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Quantum dot–mediated biosensing assays for specific nucleic acid detection
Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 115 – 121 www.nanomedjournal.com
Basic Research
Quantum dot–mediated biosensing assays for specific nucleic acid detection Hsin-Chih Yeh, MS, PhD(c), Yi-Ping Ho, MS, PhD(c), Tza-Huei Wang, PhDT Johns Hopkins University, Baltimore, Maryland Received 8 March 2005; accepted 25 March 2005
Abstract
Two new classes of quantum dot (QD) –mediated biosensing methods have been developed to detect specific DNA sequences in a separation-free format. Both methods use 2 target-specific oligonucleotide probes to recognize a specific target. The first method is based on cross-linking of 2 QDs with distinct emission wavelengths caused by probe-target hybridization. The second method uses QDs as both fluorescent tags and nanoscaffolds that capture multiple fluorescently labeled hybridization products, resulting in amplified target signals. The presence of targets is determined according to spatiotemporal coincidence of 2 different wavelength fluorescent signals emitted from the QD/DNA/probe complexes. With a single wavelength-excitation, dual wavelengthemission confocal spectroscopic system, the fluorescent signals can be measured with single-dot/ molecule sensitivity. Compared with other nanoparticle-based, separation-free assays, our method shows advantages in simplicity, testing speed, and multiplexed applications. D 2005 Elsevier Inc. All rights reserved.
Key words:
Quantum dot; Biosensor; Single-molecule detection; Multiplexed detection
The integration of nanotechnology with biology and medicine is expected to lead to major advances in molecular diagnostics, therapeutics, molecular biology, and cell biology [1-4]. For instance, nanoparticles have been recently demonstrated for the detection of specific nucleic acid sequences that are critical to the diagnosis of genetic and pathogenic diseases. These approaches take advantage of the material’s property changes (optical [5-15], electrochemical [16-18], magnetic [19], or mechanical [20]) on DNA hybridization or enzyme-mediated reactions. Among them, the optical-based, separation-free assays have raised the greatest interest due to their simplicity, automation friendliness, high analysis rate, and great potential in multiplexed assays [7,14,21,22]. Additionally, performing molecular reactions and detection in a homogeneous, separation-free format facilitates efficient binding kinetics
No financial conflict of interest was reported by the authors of this paper. T Corresponding author. Department of Mechanical Engineering and Whitaker Biomedical Engineering Institute, Johns Hopkins University, 3400 N Charles St, Baltimore, MD 21218. E-mail address: [email protected] (T.-H. Wang). 1549-9634/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2005.03.004
between sensor probes and target molecules, thereby improving detection sensitivity [10,23]. Conventional separation-free assays for specific nucleic acid detection include assays based on fluorescence correlation spectroscopy (FCS) [24,25] and fluorescence resonance energy transfer (FRET), such as molecular beacons [26-28]. Nevertheless, FCS does not have high specificity when the change in diffusion time caused by target hybridization is small. Molecular beacons are expensive and typically require sophisticated thermostability analysis during probe design. Moreover, whenever organic dyes are used as fluorescent tags, multicolor analysis is often complicated by the need of an elaborate excitation and detection system. Other common problems are poor signal-to-noise ratio, cross-talk, and photobleaching. Lately a number of nanoparticle-based, separation-free assays have been proposed to address those issues. Gold nanoparticle cross-linking aggregates with different interparticle distances appear to be different colors due to surface plasmon resonance of the gold, rendering this a method for specific polynucleotide detection [5]. The sharp melting transitions of gold aggregates are used to differentiate a perfect match target from a strand with
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Fig 1. Schematic concept of two QD-mediated biosensing methods. A, A cross-linking system using two QDs with distinct emission wavelengths. B, Organic fluorophore-labeled sandwich structures coupled to a QD. (C) Coincidence detection: The coincident signals (marked with dash lines) are detected in the presence but not the absence of specific targets.
a single base mismatch. The limitation of this approach is that it is inherently a 1-color system based on gray scale [6]. Another gold nanoparticle aggregation system induced by non– cross-linking DNA hybridization was also reported [11,12,15]. It was found that single-stranded and doublestranded oligonucleotides generated distinct nanoparticle aggregation phenomena upon adding salt to the solution. Nanoparticle aggregation was measured with a UV-visible spectrophotometer at bulk levels, making this method less quantitative in low-abundant mutation detection. Gold nanoparticles were also used as quenchers in homogeneous FRET assays [9,13]. Nevertheless, the preparation of oligonucleotide-modified gold nanoparticles is time consuming, typically from tens of hours to a few days [11]. For today’s clinical application, separation-free assays that are sensitive, capable of performing multiplexing detection, and have short sample preparation time are highly desired. Here we report a quantum dot (QD) –mediated biosensing technique that fulfills those goals. Quantum dots (2 to 10 nm), such as CdSe-ZnS core-shell nanocrystals, have size-dependent tunable photoluminescence, broad excitation, and narrow emission bandwidths as well as high quantum efficiency and photostability [1,3,4,7,14,29]. Having dimensions similar to those of biomolecules, QDs are commonly used in magnetic resonance imaging as a contrast agent, as carriers for drug delivery, and as structural scaffolds for tissue engineering. Multiplexed assays have also become feasible due to QDs’ superior photophysical properties [3,7,14]. In addition, the QDs can be surface functionalized with different probe molecules (eg, oligonucleotides, peptides, and antibodies), facilitating detection of different biomolecules including DNA, RNA, and proteins. By using a confocal fluorescence spectroscopic system, QDs can be analyzed at the single-dot level, making the
QD-based assay a viable approach for low-abundant biomolecule detection.
