Single Quantum Dot-Based Nanosensor for Multiple DNA Detection

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Anal. Chem. 2010, 82, 1921–1927

Single Quantum Dot-Based Nanosensor for Multiple DNA Detection Chun-yang Zhang* and Juan Hu Institute of Biomedical Engineering and Health Technology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China Owing to their unique optical properties, quantum dots (QDs) with different colors have been applied for simultaneous detection of multiple analytes. However, the use of single QD for multiplex detection of analytes with singlemolecule detection has not been explored. Here we report a single QD-based nanosensor for multiplex detection of HIV-1 and HIV-2 at single-molecule level in a homogeneous format. In this single QD-based nanosensor, the QD functions not only as a fluorescence pair for coincidence detection and as a fluorescence-resonance-energytransfer (FRET) donor for FRET detection but also as a local nanoconcentrator which significantly amplifies the coincidence-related fluorescence signals and the FRET signals. This single-QD-based nanosensor takes advantage of a simple ‘mix and detection’ assay with extremely low sample consumption, high sensitivity, and short analysis time and has the potential to be applied for rapid pointof-care testing, gene expression studies, high-throughput screening, and clinical diagnostics. With the enhanced security and health concern worldwide, the development of highly sensitive and selective sensors capable of simultaneous detection of multiple analytes has attracted much attention in the scientific community. In comparison with the single-target assay, multiplex assay has the ability to screen for multiple analytes in a single assay with the significant advantage of rapid, simple, low sample and reagent consumption. A variety of biosensors based on surface plasmon resonance,1 electrochemistry,2 metal nanowire,3 magnetic nanoparticles,4 chips,5 and quantum dots6,7 has been developed for multiplex detection of antibiotic residues, proteins, nucleic acids, tumors, toxins, and small molecules. Of them, the development of biosensors for multiplex detection of nucleic acids is especially desirable due to the fact that nucleic acid analysis plays an increasingly important * To whom correspondence should be addressed. E-mail: cy.zhang@ sub.siat.ac.cn. (1) Raz, S. R.; Bremer, M. G. E. G.; Haasnoot, W.; Norde, W. Anal. Chem. 2009, 81, 7743–7749. (2) Wei, F.; Patel, P.; Liao, W.; Chaudhry, K.; Zhang, L.; Arellano-Garcia, M.; Hu, S.; Elashoff, D.; Zhou, H.; Shukla, S.; Shah, F.; Ho, C. M.; Wong, D. T. Clin. Cancer Res. 2009, 15, 4446–4452. (3) Cederquist, K. B.; Golightly, R. S.; Keating, C. D. Langmuir 2008, 24, 9162–9171. (4) Wang, S. X.; Li, G. IEEE Trans. Magn. 2008, 44, 1687–1702. (5) Wilson, M. S.; Nie, W. Y. Anal. Chem. 2006, 78, 6476–6483. (6) Goldman, E. R.; Clapp, A. R.; Anderson, G. P.; Uyeda, H. T.; Mauro, J. M.; Medintz, I. L.; Mattoussi, H. Anal. Chem. 2004, 76, 684–688. (7) Liu, J.; Lee, J. H.; Lu, Y. Anal. Chem. 2007, 79, 4120–4125. 10.1021/ac9026675  2010 American Chemical Society Published on Web 02/02/2010

