You are on page 1of 5

Microchim Acta (2011) 175:355359 DOI 10.

1007/s00604-011-0657-5

SHORT COMMUNICATION

CdS quantum dots as a fluorescent sensing platform for nucleic acid detection
Wenbo Lu & Xiaoyun Qin & Yonglan Luo & Guohui Chang & Xuping Sun

Received: 23 May 2011 / Accepted: 1 July 2011 / Published online: 14 July 2011 # Springer-Verlag 2011

Abstract We demonstrate that CdS quantum dots (QDs) can be applied to fluorescence-enhanced detection of nucleic acids in a two-step protocol. In step one, a fluorescently labeled single-stranded DNA probe is adsorbed on the QDs to quench its luminescence. In step two, the hybridization of the probe with its target ssDNA produces a double-stranded DNA which detaches from the QD. This, in turn, leads to the recovery of the fluorescence of the label. The lower detection limit of the assay is as low as 1 nM. The scheme (that was applied to detect a target DNA related to the HIV) is simple and can differentiate between perfectly complementary targets and mismatches. Keyword CdS quantum dots . DNA detection . Fluorescence

Introduction It is very important to develop rapid, cost-effective, sensitive and specific methods for nucleic acid detection, owing to their potential diverse applications in gene expression profiling, clinical disease diagnostics and treatment [1, 2]. With the increasing availability of nanostructures, widespread attention has been paid to their diagnostic potential in a biotechnological system [3], and the employment of various nanostructures for this purpose has been well-documented [4]. Recently, researchers have made much effort to develop homogeneous
W. Lu : X. Qin : Y. Luo : G. Chang : X. Sun (*) Chemical Synthesis and Pollution Control Key Laboratory of Sichuan Province, School of Chemistry and Chemical Engineering, China West Normal University, Nanchong 637002, Sichuan, China e-mail: sun.xuping@hotmail.com

fluorescence assays based on FRET (fluorescence resonance energy transfer) or quenching mechanism for nucleic acid detection [5]. Eliminating the selection issue of a fluorophore quencher pair from the nanostructure-involved fluorescent sensing system has been successfully achieved, where the nanostructure is employed as a dye quencher and is able to quench dyes of different emission frequencies [5, 6]. So far, a number of structures have been employed, including gold nanoparticles [5, 711], single-walled carbon nanotube (SWCNT) [6, 12], multi-walled carbon nanotubes [13], carbon nanoparticles [14], caobon nanospheres [15], nanoC60 [16], mesoporous carbon microparticles [17], graphene oxide (GO) [18, 19], poly(p-phenylenediamine) nanobelts [20], polyaniline nanofibres [21], poly(o-phenylenediamine) colloids [22], poly(2,3-diaminonaphthalene) microspheres [23], coordination polymer colloids and nanobelts [2426], Ag@poly(m-phenylenediamine) core-shell nanoparticles [27], tetracyanoquinodimethane nanoparticles [28] and supramolecular microparticles [29]. CdS quantum dots (CdSQDs) have recently received more interest due to their unique size-dependent optical and electronic properties [3032]. In particular, CdSQDs have emerged as promising fluorescent probes in biological and medical fields such as bioassays, cell imaging, and clinic diagnosis [33, 34]. To the best of our knowledge, the use of CdSQDs as a quencher for nucleic acid detection has not been explored so far. We report on the first use of CdSQDs stabilized by sodium hexametaphosphate as an effective sensing platform for fluorescence-enhanced nucleic acid detection. The DNA detection is accomplished by two steps: (1) CdSQDs adsorbs and quenches a dye-labeled single-stranded DNA (ssDNA) probe; (2) the subsequent hybridization of the probe with its target produces a doublestranded DNA (dsDNA) which detaches from CdSQD, leading to fluorescence recovery. We also show that this

356

W. Lu et al.

sensing platform is capable of differentiating between complementary and mismatched sequences.