Methods Principle The principle of the proposed QD-mediated biosensing assays and detection scheme is illustrated in Figure 1. In the first method (Figure 1, A), 2 kinds of QDs (Quantum Dot Corp) with different peak emission wavelengths (525 QD with peak emission at 525 nm, and 605 QD with peak emission at 605 nm) are used. The QDs have been coupled to streptavidins directly through a carbodiimide-mediated coupling reaction (Streptavidin conjugates user manual, PN 90-0003 Rev 5, Quantum Dot Corp). Two biotinylated single-stranded DNA probes are each coupled to one of the 2 QDs through biotin–streptavidin interaction. The 2 QDlabeled probes are designed to hybridize at different binding sites of the same target DNA strand. In the presence of targets, cross-linking of QDs occurs upon target/probes sandwich hybridization. The second method (Figure 1, B) utilizes only 1 kind of QD (605 QD). The 2 single-stranded DNA probes used for detection are modified differently. One is biotinylated and the other is conjugated to an organic fluorophore, Oregon Green 488 (peak emission at 524 nm; Molecular Probes). These 2 probes are first mixed with a single-stranded target to form sandwich structures in solution. Quantum dots are added at the last step to capture these biotinylated sandwich structures, forming QD-sandwich nanoassemblies. In addition to being used as fluorescent tags, individual QDs also function as nanoscaffolds that locally concentrate targets and thereby amplify target signals.
H.-C. Yeh et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 115–121
In both methods the samples are rapidly driven and measured inside a microflow channel by a confocal spectroscopic system. This minimizes the time that individual analytes are exposed to the illumination region, thereby avoiding the photobleaching complication [30]. Green (525 QD or Oregon Green 488) and red (605 QD) fluorescent bursts emitted from the QD/DNA/probe complexes (crosslinking or nanoscaffold assemblies) are simultaneously detected whenever a molecular complex passes through the minuscule laser-focused measurement volume (Figure 1, C, upper), which are referred to as spatiotemporally coincident signals. On the other hand, only uncorrelated fluorescence signals are detected in absence of specific targets since the molecular complexes do not exist (Figure 1, C, lower). Instrumentation A single-wavelength excitation, dual-wavelength emission confocal spectroscopic system is incorporated to analyze these QD aggregates or QD-sandwich nanoassemblies in a microcapillary (Figure 2). The excitation laser is a 488-nm air-cooled argon laser (Melles Griot). A 100 1.3 numerical aperture oil-immersion apochromatic objective (Olympus) is used to focus the laser beam to a 1-Am spot inside the microcapillary. The emission fluorescence is collected by the same objective. Dichroic mirror 1 (Chroma Technology) is selected to allow light of wavelengths greater than 505 nm to pass through. A 50-Am pinhole (Melles Griot) is used to reject out-of-focus fluorescence and background noise, thus enhancing the signal-to-noise ratio. Dichroic mirror 2 allows light of wavelengths greater than 565 nm to pass through. Two avalanche photodiodes (EG&G) register the 2 filtered wavelengths, one for green and the other for red. A program written in LabView (National Instruments) and a digital counter are used to perform data acquisition and data analysis. The excitation laser power is kept at 150 AW during the experiments. Sample preparation High-performance liquid chromatography-purified, 35-bp oligonucleotides, 5V-GAG CCCGCAGTGGCCGAATCTCAGACTGTGGCAGG-3V (Integrated DNA Technologies) were synthesized as single-stranded target biomolecules to evaluate the QD-mediated biosensing techniques in specific nucleic acid detection. In QD cross-linking detection, 2 biotinylated 15- and 17-bp single-stranded DNA probes were coupled to streptavidin-conjugated red QDs (peak emission at 605 nm) and green QDs (peak emission at 525 nm), respectively, in a PBS buffer for 30 minutes. Single-stranded targets were then added to the system in close to 1:1 target-to-probe ratio in the hybridization buffer of 10X SSC, 20% formamide, 0.1 g/mL dextran sulfate. Incubation was carried out at room temperature for 1 hour. The reaction solution was diluted by a factor of 1000 with deionized water before testing. In the QD – organic fluorophore coupling system, the 17-bp DNA probes were labeled with Oregon Green
117 Reflecting mirror
APD2 APD2 Focusing lens Bandpass filter APD1 APD1 Dichroic mirror 2 488 notch filter Pinhole
Counter Counter
Ar-laser (488nm) Focusing lens Computer
Data Analysis
Dichroic mirror 1 Objective lens Microcapillary Biomolecules Sample
Fig 2. Schematic concept of the single-wavelength excitation, dualwavelength emission confocal spectroscopic system for coincidence detection.
488 dyes at the 5V ends rather than being biotinylated. The 35-bp targets were first mixed with these 2 probes in approximate 1:1 target-to-probe ratio in the hybridization buffer at room temperature for 1 hour. Red QDs were added to the solution to capture the sandwiches in an approximate ratio of 1 to 30 between QDs and sandwiches after hybridization. The coupling took place for 30 minutes. The reaction solution was diluted 1000 times with deionized water before testing. Preparations for both schemes require no separation. The so-called homogeneous format improves the binding kinetics, as mentioned previously. Results and Discussion Figure 3 presents 2 sets of 5-second fluorescence burst signals from the 2 detection methods individually. Coincidence of the 2 emissions is verified at a 1-millisecond temporal resolution, which is the set integration time for the photon counting during the measurements. In absence of the targets in solution, only uncorrelated fluorescent peaks are seen in both detection channels within the same measurement time period (Figure 3, A and B, lower). However, in presence of the targets in solution, many coincident signals are clearly seen (Figure 3, A and B, upper), which indicates that the coincident emissions are indeed the results of the QD cross-linking aggregates or QD-sandwich nanoassemblies passing through the minute detection volume of the confocal spectroscopic system. The principle of dual-color fluorescence coincidence detection was first proposed by Eigen and Rigler [31] using dualcolor fluorescence cross-correlation spectroscopy. Castro and Williams [32] reported the detection of unamplified genomic DNA molecules based on the coincidence detection scheme. Li et al used a dual-excitation system in single DNA molecule detection [33], and recently in quantitative counting of antibody-protein complexes [34]. By using only organic dyes as fluorophores, all these methods suffer from the aforementioned dye-associated issues. However, by incorporating semiconductor QDs, those issues have been circumvented.