role in the diagnosis of hereditary diseases, detection of infectious agents, forensic and paternity testing, veterinary medicine, and environmental monitoring.8 Recently great progress has been made on the development of biosensors capable of simultaneous determination of multiple nucleic acids,9-15 including the biosensors based on gold nanoparticle,9,10 bar-coded nanorods,11 fiber-optical DNA array,12 quantum dots-based DNA array,13electrochemical coding,14 and DNA-based fluorescence nanobarcodes.15 However, the detection sensitivity of most of these biosensors is relatively low and incapable of detecting extremely low-abundance biomolecules. Alternatively, polymerase chain reaction (PCR) provides an extremely high sensitive method for detecting low-abundance species by amplifying a single or few copies of nucleic acids across several orders of magnitude.16 However, PCR might suffer from a number of complications such as contamination-induced falsepositive signals.17 Due to its unique capability of detecting a single molecule with a high signal-to-noise ratio, the single-molecule detection technique holds great promise for direct detection of low-abundance species without amplification.18-20 So far, a series of single-molecule detection techniques has been developed for the homogeneous sequence-specific detection of nonamplified genomic DNA and mRNA, such as two-color coincident detection21-25 and dual-color fluorescence cross-correlation spectroscopy (FCS).26-28 But two-color coincident detection and dual-color FCS can only detect a singletarget analyte even though two-color fluorophores are employed in these assays. In order to screen for multiple analytes simultaneously, more than two organic fluorophores are usually needed, but their functional limitations such as the spectral cross-talk and nonuniform fluorophore photobleaching rates make subsequent (8) Kricka, L. J. Ann. Clin. Biochem. 2002, 39, 114–129. (9) Taton, T. A.; Lu, G.; Mirkin, C. A. J. Am. Chem. Soc. 2001, 123, 5164– 5165. (10) Cao, Y. W.; Jin, R.; Mirkin, C. A. Science 2002, 297, 1536–1540. (11) Reiss, B.; He, L.; Walton, I.; Cromer, R.; Keating, C.; Natan, M. Science 2001, 294, 137–141. (12) Ferguson, J. A.; Steemers, F. J.; Walt, D. R. Anal. Chem. 2000, 72, 5618– 5624. (13) Gerion, D. E.; Chen, F. Q.; Kannan, B.; Fu, A. H.; Parak, W. J.; Chen, D. J.; Majumdar, A.; Alivisatos, A. P. Anal. Chem. 2003, 75, 4766–4772. (14) Wang, J.; liu, G. D.; Merkoci, A. J. Am. Chem. Soc. 2003, 125, 3214–3215. (15) Li, Y.; Hong, Y. T.; Luo, D. Nat. Biotechnol. 2005, 23, 885–889. (16) Sambrook, J.; Russell, D. W. Molecular Cloning: A Laboratory Mannual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 2001. (17) Wilke, W. W.; Jones, R. N.; Sutton, L. D. Diagn. Microbiol. Infect. Dis. 1995, 21, 181–185. (18) Xu, X. H.; Yeung, E. S. Science 1997, 275, 1106–1109. (19) Weiss, S. Science 1999, 283, 1676–1683. (20) Hsin, T. M.; Yeung, E. S. Angew. Chem., Int. Ed. 2007, 46, 8032–8035.

