DNA captured on the substrate as described above, the p1-DNA-MNBs on the substrate were labeled with a dye FAM via the hybridization between p1-DNA on the MNBs and FAM-p2-DNA. The p1DNA-MNBs on the substrate were incubated overnight with 10 µL of 2.5 × 10-5 mol/L FAM-p2-DNA and 50 µL of 1.0 mol/L NaCl at 25 °C in the dark, followed by washing the resulting FAM-p2-DNA-p1-DNA-MNBs on the substrate eight times with 200 µL of 1.0 mol/L NaCl to remove the unhybridized FAM-p2-DNA. The fluorescence image at >510 nm of the substrate with FAM-p2-DNA-p1-DNA-MNBs was taken by an Olympus IX81 fluorescence microscope coupled with a 10× objective using an excitation wavelength of 460-490 nm. Electrochemical Detection of Single AP-p2-DNA-p1DNA-MNBs. The ECD system for determination of single AP-p2-DNA-p1-DNA-MNBs illustrated in the Supporting Information is similar to that of Jorgenson’s group34 and our previous work.35 Briefly, it consisted of three parts, a microsyringe injection device, an enzyme reaction setup, and an electrochemical detector. In the enzyme reaction setup, the 55 cm of the capillary was passed through a warm water bath of 40 °C. In order to detect single AP-p2-DNA-p1-DNA-MNBs, the AP-p2-DNA-p1DNA-MNB suspension in 0.010 mol/L borate buffer (pH 9.8) containing 1.0 × 10-3 mol/L DPP was introduced into the capillary using the microsyringe injection device and incubated for 15 min. Then, the suspension was pressured to the capillary outlet and detected by the CFDBE at 1.05 V versus a saturated calomel electrode (SCE). For high-throughput detection of the AP-p2-DNA-p1-DNA-MNBs, 10 µL of the AP-p2-DNA-p1DNA-MNB suspension in 0.010 mmol/L borate buffer (pH 9.8) was mixed with 990 µL of 1.00 × 10-3 mol/L DPP at 4 °C. Then, 100 µL of the suspension was added into the microsyringe. An amount of 5 µL or 0.5 µL of the suspension was continuously propelled by the syringe injection device through the capillary. The phenyl zones produced around every moving AP-p2-DNA-p1-DNA-MNB were detected by the CFDBE at

1.05 V versus SCE at the capillary outlet. During the run, the microsyringe was inverted for 3 min each 30 min to prevent the AP-p2-DNA-p1-DNA-MNB precipitation at the bottom of the microsyringe.

(34) Kennerdy, R. T.; St. Claire, R. L.; White, J. G.; Jorgenson, J. W. Mikrochim. Acta 1987, II, 37–45. (35) Gao, N.; Wang, W.; Zhang, X.; Jin, W.; Yin, X.; Fang, Z. Anal. Chem. 2006, 78, 3213–3220.

(36) Manufacture’s instructions of MagPrep streptavidin beads, Bangs Laboratories Inc., Fishers, IN. (37) Manufacture’s instructions of streptavidin HC coated plates, Sigma, St. Louis, MO.

RESULTS AND DISCUSSION Functionalization of MNBs with p1-DNA. To bind p1DNA-MNBs to t-DNA sequences captured on the substrate, the biotin-p1-DNA has a sequence (biotin-5′-T20-ATC CTT ATC AAT ATT-3′) with 20 thymines at the biotin end acting as a spacer to reduce the steric hindrance between the biotin-p1DNA and the surface-confined streptavidin interaction. Through the irreversible interaction between streptavidin and biotin (Ka ) 1015/mol L-1) with rapid binding kinetics and strong affinity,36 biotin-p1-DNA conjugates onto the 350 nm diameter streptavidin-MNBs, producing p1-DNA-MNBs. In this method, the primary DNA amplification factor of the final detection signal is proportional to the number of p1-DNA sequences on one p1-DNA-MNB. The average biotin binding sites on the surface of one streptavidin-MNB is ∼20 000 based on the data provided by the manufacture’s instructions. In order to ensure all binding sites on the surface of one streptavidin-MNB are conjugated by biotin-p1-DNA, the amount of biotin-p1-DNA used for reaction here is 20 times over the biotin binding sites. We determined the average number of p1-DNA bound onto the surface of one p1-DNA-MNB to be ∼7000 by using fluorescence detection after labeling them with the organic fluorophore FAM via FAM-p2-DNA. Although the measured value was different from that provided by the manufacture’s instructions, the large amplification factor was obtained. Modification of Substrate with c-DNA and Capture of t-DNA. DNA-DNA sandwich hybridization is a common technique in DNA assays. In this mode, two hybridization steps are accomplished. One is the primary hybridization between c-DNA and t-DNA and another is the secondary hybridization between t-DNA and p1-DNA on the MNBs. In order to capture t-DNA, the substrate is modified with c-DNA. Wells of the commercially available microtiter plates with streptavidin-coated substrates are used here as reactors. The biotin-c-DNA is conjugated to the streptavidin-coated substrate via the irreversible interaction between streptavidin and biotin. In this step, all biotin binding sites on the surface of streptavidin-coated substrate should be occupied by biotin-c-DNA. According to manufacture’s instructions,37 at least 300 pmol of biotin can be conjugated onto the streptavidincoated substrate with an area of 32 mm2. In the experiments, 500 pmol of biotin-c-DNA are reacted with streptavidin on the substrate. To enhance the stability of the hybrid of t-DNA with c-DNA on the substrate during capturing t-DNA, a high concentration of NaCl (>0.5 mol/L) was used as hybridization buffer. The basic assumption in this method is that one t-DNA sequence brings one p1-DNA-MNB onto the substrate. When the distance between two adjacent t-DNA sequences captured on the substrate is larger than that between two adjacent MNBs, this assumption can be satisfied. Thus, the concentration of t-DNA should be low