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Fig 3. Fluorescence burst signal raw data from confocal spectroscopy. A, QD cross-linking system. B, QD-organic fluorophore system: with targets (upper) and without targets (lower). Asterisks (*) denote the coincident signals.
Multiplexed nucleic acid detections using QD-tagged microbeads coupling with organic fluorophores have been reported [7,14]. In those assays, flow cytometry is used to sort the QD-barcoded microbeads rather than a confocal spectroscopic system. However, this method is not suitable for low-abundant nucleic acid detection, since N 105 targets are required to couple to each microbead to achieve a detectable target signal. With confocal single-molecule spectroscopy, QD can be analyzed at a single- dot level. In our second approach, strong coincident signals can be seen from only ~30 organic dyes coupled to each QD through DNA target molecules, making this a better means for lowabundant nucleic acid detection. The probes and targets first interact with each other in homogeneous solution, forming sandwiches at more efficient hybridization kinetics. If larger nucleic acid targets are used, incubation can be done at elevated temperature to further enhance the hybridization efficiency. This assay is also more compatible with enzymemediated reactions such as oligonucleotide ligation assays and single base extension in point mutation detections since no unwanted interactions between QDs and enzymes will
ever take place. Serving as nanoscaffolds or nanoconcentrators, the QDs are only added at the last step to capture the target/probe sandwiches, forming QD-sandwich nanoassemblies. Since many organic fluorophores are confined to a nanoscale domain due to biotin–streptavidin interaction, the resulting fluorescence is far brighter than the free, unbound dyes, and therefore can be easily identified and distinguished (Figure 3, B). Compared with molecular beacon-based assays for single molecule detection, whose nonspecific signals caused by the unbound random coils have the same intensity level as target signals, our QD/organic fluorophore hybrid nanobiosensors have shown much enhanced sensitivity and specificity due to this bamplification-on-concentrationQ effect. However, compared with first QD cross-linking approach, this QD/organic fluorophore hybrid biosensing technique has less potential in multiplexed detections. Crosstalk of emission spectra of organic dyes (so-called red tails, Figure 4, B) and using only single-wavelength excitation are still issues when attempting to incorporate more organic dyes to simultaneously detect more than one target in solution. Quantum dots can be used as FRET donors (unpublished
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Fig 4. Emission spectra. A, QD cross-linking system: QD 525 nm and QD 605 nm. B, QD-organic fluorophore coupling system: OG488 and QD 605 nm.
results), which broadens the selection of organic fluorophores. However, as long as the organic fluorophores are used, this technique is hampered with spectrum cross-talk. Our first approach has better potential in multiplexed detection. Quantum dots have narrow (full width at halfmaximum ~35 nm) and symmetric emission spectra. Figure 5 shows the results of simultaneous detection of 3 distinct DNA targets in solution using 3 different QDs (peak emissions at 525 nm, 605 nm, and 705 nm, respectively). Single-wavelength excitation has greatly simplified the optics in confocal setup, thus enhancing the detection reliability. With better size control during QD synthesis (ie, narrower emission spectra) and newly synthesized QDs with emission spectra in the infrared region, more target molecules can be detected simultaneously in solution. For instance, if 4 types of QDs are used, a total of 6 targets can be individually identified in a single test. However, while having greater potential in multiplexed detection, the crosslinking system shows lower hybridization efficiency, mainly due to the high negative-charge density created by extensive
Fig 5. QD cross-linking method in simultaneous detection of 3 DNA targets. A, B, and C show the results of detection of DNA targets 1, 2, and 3, respectively. Asterisks (*) denote the coincident signals.
probe loading on QD surfaces and steric hindrance [5]. Elevated temperature is often required to uncoil the target strand for better hybridization efficiency. However, QDs tend to aggregate unspecifically at temperatures higher than 808C (data not shown). Therefore, lower hybridization efficiency and longer incubation time are expected. This method is also expected to be less compatible with enzymemediated reactions, where both steric hindrance and nanoparticle-enzyme interaction may affect the enzyme activities and kinetics. The dilemma of homogeneous reaction and multiplexed detection can be overcome by incorporating more than 1 functional group, such as amine-carboxylate, rather than just single streptavidin–biotin interaction. Quantum dots with different functional groups can recognize and bind to specific probes that are correspondently functionalized after
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the hybridization or the enzymatic reaction is completed in homogeneous solution. However, carboxylate functional groups are easily hydrolyzed, making carboxylated QDs have much shorter shelf lives. Peptide conjugated QDs are currently being studied and may give 100+ high-affinity interaction pairs in the near future [4]. Regarding specificity, the 3-component, QD-based strategies should be more selective than any 2-component detection system based on single-stranded probe hybridization with the target [5]. Three-component systems, however, often required the balanced ratio between probes and targets, preferably 1:1, to get maximum sandwich hybridization efficiency. But QD cross-linking system is not limited by that because it requires only 1 target, in theory, to bridge 2 QDs. Our results not only demonstrate that specific DNA detection is achieved using QD nanobiosensors and the coincidence detection principle, but that they can also be used to quantify the target biomolecules by counting coincident events. The coincidence detection method therefore can be generally applied to many other bioanalysis areas, such as gene expression profiling, that require precise quantification of biomolecules. In addition, we took advantage of biotin–streptavidin interaction. This binding interaction is quick, reliable, and strong. It dramatically reduces the preparation time from tens of hours, for a thiolmediated, gold nanoparticle system, to just tens of minutes.
Conclusion In this article, 2 new QD-mediated separation-free biosensing methods that can be applied to identification of different nucleic acid sequences are described. We have demonstrated multiplexed detection of 3 DNA sequences by using the QD cross-linking system. However, the QD– organic fluorophore coupling system also has unique advantages in application where temperature treatment and enzymatic reactions are needed. These 2 methods have provided new platforms of biosensing with several features superior to the existing methods. In addition, 2 specific molecular probes are used, instead of only 1, to recognize a target molecule, achieving molecular identification with high specificity. Future research will aim at multiplexed applications and better understanding and control of nanoparticle cross-linking extents.