Experimental Chemical and reagents All chemically synthesized oligonucleotides were purchased from Shanghai Sangon Biotechnology Co. Ltd. (http://www. sangon.com/). DNA concentration was estimated by measuring the absorbance at 260 nm. All the other chemicals were purchased from Aladin Ltd. (http://www.aladdin-reagent.com/) and used as received without further purification. The water used throughout all experiments was purified through a Millipore system (http://www.millipore.com/). Oligonucleotide sequences used are listed below (mismatch underlined): (1) FAM dye-labeled ssDNA (PHIV): 5-FAM-AGT CAG TGT GGA AAATCT CTA GC-3 (2) Complementary target to PHIV (T0): 5-GCT AGA GAT TTT CCA CAC TGA CT-3 (3) Single-base mismatched target to PHIV (T1): 5-GCT AGA GAT TGT CCA CAC TGA CT-3 (4) Two-base mismatched target to PHIV (T2): 5-GCT AGA GAT TGT ACA CAC TGA CT-3 (5) Three-base mismatched target to PHIV (T3): 5-GCT ATA GAT TGT ACA CAC TGA CT-3 Preparation of CdS QDs The CdSQDs were prepared as follows: In a typical synthesis, 0.5 mL of 0.01 M sodium hexametaphosphate aqueous solution was added into 5 mL of 0.001 M CdCl2 aqueous solution, followed by the dropwise addition of 5 mL of 0.001 M Na2S aqueous solution at room temperature. The resulting mixture was stirred for 6 h at room temperature. Finally, the yellow solution was centrifuged and further washed with water three times. The resulting precipitates were redispersed in 10 mL of water at a concentration of 0.3 mmolL1 with the aid of ultrasound for characterization and further use. The volume of each sample for fluorescence measurement is 400 L in 20 mM Tris-HCl buffer containing 100 mM NaCl, 5 mM KCl, and 15 mM MgCl2 (pH: 7.4). Apparatus UV-vis spectra were collected on a UV5800 modal spectrophotometer (http://www.metash.com/). Transmission electron microscopy (TEM) measurements were made on a HITACHI H-8100 EM (http://www.hitachi.com/) with an accelerating

Fig. 1 UV-vis absorption spectrum of the resultant dispersion

applied potential of 200 kV. Fluorescent emission spectra were recorded on a RF-5301PC spectrofluorometer (http:// www.shimadzu.com/). Zeta potential measurements were performed on a Nano-ZS Zetasizer ZEN3600 (Malvern Instruments Ltd., U.K., http://www.malvern.com/).

Results and discussion Figure 1 shows the UV-vis absorption spectra of the resultant dispersion. It is seen that this dispersion exhibits a defined absorption edge at 473 nm, corresponding to energy gaps of 2.69 eV, which is a considerable blue-shift relative to the corresponding absorption band edge of bulk CdS (515 nm and 2.41 eV) [35].These absorption edges are assigned to the optical transition of the first excitonic state. All the observations indicate the formation of CdSQDs. Figure 2 shows typical TEM image and the corresponding particle size distribution histograms of as-formed CdSQDs, revealing that the average particle size of the resulting CdSQDs is 4 nm with a narrow size distribution. Also, it can be clearly seen that all these particles are well separated from each other. It should be pointed out that, although small CdSQDs are thermodynamically unstable due to their high surface energies and large surfaces, easily undergoing aggregation [36], the CdSQDs thus formed were free from flocculation or aggregation for several weeks, suggesting

Fig. 2 a TEM image and b corresponding size distribution histograms of the CdSQDs thus formed

CdS quantum dots as a novel fluorescent sensing platform

357

Fig. 3 Fluorescence emission spectra of PHIV (100 nM) at different conditions: a PHIV; b PHIV + 600 nM T0; c PHIV + CdSQDs; d PHIV + CdSQDs + 600 nM T0. Curve e is the emission spectra of the CdSQDs sample. Inset: fluorescence intensity ratio of PHIV-CdSQD complex with F/F0-1 (where F0 and F are the fluorescence intensity without and with the presence of T0, respectively) plotted against logarithm of the concentration of T0. All measurements were done in Tris-HCl buffer in the presence of 5 mM Mg2+ (pH: 7.4). Excitation was at 480 nm, and the emission was monitored at 517 nm

sodium hexametaphosphate serves as a very effective stabilizer for CdSQDs. We further explored the use of such CdSQDs as fluorescent sensing platform for DNA detection by choosing an oligonucleotide sequence associated with human immunodeficiency virus (HIV) labeled at the 5 end with the dye FAM as a model system. In the absence of CdSQDs, this species (PHIV) exhibit strong fluorescence emission in Tris-HCl buffer due to the presence of the fluorescein-based dye (Fig. 3, curve a). However, CdSQDs quench about 42% of the fluorescence (Fig. 3, curve c), demonstrating that the CdSQD can adsorb ssDNA and quench the dye effectively. The addition of the complementary sequence T0 in the absence of CdSQDs has a small influence on the the fluorescence of the free PHIV (Fig. 3, curve b). In contrast, the PHIV-CdSQD complex exhibits significant fluorescence enhancement upon its