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quantification analysis complicated. Consequently, there are few reports about single-molecule detection for multiple analyte assay so far. Quantum dots (QDs) are novel semiconductor nanocrystals with significant advantages over organic fluorophores.29 QDs have broad excitation and size-tunable photoluminescence spectra with narrow emission bandwidth (full-width at half-maximum of ∼25-40 nm), exceptional photochemical stability, and relative high quantum yield.29 QDs have been widely used as fluorescent markers in genomic analysis, immunoassay, fluorescence imaging, and drug delivery.30,31 Recently, QDs have been used as fluorescenceresonance-energy-transfer (FRET) donors in biosensors to detect DNA, protein, and RNA-peptide interaction.32-35 Owing to their unique optical properties, QDs with different colors have been applied for simultaneous detection of multiple toxins, mRNAs, and small molecules in the ensemble assays.6,7,36 Furthermore, onecolor immobilized QDs have just been demonstrated for multiplexed solid-phase assay of nucleic acid hybridization in the ensemble assays.37 However, the use of single QD for multiplex detection of nucleic acids in the solution with single-molecule detection has not been explored so far. In this paper, we report a single-QD-based nanosensor for multiplex detection of HIV-1 and HIV-2 based on both two-color coincidence detection and FRET detection. HIV-1 and HIV-2 cause acquired immunodeficiency syndrome (AIDS);38,39 the donated blood is routinely screened for the presence of HIV-1 and HIV-2. Therefore, a simple and ultrasensitive method for multiplex detection of HIV-1 and HIV-2 is highly desirable. The single-QDbased nanosensor we developed enables multiplex detection of HIV-1 and HIV-2 at the single-molecule level in a homogeneous (21) Castro, A.; Williams, J. G. K. Anal. Chem. 1997, 69, 3915–3920. (22) Ren, X.; Li, H.; Clarke, R. W.; Alves, D. A.; Ying, L.; Klenerman, D.; Balasubramanian, S. J. Am. Chem. Soc. 2006, 128, 4992–5000. (23) Li, H. T.; Ying, L. M.; Green, J. J.; Balasubramanian, S.; Klenerman, D. Anal. Chem. 2003, 75, 1664–1670. (24) Orte, A.; Clarke, R.; Balasubramanian, S.; Klenerman, D. Anal. Chem. 2006, 78, 7707–7715. (25) Nolan, R. L.; Cai, H.; Nolan, J. P.; Goodwin, P. M. Anal. Chem. 2003, 75, 6236–6243. (26) Camacho, A.; Korn, K.; Damond, M.; Cajot, J. F.; Litborn, E.; Liao, B. H.; Thyberg, P.; Winter, H.; Honegger, A.; Gardellin, P.; Rigler, R. J. Biotechnol. 2004, 107, 107–114. (27) Rigler, R.; Foldes-Papp, Z.; Meyer-Alme, F. J.; Sammet, C.; Volcker, M.; Schnetz, A. J. Biotechnol. 1998, 63, 97–109. (28) Maiti, S.; Haupts, U.; Webb, W. W. Proc. Natl Acad. Sci. U.S.A. 1997, 94, 11753–11757. (29) Pons, T.; Mattoussi, H. Ann. Biomed. Eng. 2009, 37, 1934–1959. (30) Medintz, I. L.; Uyeda, H. T.; Goldman, E.; Mattoussi, H. Nat. Mater. 2005, 4, 437–446. (31) Alivisatos, A. P. Nat. Biotechnol. 2004, 22, 47–52. (32) Patolsky, F.; Gill, R.; Weizmann, Y.; Mokari, T.; Banin, U.; Willner, I. J. Am. Chem. Soc. 2003, 125, 13918–13919. (33) Medintz, I. L.; Clapp, A. R.; Mattoussi, H.; Goldman, E. R.; Fisher, B.; Mauro, J. M. Nat. Mater. 2003, 2, 630–638. (34) Medintz, I. L.; Konnert, J. H.; Clapp, A. R.; Stanish, I.; Twigg, M. E.; Mattoussi, H.; Mauro, J. M.; Deschamps, J. R. Proc. Natl Acad. Sci. U.S.A. 2004, 101, 9612–9617. (35) Zhang, C. Y.; Johnson, L. W. J. Am. Chem. Soc. 2006, 128, 5324–5325. (36) Chan, P.; Yuen, T.; Ruf, F.; Gonzalez-Maeso, J.; Sealfon, S. C. Nucleic Acids Res. 2005, 33, e161. (37) Algar, W. R.; Krull, U. J. Anal. Chem. 2010, 82, 400–405. (38) Popovic, M.; Sarngadharan, M. G.; Read, E.; Gallo, R. C. Science 1984, 224, 497–500. (39) Clavel, F.; Guetard, D.; Brun-Vezinet, F.; Chamaret, S.; Rey, M. A.; SantosFerreira, M. O.; Laurent, A. G.; Dauguet, C.; Katlama, C.; Rouzoux, C.; Klatzmann, D.; Champalimaud, J. L.; Montagnier, L. Science 1986, 233, 343–346.