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Figure 3. Epifluorescence images of the substrate with FAM-p2DNA-p1-DNA-MNBs fabricated using 1.0 × 10-13 mol/L t-DNA (A) before and (B) after dehybridization using 50% (w/w) urea solution. Figure 2. TIRFM fluorescence image of single QD-labeled t-DNA sequences on the substrate fabricated using 1.0 × 10-13 mol/L DNA.

enough. It can be roughly calculated that 3 × 108 MNBs with a diameter of 350 nm can completely occupy the substrate within an area of 32 mm2. This means that the amount of t-DNA should be lower than 5 × 10-16 mol. In our experiments, 10 µL of t-DNA solutions with concentrations lower than 1.0 × 10-13 mol/L was used. Thus, if all t-DNA are captured on the substrate, the average distance between two t-DNAs is 10 times longer than the diameter of the MNB. Although there are many p1-DNAs on each MNB, it is not impossible that one MNB is bound to more than two t-DNA sequences because the space between two adjacent t-DNA sequences is big enough. In order to prove this assumption, we measured the average distance between two adjacent t-DNA sequences using TIRFM that can take a single-molecule image.35 To do so, the t-DNA captured on the substrate was labeled with QDs using biotin-p1-DNA and SAQDs. The fluorescence image for 1.0 × 10-13 mol/L t-DNA is shown in Figure 2. In the image, each bright spot is corresponding to a single t-DNA. It is obvious that the interspot distance is greater than 5 µm, much bigger than the distance between two adjacent MNBs. Thus, we can conclude that one t-DNA sequence should bring one MNB onto the substrate when the concentrations of t-DNA are lower than 1.0 × 10-13 mol/L. The conclusion was demonstrated by a control experiment, in which the t-DNA solution was replaced by a buffer. In this case, only 7-8 bright spots were observed in 30 images, i.e., 1 × 10-13 mol/L led to overlapping peaks (Figure 7, curve 8). In this case, the peak was more 2-fold wider than that of a single peak, which was obtained from the elution curve at lower concentrations. CONCLUSION ECD can quantitatively detect single MNBs corresponding to single DNA sequences by a combination of DNA amplification and enzyme amplification. The method offers several significant advantages. First, the signal detected is very strong due to DNA and enzyme amplification. Second, ultrahigh sensitivity can be reached thanks to counting single MNBs. Third, since quantification relies on the number of peaks rather than their signal size, the reproducibility of the detected signal intensity, which is a problem often found with ECD methods due to chemical passivation and/or fouling of the electrode surface, becomes irrelevant. Our method allows an elution curve to be recorded by several electrodes without loss in reproducibility of the determined results. Fourth, no expensive instrument is required and MNBs and streptavidin-coated microtiter plates are commercially available. ACKNOWLEDGMENT This project was supported by the National Natural Science Foundation of China (Grant Nos. 20475033, 20675047, 90713016, and 20705016), the National Basic Research Program of China (Grant No. 2007CB936602), the Natural Science Foundation of Shandong Province in China (Grant No. Y2008B20), and the State Key Laboratory of Electroanalytical Chemistry, Changchun, Institute of Applied Chemistry, Chinese Academy of Science (Grant No. 2008009). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review December 31, 2008. AC802183U

October

15,

2008.

Accepted