incubation with T0 over a period of 30 min, as is evidenced by a 73% fluorescence recovery (Fig. 3, curve d). Note that CdSQD itself exhibits no fluorescence emission (Fig. 3, curve e) and thus makes no contribution to the fluorescence intensity of each CdS-involved sample measured. The inset in Fig. 3 illustrates the fluorescence intensity changes (F/F0-1) of PHIV-CdSQD complex upon addition of different concentrations of T0, where F0 and F are FAM fluorescence intensities at 517 nm in the absence and the presence of T0, respectively. In the DNA concentration range of 1.0300 nM, a dramatic increase of FAM fluorescence intensity is observed, suggesting that the CdSQD/DNA assembly approach is effective in probing biomolecular interactions. The zeta potential of CdSQDs was measured to be about 9.2 mV, indicating that the CdSQD has a low negatively charged surface. So, there are slight electrostatic repulsions between CdSQD and the negatively charged backbone of ssDNA. These repulsions are offset by stronger coordination interaction between the nitrogen atom of DNA base and CdSQDs [37]. In contrast, CdSQD should have weak or no binding with dsDNA due to the absence of unpaired DNA bases and the rigid conformation of dsDNA. Scheme 1 presents a schematic to illustrate the fluorescence-enhanced DNA detection using CdSQDs as a sensing platform. The DNA detection is accomplished by the following two steps: (1) the adsorption of fluorescent FAM-labeled ssDNA onto CdSQD via coordination interactions leads to fluorescence quenching because of their close proximity. (2) in the presence of target, the specific hybridization between the dye-labeled DNA and its target disturbs the interaction between the dye-labeled ssDNA and CdSQD, producing a dsDNA which detaches from PDIM and leading to fluorescence recovery. To get further insight into the kinetics of the fluorescence quenching and recovery, we collected the time-dependent

Scheme 1 A schematic (not to scale) illustrating the fluorescence-enhanced DNA detection using CdSQDs as a sensing platform

358

W. Lu et al.

fluorescence spectra of PHIV and CdSQDs, as well as of the PHIV-CdSQD complex with T0, as shown in Fig. 4. The curve a shows the fluorescence quenching of PHIV by CdSQDs as a function of incubation time. In the absence of the target, the curve a exhibits a rapid reduction in the first 5 min and reaches equilibrium over a period of 10 min, indicating that the adsorption process of ssDNA on CdSQD is very fast. The curve b shows the fluorescence recovery of PHIV-CdSQD by T0 as a function of time. In the presence of the target T0, the curve shows a fast increase in the first 3 min, and the best fluorescence response is obtained over a period of 15 min. All the above observations indicate that the CdSQD is superior to SWCNT and GO in detection speed [5, 11, 18]. It is worthwhile mentioning that this sensing platform can differentiate complementary and mismatched sequences. Figure 5 shows the fluorescence responses of PHIVCdSQD complex toward perfect complementary target T0 and single, two, and three-base mismatched targets T1, T2, and T 3. The F/F0 value (where F0 and F are the fluorescence intensity without and with the presence of target, respectively) obtained upon addition of 600 nM of T1, T2, and T3 is about 66%, 61%, and 49% of the value obtained upon addition of 600 nM of T0 into PHIV-CdSQD complex, respectively. Compared to the complementary target, the mismatched target should have lower hybridization ability toward the adsorbed dye-labeled ssDNA probe leading to a decreased hybridization and thus fluorescence recovery efficiency. The inset in Fig. 5 presents the corresponding fluorescence intensity histograms. All the above observations suggest that the results exhibit good reproducibility and the CdSQD-based sensing system is likely to be capable of practically useful mismatch detection upon further development. It was found that the amount of CdSQDs has heavy influence on the fluorescence quenching and recovery.

Fig. 5 Fluorescence emission spectra of PHIV (100 nM) at different conditions: a PHIV-CdSQD complex; b PHIV-CdSQD complex + 600 nM T0; c PHIV-CdSQD complex + 600 nM T1; d PHIV-CdSQD complex + 600 nM T2; e PHIV-CdSQD complex + 600 nM T3. Inset: the corresponding fluorescence intensity histograms with error bar. All measurements were done in Tris-HCl buffer in the presence of 5 mM Mg2+ (pH: 7.4). Excitation was at 480 nm, and the emission was monitored at 517 nm

Figure 6 shows the fluorescence intensity histograms of PHIV + CdSQDs and PHIV + CdSQDs + T0 for four samples measured with the using of 0, 5, 15 and 20-L CdSQDs dispersion (0.3 mmolL1), respectively. Obviously, the increase of CdS NPs in amount leads to increased quenching efficiency but decreased recovery efficiency. We can attribute such observation to the following reasons: CdSQD has a strong affinity for ssDNA and thus the use of increased amount of CdSQDs leads to more efficient adsorption of ssDNA on them, giving a higher quenching efficiency. During the subsequent recovery process, however, the adsorption of targets on excessive CdSQDs will compete with the hybridization of target with ssDNA absorbed on CdSQD, leading to decreased hybridization and thus recovery efficiency. Based on these observations, an optimal volume of 10 L was chosen in our present study.