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format. In this nanosensor, the QD functions not only as a fluorescence pair for the coincidence detection and as a FRET donor for the FRET assay but also as a local nanoconcentrator which significantly amplifies the coincidence-related fluorescence signals and the FRET signals. EXPERIMENTAL SECTION Sample Preparation. We designed two biotinylated capture probes, one Alexa Fluor 488- and one Alexa Fluor 647-labeled reporter probe, and two target oligonucleotides which were conserved sequences of the gap gene of HIV-1 and the env gene of HIV-2, as models for demonstrating the single QD-based nanosensor for multiple DNA detection. Each target oligonucleotide was complementary to one biotinylated capture probes and one Alexa Fluor 488- (or Alexa Fluor 647-) labeled reporter probe by sandwich reaction. The sequences of two biotinylated capture probes are as follows: capture probe 1, 5′-TAG TAA GAA TGT ATA GC-3′-TEG-Biotin and capture probe 2, 5′-GCA ACT AAA TTC A-3′-TEG-Biotin. The sequences of Alexa Fluor 488- and Alexa Fluor 647-labeled reporter probes are as follows: reporter probe 1, Alexa Fluor 488-5′-CTG GGA TTA AAT AAA A-3′ and reporter probe 2, Alexa Fluor 647-5′-AAA GGA CCA GGC-3′. The sequences of two target oligonucleotides are as follows: target DNA 1 for HIV-1, 5′-GCT ATA CAT TCT TAC TAT TTT ATT TAA TCC CAG3′ and target DNA 2 for HIV-2, 5′-TGA ATT TAG TTG CGC CTG GTC CTT T-3′. All above oligonucleotides were purified by highperformance liquid chromatography and purchased from Integrated DNA Technology Inc. (Coralville, IA, USA). The hybridization experiments were performed in a buffered solution containing 100 mM Tris-HCl, 10 mM (NH4)2SO4, 3 mM MgCl2, pH 8.0. The reactions were carried out by mixing biotinylated capture probes, Alexa Fluor 488- and Alexa Fluor 647-labeled reporter probes, and the target DNAs at 40 °C for 30 min (the molecular ratio of capture probes to corresponding reporter probes was kept at the ratio of 1:1). After cooling to room temperature, the streptavidin-coated 605-nm-emission QDs (605QDs) (Quantum Dot Corp., Hayward, CA) were added to capture the sandwiched hybrids prior to detection. Finally the solution was subjected to single-molecule detection at the 605QD concentration of 2.5 × 10-11 M. Experimental Setup for Single-Molecule Detection. The experimental setup for single-molecule detection was shown in Figure 1. A 488-nm Argon laser was used as an excitation light source for Alexa Fluor 488 and the 605QDs. The 488-nm beam was collimated, reflected by a dichroic mirror (Z488RDC, Chroma Technology Corp, Rockingham, VT), and then focused by an oil immersion 100 × /1.30 NA objective lens (Olympus America, Inc., Melville, NY) to excite a sample in a 50-µm ID capillary, and the sample passed a laser-focused detection volume through pressuredriven flow with a syringe pump (Harvard Apparatus, Holliston, MA). Photons emitted from three fluorophores were collected through the same objective, passed through the first dichroic mirror followed by a 50 µm pinhole (Melles Griot Co, Irvine, CA), and then separated by a second dichroic mirror (645DCLP, Chroma Technology Corp, Rockingham, VT). After separation, the signals emitted from Alexa Fluor 647 were filtered by a bandpass filter (D680/30M, Chroma Technology Corp, Rockingham, VT) and detected by an avalanche photodiode (Model SPCM-AQR13, EG&G Canada, Vaudreuil, PQ, Canada) in the first channel.

Figure 1. Schematic view of the experimental apparatus used for simultaneous detection of the photons emitted from the 605QD, Alexa Fluor 488, and Alexa Fluor 647. Photons emitted from the 605QD, Alexa Fluor 488, and Alexa Fluor 647 were separated by three dichroic mirrors and detected by three avalanche photodiodes (APDs), respectively.

At the same time, photons emitted from the 605QD and Alexa Fluor 488 were separated by a third dichroic mirror (565DCLP, Chroma Technology Corp, Rockingham, VT). After separation, the signals emitted from Alexa Fluor 488 were filtered by a bandpass filter (D535/40M, Chroma Technology Corp, Rockingham, VT) and detected by an avalanche photodiode in the second channel; photons emitted from the 605QD were filtered by a bandpass filter (D605/20M, Chroma Technology Corp, Rockingham, VT) and detected with another avalanche photodiode in the third channel. A program written with Labview (National Instruments, Austin, TX) and a digital counter (National Instruments, Austin, TX) were used to perform data acquisition and online data analysis. Fluorescence signals from all three channels were integrated in 0.5-ms interval for a total running time of 100 s for each experiment. In single-molecule detection, a threshold was used to distinguish single-molecule fluorescence signals from random fluctuation in the background. The threshold value was determined by evaluating data from the control sample. In this study, a threshold of 12 photon counts · ms-1 was set for Alexa Fluor 488, a threshold of 10 photon counts · ms-1 was set for Alexa Fluor 647, and a threshold of 10 photon counts · ms-1 was set for the 605QD. RESULTS AND DISCUSSION Principle of Single-QD-Based Nanosensor for Multiple DNA Detection. Alexa Fluor 488, Alexa Fluor 647, and the 605QD were used as fluorophores in this single-QD-based nanosensor. Their absorption and emission spectra were shown in Figure 2. They formed an Alexa Fluor 488-DNA-605QD-DNAAlexa Fluor 647 complex through DNA sandwich hybridization and streptavidin-biotin binding (Figure 3A). In this complex, Alexa Fluor 488 and the 605QD might be excited simultaneously under the excitation wavelength of 488 nm; there were no spectral overlap and cross-talk between the emission of