Acknowledgment This work is supported by the National Science Foundation (DBI-0352407) and the Whitaker Foundation.
References [1] Alivisatos P. The use of nanocrystals in biological detection. Nat Biotechnol 2004;22:47 - 52.
[2] Penn SG, He L, Natan MJ. Nanoparticles for bioanalysis. Curr Opin Chem Biol 2003;7:609 - 15. [3] Chan WCW, Maxwell DJ, Gao XH, Bailey RE, Han MY, Nie SM. Luminescent quantum dots for multiplexed biological detection and imaging. Curr Opin Biotechnol 2002;13:40 - 6. [4] Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, et al. Quantum dots for live cells, in vivo imaging, and diagnostics. Science 2005;307:538 - 44. [5] Elghanian R, Storhoff JJ, Mucic RC, Letsinger RL, Mirkin CA. Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles. Science 1997;277:1078 - 81. [6] Cao YWC, Jin RC, Mirkin CA. Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 2002;297:1536 - 40. [7] Han MY, Gao XH, Su JZ, Nie S. Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nat Biotechnol 2001;19: 631 - 5. [8] Taton TA, Mirkin CA, Letsinger RL. Scanometric DNA array detection with nanoparticle probes. Science 2000;289:1757 - 60. [9] Dubertret B, Calame M, Libchaber AJ. Single-mismatch detection using gold-quenched fluorescent oligonucleotides. Nat Biotechnol 2001;19:365 - 70. [10] Storhoff JJ, Lucas AD, Garimella V, Bao YP, Muller UR. Homogeneous detection of unamplified genomic DNA sequences based on colorimetric scatter of gold nanoparticle probes. Nat Biotechnol 2004;22:883 - 7. [11] Sato K, Sawayanagi M, Hosokawa K, Maeda M. Single-base mutation detection using neutravidin-modified polystyrene nanoparticle aggregation. Anal Sci 2004;20:893 - 4. [12] Sato K, Hosokawa K, Maeda M. Rapid aggregation of gold nanoparticles induced by non-cross-linking DNA hybridization. J Am Chem Soc 2003;125:8102 - 3. [13] Maxwell DJ, Taylor JR, Nie SM. Self-assembled nanoparticle probes for recognition and detection of biomolecules. J Am Chem Soc 2002; 124:9606 - 12. [14] Xu HX, Sha MY, Wong EY, Uphoff J, Xu YH, Treadway JA, et al. Multiplexed SNP genotyping using the Qbead (TM) system: a quantum dot-encoded microsphere-based assay. Nucleic Acids Res 2003;31:e43. [15] Li HX, Rothberg L. Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles. Proc Natl Acad Sci U S A 2004;101:14036 - 9. [16] Park SJ, Taton TA, Mirkin CA. Array-based electrical detection of DNA with nanoparticle probes. Science 2002;295:1503 - 6. [17] Wang J, Liu GD, Merkoci A. Electrochemical coding technology for simultaneous detection of multiple DNA targets. J Am Chem Soc 2003;125:3214 - 5. [18] Kerman K, Saito M, Morita Y, Takamura Y, Ozsoz M, Tamiya E. Electrochemical coding of single-nucleotide polymorphisms by monobase-modified gold nanoparticles. Anal Chem 2004;76: 1877 - 84. [19] Wang J, Xu DK, Polsky R. Magnetically-induced solid-state electrochemical detection of DNA hybridization. J Am Chem Soc 2002;124:4208 - 9. [20] Su M, Li SU, Dravid VP. Microcantilever resonance-based DNA detection with nanoparticle probes. Appl Phys Lett 2003;82:3562 - 4. [21] Vaarno J, Ylikoski E, Meltola NJ, Soini JT, Hanninen P, Lahesmaa R, et al. New separation-free assay technique for SNPs using two-photon excitation fluorophore. Nucleic Acids Res 2004;32:e108. [22] Cai H, White PS, Torney D, Deshpande A, Wang ZL, Marrone B, et al. Flow cytometry-based minisequencing: a new platform for high-throughput single-nucleotide polymorphism scoring. Genomics 2000;66:135 - 43. [23] Nam JM, Thaxton CS, Mirkin CA. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science 2003;301: 1884 - 6.
H.-C. Yeh et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 115–121 [24] Kettling U, Koltermann A, Schwille P, Eigen M. Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy. Proc Natl Acad Sci U S A 1998;95:1416 - 20. [25] Edman L, Mets U, Rigler R. Conformational transitions monitored for single molecules in solution. Proc Natl Acad Sci U S A 1996;93: 6710 - 5. [26] Zhang C, Chao SY, Wang TH. Comparative quantification of nucleic acids using single-molecule detection and molecular beacons. Analyst 2005;130:483 - 8. [27] Tyagi S, Bratu DP, Kramer FR. Multicolor molecular beacons for allele discrimination. Nat Biotechnol 1998;16:49 - 53. [28] Tyagi S, Kramer FR. Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 1996;14:303 - 8. [29] Chan WCW, Nie SM. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 1998;281:2016 - 8.
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[30] Wang TH, Peng YH, Wong PK, Ho CM. Single-molecule tracing on a fluidic microchip for quantitative detection of low-abundance nucleic acids. J Am Chem Soc 2005;127:5354 - 9. [31] Eigen M, Rigler R. Sorting single molecules — application to diagnostics and evolutionary biotechnology. Proc Natl Acad Sci U S A 1994;91:5740 - 7. [32] Castro A, Williams JGK. Single-molecule detection of specific nucleic acid sequences in unamplified genomic DNA. Anal Chem 1997;69: 3915 - 20. [33] Li HT, Ying LM, Green JJ, Balasubramanian S, Klenerman D. Ultrasensitive coincidence fluorescence detection of single DNA molecules. Anal Chem 2003;75:1664 - 70. [34] Li HT, Zhou DJ, Browne H, Balasubramanian S, Klenerman D. Molecule by molecule direct and quantitative counting of antibodyprotein complexes in solution. Anal Chem 2004;76:4446 - 51.