Fig. 4 a Fluorescence quenching of PHIV (100 nM) by CdSQDs and b fluorescence recovery of PHIV-CdS by T0 (600 nM) in Tris-HCl buffer as a function of time. All measurements were done in Tris-HCl buffer in the presence of 5 mM Mg2+ (pH: 7.4).. Excitation was at 480 nm, and the emission was monitored at 517 nm

Fig. 6 Fluorescence intensity histograms of PHIV + CdSQDs and PHIV + CdSQDs + T0 with the using of 0, 5, 10, 15 and 20-L CdSQDs sample in this system ([PHIV]=100 nM; [T0]=600 nM). Excitation was at 480 nm, and the emission was monitored at 517 nm. All measurements were done in 20 mM TrisHCl buffer (pH 7.4, containing 100 mM NaCl, 5 mM KCl and 5 mM MgCl2)

CdS quantum dots as a novel fluorescent sensing platform

359 17. Liu S, Li H, Wang L, Tian J, Sun X (2010) New application of mesoporous carbon microparticles to nucleic acid detection. J Mater Chem 21:339341 18. Lu C, Yang H, Zhu C, Chen X, Chen G (2009) A graphene platform for sensing biomolecules. Angew Chem Int Ed 48:47854787 19. He S, Song B, Li D, Zhu C, Qi W, Wen W, Wang L, Song S, Fang H, Fan C (2010) A graphene nanoprobe for rapid, sensitive, and multicolor fluorescent DNA analysis. Adv Funct Mater 20:453459 20. Wang L, Zhang Y, Tian J, Li H, Sun X (2010) Conjugation polymer nanobelts: a novel fluorescent sensing platform for nucleic acid detection. Nucleic Acids Res 39:e37e42 21. Liu S, Wang L, Luo Y, Tian J, Li H, Sun X (2010) Polyaniline nanofibres for fluorescent nucleic acid detection. Nanoscale 3:967969 22. Tian J, Li H, Luo Y, Wang L, Zhang Y, Sun X (2011) Poly(ophenylenediamine) colloid-quenched fluorescent oligonucleotide as a probe for fluorescence-enhanced nucleic acid detection. Langmuir 27:874877 23. Tian J, Zhang Y, Luo Y, Li H, Zhai J, Sun X (2011) Poly(2,3diaminonaphthalene) microspheres as a novel quencher for fluorescence-enhanced nucleic acid detection. Analyst 136:2221 2224 24. Li H, Sun X (2011) Fluorescence-enhanced nucleic acid detection: using coordination Polymer colloids as a sensing platform. Chem Commum 47:26252627 25. Luo Y, Liao F, Lu W, Chang G, Sun X (2011) Coordination polymer nanobelts for nucleic acid detection. Nanotechnol 22:195502 195506 26. Li H, Wang L, Zhang Y, Tian J, Sun X (2011) Coordination polymer nanobelts as an effective sensing platform for fluorescence-enhanced nucleic acid detection. Macromol Rapid Commun 32:899904 27. Zhang Y, Wang L, Tian J, Li H, Luo Y, Sun X (2011) Ag@poly(mphenylenediamine) coreshell nanoparticles for highly selective, multiplex nucleic acid detection. Langmuir 27:21702175 28. Li H, Wang L, Zhai J, Luo Y, Zhang Y, Tian J, Sun X (2011) Tetracyanoquinodimethane nanoparticles as an effective sensing platform for fluorescent nucleic acid detection. Anal Methods 3:10511055 29. Li H, Zhai J, Sun X (2011) Electrostatic-assembly-driven formation of supramolecular rhombus microparticles and their application for fluorescent nucleic acid detection. PLoS One 6:e18958 30. Alivisatos AP (1996) Semiconductor clusters, nanocrystals, and quantum dots. Science 271:933937 31. Coe S, Woo WK, Bawendi M, Bulovic V (2002) Electroluminescence from single monolayers of nanocrystals in molecular organic devices. Nature 420:800803 32. Atay Z, Biver T, Corti A, Eltugral N, Lorenzini E, Masini M, Paolicchi A, Pucci A, Ruggeri G, Secco F et al (2009) Noncovalent interactions of cadmium sulphide and gold nanoparticles with DNA. J Nanopart Res 12:22412253 33. Kim T, Noh M, Lee H, Joo SW, Lee SY, Lee K (2009) Fluorescence-based detection of point mutation in DNA sequences by CdS quantum dot aggregation. J Phys Chem B 113:14487 14490 34. Liu Y, Ai K, Yuan Q, Lu L (2011) Fluorescence-enhanced gadolinium-doped zinc oxide quantum dots for magnetic resonance and fluorescence imaging. Biomaterial 32:11851192 35. Balu AM, Campelo JM, Luque R, Rajabi F, Romero AA (2010) Biomaterials supported CdS nanocrystals. Mater Chem Phys 124:5254 36. Nair PS, Revaprasadu N, Radhakrishnan T, Kolawole GA (2001) Preparation of CdS nanoparticles using the cadmium(II) complex of N, N-bis(thiocarbamoyl)hydrazine as a simple single-source precursor. J Mater Chem 11:15551556 37. Jin J, Jiang L, Chen X, Yang W, Li T (2010) Construction of CdS nanoparticle chain on DNA template. Chin J Chem 21:208210