Figure 2. The normalized absorption and emission spectra of the 605QD, Alexa Fluor 488, and Alexa Fluor 647. Blue line, absorption spectrum of Alexa Fluor 488; green line, emission spectrum of Alexa Fluor 488; black line, absorption spectrum of the 605QD; magenta line, emission spectrum of the 605QD; cyan line, absorption spectrum of Alexa Fluor 647; red line, emission spectrum of Alexa Fluor 647.

Alexa Fluor 488 and that of the 605QD (Figure 2). In contrast, Alexa Fluor 647 cannot be excited by the 488-nm laser. But due to the significant spectral overlap between the emission spectrum of the 605QD and the absorption spectrum of Alexa Fluor 647 (Figure 2), FRET might happen between the 605QD and Alexa Fluor 647;40 as a result, the emission of Alexa Fluor 647 might still be observed in this complex under the excitation of 488-nm laser. Notably, there were no spectral overlap and cross-talk between the emission of the 605QD and that of Alexa Fluor 647(Figure 2). Therefore, in this Alexa Fluor 488-DNA605QD-DNA-Alexa Fluor 647 complex, the emissions of Alexa Fluor 488, Alexa Fluor 647, and the 605QD might be observed simultaneously without either spectral overlap or cross-talk under the excitation of 488-nm laser (Figure 3A), suggesting that this single-QD-based nanosensor might be suitable for multiple DNA detection. It should be noted that a single 605QD might efficiently couple to multiple Alexa Fluor 488-labeled and Alexa Fluor 647-labeled sandwich hybrids; therefore, the 605QD also served as a nanoscaffold to amplify both Alexa Fluor 488 and Alexa Fluor 647 fluorescence signals. Design of Single-QD-Based Nanosensor for Multiple DNA Detection. This single QD-based DNA nanosensor consisted of a streptavidin-coated 605QD, two biotinylated capture probes, and two fluorescent dyes (Alexa Fluor 488 and Alexa Fluor 647)labeled report probes (Figure 3B-D). Two kinds of oligonucleotide probes, reporter probe 1 and capture probe 1, were used to recognize and detect target DNA 1 for HIV-1 (Figure 3B). Another two kinds of oligonucleotide probes, reporter probe 2 and capture probe 2, were used to recognize and detect target DNA 2 for HIV-2 (Figure 3C). First the mixture of reporter probe 1, capture probe 1, reporter probe 2, and capture probe 2 was prepared, then the samples containing the target DNAs were added to specifically hybridize with their corresponding probes to form the sandwich hybrids, and at last the streptavidin-coated 605QDs were added to capture the sandwich hybrids and form different complexes; these complexes were further subjected to the single-molecule Analytical Chemistry, Vol. 82, No. 5, March 1, 2010

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Figure 3. Design of the single-QD-based nanosensor for multiple DNA detection. (A) Principle of the single QD-based nanosensor for multiple DNA detection. In the Alexa Fluor 488-DNA-605QD-DNA-Alexa Fluor 647 complex, Alexa Fluor 488 and the 605QD might be excited simultaneously under the excitation wavelength of 488 nm; Alexa Fluor 647 might emit fluorescence as a result of FRET between the 605QD and Alexa Fluor 647. Consequently, the emissions of Alexa Fluor 488, Alexa Fluor 647, and the 605QD might be observed simultaneously without either spectral overlap or cross-talk under the excitation of 488-nm laser. (B) In the presence of target DNA 1, only Alexa Fluor 488 was able to assemble on the surface of the 605QD to form a 605QD-DNA-Alexa Fluor 488 complex through DNA sandwich hybridization and streptavidin-biotin binding. The signals from Alexa Fluor 488 and the 605QD might be obtained simultaneously from the green and blue channels. (C) In the presence of target DNA 2, only Alexa Fluor 647 was able to assemble on the surface of the 605QD to form a 605QD-DNA-Alexa Fluor 647 complex through DNA sandwich hybridization and streptavidin-biotin binding. The signals from the 605QD and Alexa Fluor 647 might be obtained simultaneously from the blue and red channels. (D) In the presence of both target DNA 1 and target DNA 2, both Alexa Fluor 488 and Alexa Fluor 647 were able to assemble on the surface of the 605QD to form an Alexa Fluor 488-DNA-605QD-DNA-Alexa Fluor 647 complex through DNA sandwich hybridization and streptavidin-biotin binding. The signals from Alexa Fluor 488, the 605QD, and Alexa Fluor 647 might be obtained simultaneously from the green, blue, and red channels.