Basic Research
Quantum dot–mediated biosensing assays for specific nucleic acid detection Hsin-Chih Yeh, MS, PhD(c), Yi-Ping Ho, MS, PhD(c), Tza-Huei Wang, PhDT Johns Hopkins University, Baltimore, Maryland Received 8 March 2005; accepted 25 March 2005
Abstract
Two new classes of quantum dot (QD) –mediated biosensing methods have been developed to detect specific DNA sequences in a separation-free format. Both methods use 2 target-specific oligonucleotide probes to recognize a specific target. The first method is based on cross-linking of 2 QDs with distinct emission wavelengths caused by probe-target hybridization. The second method uses QDs as both fluorescent tags and nanoscaffolds that capture multiple fluorescently labeled hybridization products, resulting in amplified target signals. The presence of targets is determined according to spatiotemporal coincidence of 2 different wavelength fluorescent signals emitted from the QD/DNA/probe complexes. With a single wavelength-excitation, dual wavelengthemission confocal spectroscopic system, the fluorescent signals can be measured with single-dot/ molecule sensitivity. Compared with other nanoparticle-based, separation-free assays, our method shows advantages in simplicity, testing speed, and multiplexed applications. D 2005 Elsevier Inc. All rights reserved.
Key words:
Quantum dot; Biosensor; Single-molecule detection; Multiplexed detection
The integration of nanotechnology with biology and medicine is expected to lead to major advances in molecular diagnostics, therapeutics, molecular biology, and cell biology [1-4]. For instance, nanoparticles have been recently demonstrated for the detection of specific nucleic acid sequences that are critical to the diagnosis of genetic and pathogenic diseases. These approaches take advantage of the material’s property changes (optical [5-15], electrochemical [16-18], magnetic [19], or mechanical [20]) on DNA hybridization or enzyme-mediated reactions. Among them, the optical-based, separation-free assays have raised the greatest interest due to their simplicity, automation friendliness, high analysis rate, and great potential in multiplexed assays [7,14,21,22]. Additionally, performing molecular reactions and detection in a homogeneous, separation-free format facilitates efficient binding kinetics
No financial conflict of interest was reported by the authors of this paper. T Corresponding author. Department of Mechanical Engineering and Whitaker Biomedical Engineering Institute, Johns Hopkins University, 3400 N Charles St, Baltimore, MD 21218. E-mail address: [email protected] (T.-H. Wang). 1549-9634/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2005.03.004
between sensor probes and target molecules, thereby improving detection sensitivity [10,23]. Conventional separation-free assays for specific nucleic acid detection include assays based on fluorescence correlation spectroscopy (FCS) [24,25] and fluorescence resonance energy transfer (FRET), such as molecular beacons [26-28]. Nevertheless, FCS does not have high specificity when the change in diffusion time caused by target hybridization is small. Molecular beacons are expensive and typically require sophisticated thermostability analysis during probe design. Moreover, whenever organic dyes are used as fluorescent tags, multicolor analysis is often complicated by the need of an elaborate excitation and detection system. Other common problems are poor signal-to-noise ratio, cross-talk, and photobleaching. Lately a number of nanoparticle-based, separation-free assays have been proposed to address those issues. Gold nanoparticle cross-linking aggregates with different interparticle distances appear to be different colors due to surface plasmon resonance of the gold, rendering this a method for specific polynucleotide detection [5]. The sharp melting transitions of gold aggregates are used to differentiate a perfect match target from a strand with
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Fig 1. Schematic concept of two QD-mediated biosensing methods. A, A cross-linking system using two QDs with distinct emission wavelengths. B, Organic fluorophore-labeled sandwich structures coupled to a QD. (C) Coincidence detection: The coincident signals (marked with dash lines) are detected in the presence but not the absence of specific targets.
a single base mismatch. The limitation of this approach is that it is inherently a 1-color system based on gray scale [6]. Another gold nanoparticle aggregation system induced by non– cross-linking DNA hybridization was also reported [11,12,15]. It was found that single-stranded and doublestranded oligonucleotides generated distinct nanoparticle aggregation phenomena upon adding salt to the solution. Nanoparticle aggregation was measured with a UV-visible spectrophotometer at bulk levels, making this method less quantitative in low-abundant mutation detection. Gold nanoparticles were also used as quenchers in homogeneous FRET assays [9,13]. Nevertheless, the preparation of oligonucleotide-modified gold nanoparticles is time consuming, typically from tens of hours to a few days [11]. For today’s clinical application, separation-free assays that are sensitive, capable of performing multiplexing detection, and have short sample preparation time are highly desired. Here we report a quantum dot (QD) –mediated biosensing technique that fulfills those goals. Quantum dots (2 to 10 nm), such as CdSe-ZnS core-shell nanocrystals, have size-dependent tunable photoluminescence, broad excitation, and narrow emission bandwidths as well as high quantum efficiency and photostability [1,3,4,7,14,29]. Having dimensions similar to those of biomolecules, QDs are commonly used in magnetic resonance imaging as a contrast agent, as carriers for drug delivery, and as structural scaffolds for tissue engineering. Multiplexed assays have also become feasible due to QDs’ superior photophysical properties [3,7,14]. In addition, the QDs can be surface functionalized with different probe molecules (eg, oligonucleotides, peptides, and antibodies), facilitating detection of different biomolecules including DNA, RNA, and proteins. By using a confocal fluorescence spectroscopic system, QDs can be analyzed at the single-dot level, making the
QD-based assay a viable approach for low-abundant biomolecule detection.