Conclusions In summary, CdSQDs have been proven to be a very effective sensing platform for fluorescence-enhanced DNA detection with a high selectivity down to single-base mismatch. Our observations are significant for the following two reasons: (1) it is a new application of CdSQDs for fluorescent DNA detection; (2) this sensing platform is promising for effective fluorescence-enhanced detection sensitive and selective to the target molecule studied.

References
1. Gresham D, Ruderfer DM, Pratt SC, Schacherer J, Dunham MJ, Botstein D, Kruglyak L (2006) Genome-wide detection of polymorphisms at nucleotide resolution with a single DNA microarray. Science 311:19321936 2. Wei M, Guo L, Famouri P (2011) DNA biosensors based on metallo-intercalator probes and electrocatalytic amplification. Microchim Acta 172:247260 3. Brayner R (2008) The toxicological impact of nanoparticles. Nano Today 3:4855 4. Rosi NL, Mirkin CA (2005) Nanostructures in biodiagnostics. Chem Rev 105:15471562 5. Ray PC, Darbha GK, Ray A, Walker J, Hardy W (2007) Gold nanoparticle based FRET for DNA detection. Plasmonics 2:173 183 6. Yang R, Tang Z, Yan J, Kang H, Kim Y, Zhu Z, Tan W (2008) Noncovalent assembly of carbon nanotubes and single-stranded DNA: an effective sensing platform for probing biomolecular interactions. Anal Chem 80:74087413 7. Dubertret B, Calame M, Libchaber AJ (2001) Single-mismatch detection using gold-quenched fluorescent oligonucleotides. Nat Biotechnol 19:365370 8. Maxwell DJ, Taylor JR, Nie S (2002) Self-assembled nanoparticle probes for recognition and detection of biomolecules. J Am Chem Soc 124:96069612 9. Li H, Rothberg L (2004) DNA sequence detection using selective fluorescence quenching of tagged oligonucleotide probes by gold nanoparticles. Anal Chem 76:54145417 10. Song S, Liang Z, Zhang J, Wang L, Li G, Fan C (2009) Goldnanoparticle-based multicolor nanobeacons for sequence-specific DNA analysis. Angew Chem Int Ed 48:86708674 11. Li D, Song S, Fan C (2010) Target-responsive structural switching for nucleic acid-based sensors. Acc Chem Res 43:631641 12. Yang R, Jin J, Chen Y, Shao N, Kang H, Xiao Z, Tang Z, Wu Y, Zhu Z, Tan W (2008) Carbon nanotube-quenched fluorescent oligonucleotides: probes that fluoresce upon hybridization. J Am Chem Soc 130:83518358 13. Li H, Tian J, Wang L, Zhang Y, Sun X (2010) Multi-walled carbon nanotubes as an effective fluorescent sensing platform for nucleic acid detection. J Mater Chem 21:824828 14. Li H, Zhang Y, Wang L, Tian J, Sun X (2011) Nucleic acid detection using carbon nanoparticles as an fluorescent sensing platform. Chem Commun 47:961963 15. Li H, Zhang Y, Wu T, Liu S, Wang L, Sun X (2011) Carbon nanospheres for fluorescent biomolecular detection. J Mater Chem 21:46634668 16. Li H, Zhang Y, Luo Y, Sun X (2011) Nano-C60: a novel, effective fluorescent sensing platform for biomolecular detection. Small 7:15621568