detection (Figure 3B-D). One significant advantage of this single QD-based nanosensor was that it was a homogeneous assay without separating the binding probes from the free probes. In the presence of target DNA 1, only Alexa Fluor 488 was able to assemble on the surface of the 605QD to form a 605QD-DNAAlexa Fluor 488 complex through DNA sandwich hybridization and streptavidin-biotin binding. As a result, the 605QD and Alexa Fluor 488 might emit simultaneously under the excitation of 4881924

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nm laser; the simultaneous presence of both the 605QD and Alexa Fluor 488 fluorescence signals in a single-molecule detection was denoted as a coincidence signal. The appearance of such coincidence signals indicated the presence of target DNA 1 in the sample (Figure 3B). No coincidence signal might be obtained in the absence of target DNA 1, because Alexa Fluor 488 could not be linked to the 605QD and far away from each other in the singlemolecule detection. While in the presence of target DNA 2, only

Figure 4. Representative traces of the fluorescence bursts from Alexa Fluor 488, the 605QD, and Alexa Fluor 647 detected with a single QD-based nanosensors for multiple DNA detection in a microfluidic flow. The fluorescence signals of Alexa Fluor 488, the 605QD, and Alexa Fluor 647 were obtained simultaneously from three separate channels in the presence of HIV-1(A), HIV-2 (B), both of HIV-1 and HIV-2 (C). The fluorescence signals of Alexa Fluor 488 were shown in green. The fluorescence signals of the 605QD were shown in blue. The fluorescence signals of Alexa Fluor 647 were shown in red.

Alexa Fluor 647 was able to assemble on the surface of 605QD to form a 605QD-DNA-Alexa Fluor 647 complex through DNA sandwich hybridization and streptavidin-biotin binding. The emission of Alexa Fluor 647 might be observed as a result of FRET between the 605QD and Alexa Fluor 647 in this complex under the excitation of 488-nm laser.40 The appearance of Alexa Fluor 647 fluorescence signal indicated the presence of target DNA 2 in the sample (Figure 3C). No Alexa Fluor 647 fluorescence signals might be observed in the absence of target DNA 2 because Alexa Fluor 647 could not be linked to the 605QD and far away from each other in the single-molecule detection. In the presence of both target DNA 1 and target DNA 2, both Alexa Fluor 488 and Alexa Fluor 647 were able to assemble on the surface of 605QD to form an Alexa Fluor 488-DNA-605QDDNA-Alexa Fluor 647 complex through DNA sandwich hybridization and streptavidin-biotin binding. The emission of Alexa Fluor 488, the 605QD, and Alexa Fluor 647 might be observed simultaneously in this complex under the excitation of 488-nm laser. The appearance of both the coincidence signal and Alexa Fluor 647 fluorescence signals indicated the presence of both target DNA 1 and target DNA 2 in the sample (Figure 3D). An important requirement of two-color coincidence detection was that the wavelengths of the two fluorescent probes had very low cross-talk to avoid false-coincidence. Since the Stokes’ shift of the organic fluorophores was typically very small (20-30 nm), it was difficult to find two fluorophores that could be excited by one single-wavelength laser and in the meantime had no crosstalk. Selecting two fluorophores excited by two lasers with different wavelengths might maximize the separation between the two emission wavelengths and minimize the cross-talk. However,