Methods Principle The principle of the proposed QD-mediated biosensing assays and detection scheme is illustrated in Figure 1. In the first method (Figure 1, A), 2 kinds of QDs (Quantum Dot Corp) with different peak emission wavelengths (525 QD with peak emission at 525 nm, and 605 QD with peak emission at 605 nm) are used. The QDs have been coupled to streptavidins directly through a carbodiimide-mediated coupling reaction (Streptavidin conjugates user manual, PN 90-0003 Rev 5, Quantum Dot Corp). Two biotinylated single-stranded DNA probes are each coupled to one of the 2 QDs through biotin–streptavidin interaction. The 2 QDlabeled probes are designed to hybridize at different binding sites of the same target DNA strand. In the presence of targets, cross-linking of QDs occurs upon target/probes sandwich hybridization. The second method (Figure 1, B) utilizes only 1 kind of QD (605 QD). The 2 single-stranded DNA probes used for detection are modified differently. One is biotinylated and the other is conjugated to an organic fluorophore, Oregon Green 488 (peak emission at 524 nm; Molecular Probes). These 2 probes are first mixed with a single-stranded target to form sandwich structures in solution. Quantum dots are added at the last step to capture these biotinylated sandwich structures, forming QD-sandwich nanoassemblies. In addition to being used as fluorescent tags, individual QDs also function as nanoscaffolds that locally concentrate targets and thereby amplify target signals.
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In both methods the samples are rapidly driven and measured inside a microflow channel by a confocal spectroscopic system. This minimizes the time that individual analytes are exposed to the illumination region, thereby avoiding the photobleaching complication [30]. Green (525 QD or Oregon Green 488) and red (605 QD) fluorescent bursts emitted from the QD/DNA/probe complexes (crosslinking or nanoscaffold assemblies) are simultaneously detected whenever a molecular complex passes through the minuscule laser-focused measurement volume (Figure 1, C, upper), which are referred to as spatiotemporally coincident signals. On the other hand, only uncorrelated fluorescence signals are detected in absence of specific targets since the molecular complexes do not exist (Figure 1, C, lower). Instrumentation A single-wavelength excitation, dual-wavelength emission confocal spectroscopic system is incorporated to analyze these QD aggregates or QD-sandwich nanoassemblies in a microcapillary (Figure 2). The excitation laser is a 488-nm air-cooled argon laser (Melles Griot). A 100 1.3 numerical aperture oil-immersion apochromatic objective (Olympus) is used to focus the laser beam to a 1-Am spot inside the microcapillary. The emission fluorescence is collected by the same objective. Dichroic mirror 1 (Chroma Technology) is selected to allow light of wavelengths greater than 505 nm to pass through. A 50-Am pinhole (Melles Griot) is used to reject out-of-focus fluorescence and background noise, thus enhancing the signal-to-noise ratio. Dichroic mirror 2 allows light of wavelengths greater than 565 nm to pass through. Two avalanche photodiodes (EG&G) register the 2 filtered wavelengths, one for green and the other for red. A program written in LabView (National Instruments) and a digital counter are used to perform data acquisition and data analysis. The excitation laser power is kept at 150 AW during the experiments. Sample preparation High-performance liquid chromatography-purified, 35-bp oligonucleotides, 5V-GAG CCCGCAGTGGCCGAATCTCAGACTGTGGCAGG-3V (Integrated DNA Technologies) were synthesized as single-stranded target biomolecules to evaluate the QD-mediated biosensing techniques in specific nucleic acid detection. In QD cross-linking detection, 2 biotinylated 15- and 17-bp single-stranded DNA probes were coupled to streptavidin-conjugated red QDs (peak emission at 605 nm) and green QDs (peak emission at 525 nm), respectively, in a PBS buffer for 30 minutes. Single-stranded targets were then added to the system in close to 1:1 target-to-probe ratio in the hybridization buffer of 10X SSC, 20% formamide, 0.1 g/mL dextran sulfate. Incubation was carried out at room temperature for 1 hour. The reaction solution was diluted by a factor of 1000 with deionized water before testing. In the QD – organic fluorophore coupling system, the 17-bp DNA probes were labeled with Oregon Green
117 Reflecting mirror
APD2 APD2 Focusing lens Bandpass filter APD1 APD1 Dichroic mirror 2 488 notch filter Pinhole
Counter Counter
Ar-laser (488nm) Focusing lens Computer
Data Analysis
Dichroic mirror 1 Objective lens Microcapillary Biomolecules Sample
Fig 2. Schematic concept of the single-wavelength excitation, dualwavelength emission confocal spectroscopic system for coincidence detection.
488 dyes at the 5V ends rather than being biotinylated. The 35-bp targets were first mixed with these 2 probes in approximate 1:1 target-to-probe ratio in the hybridization buffer at room temperature for 1 hour. Red QDs were added to the solution to capture the sandwiches in an approximate ratio of 1 to 30 between QDs and sandwiches after hybridization. The coupling took place for 30 minutes. The reaction solution was diluted 1000 times with deionized water before testing. Preparations for both schemes require no separation. The so-called homogeneous format improves the binding kinetics, as mentioned previously. Results and Discussion Figure 3 presents 2 sets of 5-second fluorescence burst signals from the 2 detection methods individually. Coincidence of the 2 emissions is verified at a 1-millisecond temporal resolution, which is the set integration time for the photon counting during the measurements. In absence of the targets in solution, only uncorrelated fluorescent peaks are seen in both detection channels within the same measurement time period (Figure 3, A and B, lower). However, in presence of the targets in solution, many coincident signals are clearly seen (Figure 3, A and B, upper), which indicates that the coincident emissions are indeed the results of the QD cross-linking aggregates or QD-sandwich nanoassemblies passing through the minute detection volume of the confocal spectroscopic system. The principle of dual-color fluorescence coincidence detection was first proposed by Eigen and Rigler [31] using dualcolor fluorescence cross-correlation spectroscopy. Castro and Williams [32] reported the detection of unamplified genomic DNA molecules based on the coincidence detection scheme. Li et al used a dual-excitation system in single DNA molecule detection [33], and recently in quantitative counting of antibody-protein complexes [34]. By using only organic dyes as fluorophores, all these methods suffer from the aforementioned dye-associated issues. However, by incorporating semiconductor QDs, those issues have been circumvented.
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Fig 3. Fluorescence burst signal raw data from confocal spectroscopy. A, QD cross-linking system. B, QD-organic fluorophore system: with targets (upper) and without targets (lower). Asterisks (*) denote the coincident signals.
Multiplexed nucleic acid detections using QD-tagged microbeads coupling with organic fluorophores have been reported [7,14]. In those assays, flow cytometry is used to sort the QD-barcoded microbeads rather than a confocal spectroscopic system. However, this method is not suitable for low-abundant nucleic acid detection, since N 105 targets are required to couple to each microbead to achieve a detectable target signal. With confocal single-molecule spectroscopy, QD can be analyzed at a single- dot level. In our second approach, strong coincident signals can be seen from only ~30 organic dyes coupled to each QD through DNA target molecules, making this a better means for lowabundant nucleic acid detection. The probes and targets first interact with each other in homogeneous solution, forming sandwiches at more efficient hybridization kinetics. If larger nucleic acid targets are used, incubation can be done at elevated temperature to further enhance the hybridization efficiency. This assay is also more compatible with enzymemediated reactions such as oligonucleotide ligation assays and single base extension in point mutation detections since no unwanted interactions between QDs and enzymes will
ever take place. Serving as nanoscaffolds or nanoconcentrators, the QDs are only added at the last step to capture the target/probe sandwiches, forming QD-sandwich nanoassemblies. Since many organic fluorophores are confined to a nanoscale domain due to biotin–streptavidin interaction, the resulting fluorescence is far brighter than the free, unbound dyes, and therefore can be easily identified and distinguished (Figure 3, B). Compared with molecular beacon-based assays for single molecule detection, whose nonspecific signals caused by the unbound random coils have the same intensity level as target signals, our QD/organic fluorophore hybrid nanobiosensors have shown much enhanced sensitivity and specificity due to this bamplification-on-concentrationQ effect. However, compared with first QD cross-linking approach, this QD/organic fluorophore hybrid biosensing technique has less potential in multiplexed detections. Crosstalk of emission spectra of organic dyes (so-called red tails, Figure 4, B) and using only single-wavelength excitation are still issues when attempting to incorporate more organic dyes to simultaneously detect more than one target in solution. Quantum dots can be used as FRET donors (unpublished
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Fig 4. Emission spectra. A, QD cross-linking system: QD 525 nm and QD 605 nm. B, QD-organic fluorophore coupling system: OG488 and QD 605 nm.
results), which broadens the selection of organic fluorophores. However, as long as the organic fluorophores are used, this technique is hampered with spectrum cross-talk. Our first approach has better potential in multiplexed detection. Quantum dots have narrow (full width at halfmaximum ~35 nm) and symmetric emission spectra. Figure 5 shows the results of simultaneous detection of 3 distinct DNA targets in solution using 3 different QDs (peak emissions at 525 nm, 605 nm, and 705 nm, respectively). Single-wavelength excitation has greatly simplified the optics in confocal setup, thus enhancing the detection reliability. With better size control during QD synthesis (ie, narrower emission spectra) and newly synthesized QDs with emission spectra in the infrared region, more target molecules can be detected simultaneously in solution. For instance, if 4 types of QDs are used, a total of 6 targets can be individually identified in a single test. However, while having greater potential in multiplexed detection, the crosslinking system shows lower hybridization efficiency, mainly due to the high negative-charge density created by extensive
Fig 5. QD cross-linking method in simultaneous detection of 3 DNA targets. A, B, and C show the results of detection of DNA targets 1, 2, and 3, respectively. Asterisks (*) denote the coincident signals.
probe loading on QD surfaces and steric hindrance [5]. Elevated temperature is often required to uncoil the target strand for better hybridization efficiency. However, QDs tend to aggregate unspecifically at temperatures higher than 808C (data not shown). Therefore, lower hybridization efficiency and longer incubation time are expected. This method is also expected to be less compatible with enzymemediated reactions, where both steric hindrance and nanoparticle-enzyme interaction may affect the enzyme activities and kinetics. The dilemma of homogeneous reaction and multiplexed detection can be overcome by incorporating more than 1 functional group, such as amine-carboxylate, rather than just single streptavidin–biotin interaction. Quantum dots with different functional groups can recognize and bind to specific probes that are correspondently functionalized after
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the hybridization or the enzymatic reaction is completed in homogeneous solution. However, carboxylate functional groups are easily hydrolyzed, making carboxylated QDs have much shorter shelf lives. Peptide conjugated QDs are currently being studied and may give 100+ high-affinity interaction pairs in the near future [4]. Regarding specificity, the 3-component, QD-based strategies should be more selective than any 2-component detection system based on single-stranded probe hybridization with the target [5]. Three-component systems, however, often required the balanced ratio between probes and targets, preferably 1:1, to get maximum sandwich hybridization efficiency. But QD cross-linking system is not limited by that because it requires only 1 target, in theory, to bridge 2 QDs. Our results not only demonstrate that specific DNA detection is achieved using QD nanobiosensors and the coincidence detection principle, but that they can also be used to quantify the target biomolecules by counting coincident events. The coincidence detection method therefore can be generally applied to many other bioanalysis areas, such as gene expression profiling, that require precise quantification of biomolecules. In addition, we took advantage of biotin–streptavidin interaction. This binding interaction is quick, reliable, and strong. It dramatically reduces the preparation time from tens of hours, for a thiolmediated, gold nanoparticle system, to just tens of minutes.
Conclusion In this article, 2 new QD-mediated separation-free biosensing methods that can be applied to identification of different nucleic acid sequences are described. We have demonstrated multiplexed detection of 3 DNA sequences by using the QD cross-linking system. However, the QD– organic fluorophore coupling system also has unique advantages in application where temperature treatment and enzymatic reactions are needed. These 2 methods have provided new platforms of biosensing with several features superior to the existing methods. In addition, 2 specific molecular probes are used, instead of only 1, to recognize a target molecule, achieving molecular identification with high specificity. Future research will aim at multiplexed applications and better understanding and control of nanoparticle cross-linking extents.