the confocal volumes of the two lasers could not have perfect overlap (typically less than 30%23 due to the wavelength difference of two excitation lasers), thereby decreasing the detection efficiency. The emerging of QDs might efficiently overcome the above limitations, because QDs exhibited size-dependent tunable photoluminescence with narrow emission bandwidths, and QDs with different emission wavelength might be excited by a singlewavelength laser.29 Two different-color QDs had been used as fluorescent probes for two-color coincidence detection.41 In the current study, both Alexa Fluor 488 and the 605QD might be excited by a single-wavelength of 488-nm laser simultaneously, making them ideal fluorescent probes for two-color coincidence detection. In addition, an important requirement of FRET was the significant spectral overlap between the donor and the acceptor. The 605QDs not only met the above requirement but also kept good spectral resolution from both Alexa Fluor 647 emission and Alexa Fluor 488 emission (Figure 2). Therefore, the 605QD was an ideal fluorophore as a fluorescence pair for the two-color coincidence detection and as a FRET donor for the FRET assay. Detection of HIV-1 and HIV-2 with a Single-QD-Based Nanosensor. Figure 4 showed the representative fluorescence signals for multiple DNA detection in a microfluidic flow. The fluorescence signals from Alexa Fluor 488, the 605QD, and Alexa Fluor 647 were obtained simultaneously from three separate channels. In the presence of HIV-1, only coincidence signals but no Alexa Fluor 647 fluorescence signals were observed (Figure 4A). In the presence of HIV-2, distinct Alexa Fluor 647 fluorescence signals were observed with each Alexa Fluor 647 fluorescence signal corresponding to one 605QD signal, but no coinci-

(40) Zhang, C. Y.; Johnson, L. W. Angew. Chem., Int. Ed. 2007, 46, 3482–3485.

(41) Zhang, C. Y.; Johnson, L. W. Analyst 2006, 131, 484–488.

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Figure 5. Single-QD-based nanosensor for multiple DNA detection. (A) Representative traces of fluorescence bursts from Alexa Fluor 488, the 605QD, and Alexa Fluor 647 in the control groups in the absence of specific target DNAs. The fluorescence signals of Alexa Fluor 488 were shown in green. The fluorescence signals of the 605QD were shown in blue. The fluorescence signals of Alexa Fluor 647 were shown in red. (B) Variance of coincidence events as a function of increasing Alexa Fluor 488-to-605QD ratio in the presence of target DNA 1 (b) and in the absence of target DNA 1 (9). (C) Variance of burst counts of Alexa Fluor 647 (b) and burst counts of 605QD (9) as a function of increasing Alexa Fluor 647-to-605QD ratio in the presence of target DNA 2. Error bars showed the standard deviation of three experiments. (D) Multiplex detection of HIV-1 and HIV-2 using single QD-based nanosensors. Error bars showed the standard deviation of three experiments.

dence signal was observed (Figure 4B). In contrast, the presence of both HIV-1 and HIV-2 resulted in the appearance of both the coincidence signals and Alexa Fluor 647 fluorescence signals (Figure 4C). Since this assay was carried out in a microfluidic flow at a single-molecule level, the free Alexa Fluor 488- and Alexa Fluor 647-labeled probes were far away from the 605QD, and no false coincidence and Alexa Fluor 647 fluorescence signals were observed in the control groups in the absence of specific target DNAs (Figure 5A). The good control in Figure 5A also indicated no significant nonspecific binding of Alexa Fluor 488 and Alexa Fluor 647 on the surface of the 605QD, even no direct excitation of Alexa Fluor 647, and no leaking of the 605QD emission into either the Alexa Fluor 488 channel or the Alexa Fluor 647 channel under the current experimental condition. It was worth noting that the coincidence-related Alexa Fluor 488 fluorescence signals (Figure 4A) were much higher than those free Alexa Fluor 488 signals (Figure 4B), indicating that the 605QD functioned as a local nanoconcentrator which significantly amplified the coincidence-related Alexa Fluor 488 signals. The extremely low fluorescence signals for free Alexa Fluor 488 in 1926