Acknowledgment This work is supported by the National Science Foundation (DBI-0352407) and the Whitaker Foundation.
References [1] Alivisatos P. The use of nanocrystals in biological detection. Nat Biotechnol 2004;22:47 - 52.
[2] Penn SG, He L, Natan MJ. Nanoparticles for bioanalysis. Curr Opin Chem Biol 2003;7:609 - 15. [3] Chan WCW, Maxwell DJ, Gao XH, Bailey RE, Han MY, Nie SM. Luminescent quantum dots for multiplexed biological detection and imaging. Curr Opin Biotechnol 2002;13:40 - 6. [4] Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, et al. Quantum dots for live cells, in vivo imaging, and diagnostics. Science 2005;307:538 - 44. [5] Elghanian R, Storhoff JJ, Mucic RC, Letsinger RL, Mirkin CA. Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles. Science 1997;277:1078 - 81. [6] Cao YWC, Jin RC, Mirkin CA. Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 2002;297:1536 - 40. [7] Han MY, Gao XH, Su JZ, Nie S. Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nat Biotechnol 2001;19: 631 - 5. [8] Taton TA, Mirkin CA, Letsinger RL. Scanometric DNA array detection with nanoparticle probes. Science 2000;289:1757 - 60. [9] Dubertret B, Calame M, Libchaber AJ. Single-mismatch detection using gold-quenched fluorescent oligonucleotides. Nat Biotechnol 2001;19:365 - 70. [10] Storhoff JJ, Lucas AD, Garimella V, Bao YP, Muller UR. Homogeneous detection of unamplified genomic DNA sequences based on colorimetric scatter of gold nanoparticle probes. Nat Biotechnol 2004;22:883 - 7. [11] Sato K, Sawayanagi M, Hosokawa K, Maeda M. Single-base mutation detection using neutravidin-modified polystyrene nanoparticle aggregation. Anal Sci 2004;20:893 - 4. [12] Sato K, Hosokawa K, Maeda M. Rapid aggregation of gold nanoparticles induced by non-cross-linking DNA hybridization. J Am Chem Soc 2003;125:8102 - 3. [13] Maxwell DJ, Taylor JR, Nie SM. Self-assembled nanoparticle probes for recognition and detection of biomolecules. J Am Chem Soc 2002; 124:9606 - 12. [14] Xu HX, Sha MY, Wong EY, Uphoff J, Xu YH, Treadway JA, et al. Multiplexed SNP genotyping using the Qbead (TM) system: a quantum dot-encoded microsphere-based assay. Nucleic Acids Res 2003;31:e43. [15] Li HX, Rothberg L. Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles. Proc Natl Acad Sci U S A 2004;101:14036 - 9. [16] Park SJ, Taton TA, Mirkin CA. Array-based electrical detection of DNA with nanoparticle probes. Science 2002;295:1503 - 6. [17] Wang J, Liu GD, Merkoci A. Electrochemical coding technology for simultaneous detection of multiple DNA targets. J Am Chem Soc 2003;125:3214 - 5. [18] Kerman K, Saito M, Morita Y, Takamura Y, Ozsoz M, Tamiya E. Electrochemical coding of single-nucleotide polymorphisms by monobase-modified gold nanoparticles. Anal Chem 2004;76: 1877 - 84. [19] Wang J, Xu DK, Polsky R. Magnetically-induced solid-state electrochemical detection of DNA hybridization. J Am Chem Soc 2002;124:4208 - 9. [20] Su M, Li SU, Dravid VP. Microcantilever resonance-based DNA detection with nanoparticle probes. Appl Phys Lett 2003;82:3562 - 4. [21] Vaarno J, Ylikoski E, Meltola NJ, Soini JT, Hanninen P, Lahesmaa R, et al. New separation-free assay technique for SNPs using two-photon excitation fluorophore. Nucleic Acids Res 2004;32:e108. [22] Cai H, White PS, Torney D, Deshpande A, Wang ZL, Marrone B, et al. Flow cytometry-based minisequencing: a new platform for high-throughput single-nucleotide polymorphism scoring. Genomics 2000;66:135 - 43. [23] Nam JM, Thaxton CS, Mirkin CA. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science 2003;301: 1884 - 6.
H.-C. Yeh et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 115–121 [24] Kettling U, Koltermann A, Schwille P, Eigen M. Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy. Proc Natl Acad Sci U S A 1998;95:1416 - 20. [25] Edman L, Mets U, Rigler R. Conformational transitions monitored for single molecules in solution. Proc Natl Acad Sci U S A 1996;93: 6710 - 5. [26] Zhang C, Chao SY, Wang TH. Comparative quantification of nucleic acids using single-molecule detection and molecular beacons. Analyst 2005;130:483 - 8. [27] Tyagi S, Bratu DP, Kramer FR. Multicolor molecular beacons for allele discrimination. Nat Biotechnol 1998;16:49 - 53. [28] Tyagi S, Kramer FR. Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 1996;14:303 - 8. [29] Chan WCW, Nie SM. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 1998;281:2016 - 8.
121
[30] Wang TH, Peng YH, Wong PK, Ho CM. Single-molecule tracing on a fluidic microchip for quantitative detection of low-abundance nucleic acids. J Am Chem Soc 2005;127:5354 - 9. [31] Eigen M, Rigler R. Sorting single molecules — application to diagnostics and evolutionary biotechnology. Proc Natl Acad Sci U S A 1994;91:5740 - 7. [32] Castro A, Williams JGK. Single-molecule detection of specific nucleic acid sequences in unamplified genomic DNA. Anal Chem 1997;69: 3915 - 20. [33] Li HT, Ying LM, Green JJ, Balasubramanian S, Klenerman D. Ultrasensitive coincidence fluorescence detection of single DNA molecules. Anal Chem 2003;75:1664 - 70. [34] Li HT, Zhou DJ, Browne H, Balasubramanian S, Klenerman D. Molecule by molecule direct and quantitative counting of antibodyprotein complexes in solution. Anal Chem 2004;76:4446 - 51.