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Figure 4B might result from the short duration time of Alexa Fluor 488 in the focus volume in a microfluidic flow. It should be noted that detecting molecules in a microfluidic flow might effectively prevent the photobleaching of Alexa Fluor 488 and Alexa Fluor 647 and consequently improved the detection accuracy, because each molecule rapidly flowed into and left the laser illumination region only once, and the duration that a molecule stayed in the detection region was very short. In addition, the performance of single-QD-based nanosensor in a microfluidic flow had another advantage of improved FRET efficiency, which might significantly amplify Alexa Fluor 647 fluorescence signals.40 We further addressed the issue associated with the conjugation of nanoparticles, i.e. the number of dye-labeled DNA sandwich complexes per QD. In theory, the higher the Alexa Fluor 488-to605QD ratio, the higher the signal amplification for the coincidence detection and the more coincidence signals being detected. However, the high Alexa Fluor 488-to-605QD ratio might also increase the probability of both Alexa Fluor 488 and the 605QD simultaneously locating in the focus volume and led to more false coincidence signals in the absence of target DNA (Figure 5B) and consequently compromised the detection sensitivity of two-

color coincidence detection. In addition, the function of the 605QD as a local nanoconcentrator might significantly amplify the FRETrelated Alexa Fluor 647 fluorescence signals as well.40 In principle, the higher the Alexa Fluor 647-to-605QD ratio, the higher FRET efficiency, consequently the more and the higher Alexa Fluor 647 fluorescence signals being detected. However, the high Alexa Fluor 647-to-605QD ratio might also result in the FRET-induced quenching of 605QD fluorescence, consequently less 605QD signals being detected (Figure 5C); this might eventually compromise the detection sensitivity of two-color coincidence detection when both target DNAs were present in the sample. To ensure the accuracy of both coincidence and FRET detection, the ratio of Alexa Fluor 488-labeled reporter probe 1: Alexa Fluor 647labeled reporter probe 2: 605QD must be optimized so as to not only obtain the high fluorescence signals of Alexa Fluor 488 and Alexa Fluor 647 but also to prevent the missing of true coincidence signals and the increase of false coincidence signals. Based on Figure 5B,C, the optimal ratio of 6:12:1 for Alexa Fluor 488-labeled reporter probe 1: Alexa Fluor 647-labeled reporter probe 2: 605QD was chosen in this study. Figure 5D showed the quantitative result of multiple DNA detection with a single QD-based nanosensor. In the presence of HIV-1, only coincidence signals were detected, but no Alexa Fluor 647 signals were observed. While in the presence of HIV-2, only Alexa Fluor 647 signals were detected, but no coincidence signals were observed. When both HIV-1 and HIV-2 presented in the sample, both coincidence and Alexa Fluor 647 signals were detected simultaneously. This homogeneous assay was very simple without the need of separation, and positive or negative results might be obtained by just counting the fluorescence bursts (42) Vet, J. A. E.; Majithia, A. R.; Marras, S. A. E.; Dube, S.; Poiesz, B. J.; Kramer, F. R. Proc. Natl Acad. Sci. U.S.A. 1999, 96, 6394–6399.

of coincidence and FRET without further data processing. Because this single-QD-based nanosensor took advantage of a simple ‘mix and detection’ assay with extremely low sample consumption, high sensitivity and short analysis time, it had potential to be applied for rapid point-of-care testing and clinical diagnostics. CONCLUSIONS In conclusion, a single QD-based nanosensor for multiple DNA detection was developed based on both coincidence and FRET detection. In this single-QD based nanosensor, the QD functioned as both a fluorescence pair for coincidence detection and a FRET donor for FRET detection. The QD also functioned as a local nanoconcentrator which significantly amplified the coincidencerelated fluorescence signals and the FRET signals. In comparison with the previous multiplex nucleic acid assay of HIV-1 and HIV-2 based on amplicon-specific molecular beacons and simultaneous PCR,42 our approach had significant advantages of simple probepreparation, PCR-free, high sensitivity, and low cost. This single QD-based nanosensor offered a simple and ultrasensitive approach for multiple DNA detection at single-molecule level in a homogeneous format and might find wide applications in gene expression studies, point-of-care testing, high-throughput screening, and clinical diagnostics. ACKNOWLEDGMENT This work was supported by the Knowledge Innovation Project of the Chinese Academy of Science (KGCX2-YW-130) and by the National Basic Research Program 973 (Grant No. 2010CB732600). Received for review November 21, 2009. Accepted January 23, 2010. AC9026675

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