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Microchim Acta (2011) 175:355–359 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, 7–11], 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 [24–26], 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 [30–32]. 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

metash.com/).shimadzu. easily undergoing aggregation [36]. Transmission electron microscopy (TEM) measurements were made on a HITACHI H-8100 EM (http://www. suggesting Fig. which is a considerable blue-shift relative to the corresponding absorption band edge of bulk CdS (515 nm and 2.com/) with an accelerating Fig. Lu et al. Fluorescent emission spectra were recorded on a RF-5301PC spectrofluorometer (http:// www. Ltd. revealing that the average particle size of the resulting CdSQDs is 4 nm with a narrow size distribution. Finally.01 M sodium hexametaphosphate aqueous solution was added into 5 mL of 0. All the other chemicals were purchased from Aladin Ltd.5 mL of 0.001 M Na2S aqueous solution at room temperature. 5 mM KCl. Experimental Chemical and reagents All chemically synthesized oligonucleotides were purchased from Shanghai Sangon Biotechnology Co.com/). 0. and 15 mM MgCl2 (pH: 7. The resulting precipitates were redispersed in 10 mL of water at a concentration of 0. U.. sensing platform is capable of differentiating between complementary and mismatched sequences. http://www. sangon. followed by the dropwise addition of 5 mL of 0. (http://www.41 eV) [35]. DNA concentration was estimated by measuring the absorbance at 260 nm. 2 a TEM image and b corresponding size distribution histograms of the CdSQDs thus formed . (http://www. Zeta potential measurements were performed on a Nano-ZS Zetasizer ZEN3600 (Malvern Instruments Ltd.com/). the CdSQDs thus formed were free from flocculation or aggregation for several weeks.com/). Figure 2 shows typical TEM image and the corresponding particle size distribution histograms of as-formed CdSQDs. It should be pointed out that. 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..hitachi.These absorption edges are assigned to the optical transition of the first excitonic state.malvern. The volume of each sample for fluorescence measurement is 400 μL in 20 mM Tris-HCl buffer containing 100 mM NaCl. The water used throughout all experiments was purified through a Millipore system (http://www.69 eV.com/).001 M CdCl2 aqueous solution.millipore. corresponding to energy gaps of 2. It is seen that this dispersion exhibits a defined absorption edge at 473 nm.K. it can be clearly seen that all these particles are well separated from each other.aladdin-reagent.4).com/) and used as received without further purification. The resulting mixture was stirred for 6 h at room temperature. 1 UV-vis absorption spectrum of the resultant dispersion applied potential of 200 kV. Results and discussion Figure 1 shows the UV-vis absorption spectra of the resultant dispersion.3 mmol·L−1 with the aid of ultrasound for characterization and further use. Apparatus UV-vis spectra were collected on a UV5800 modal spectrophotometer (http://www. All the observations indicate the formation of CdSQDs. although small CdSQDs are thermodynamically unstable due to their high surface energies and large surfaces. Also.356 W. the yellow solution was centrifuged and further washed with water three times.

All measurements were done in Tris-HCl buffer in the presence of 5 mM Mg2+ (pH: 7. In the absence of CdSQDs. d PHIV + CdSQDs + 600 nM T0.0–300 nM. In contrast. To get further insight into the kinetics of the fluorescence quenching and recovery. 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. 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. curve d). However. The zeta potential of CdSQDs was measured to be about −9. 3 illustrates the fluorescence intensity changes (F/F0-1) of PHIV-CdSQD complex upon addition of different concentrations of T0. curve a). c PHIV + CdSQDs. 3. and the emission was monitored at 517 nm sodium hexametaphosphate serves as a very effective stabilizer for CdSQDs. suggesting that the CdSQD/DNA assembly approach is effective in probing biomolecular interactions. 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. curve c). 3. In the DNA concentration range of 1. The inset in Fig.4). curve b). producing a dsDNA which detaches from PDIM and leading to fluorescence recovery. Curve e is the emission spectra of the CdSQDs sample. b PHIV + 600 nM T0. Scheme 1 presents a schematic to illustrate the fluorescence-enhanced DNA detection using CdSQDs as a sensing platform. a dramatic increase of FAM fluorescence intensity is observed. CdSQD should have weak or no binding with dsDNA due to the absence of unpaired DNA bases and the rigid conformation of dsDNA. we collected the time-dependent Scheme 1 A schematic (not to scale) illustrating the fluorescence-enhanced DNA detection using CdSQDs as a sensing platform . CdSQDs quench about 42% of the fluorescence (Fig. Note that CdSQD itself exhibits no fluorescence emission (Fig. where F0 and F are FAM fluorescence intensities at 517 nm in the absence and the presence of T0. 3 Fluorescence emission spectra of PHIV (100 nM) at different conditions: a PHIV. Excitation was at 480 nm. 3. respectively. indicating that the CdSQD has a low negatively charged surface.CdS quantum dots as a novel fluorescent sensing platform 357 Fig. as is evidenced by a 73% fluorescence recovery (Fig. the PHIV-CdSQD complex exhibits significant fluorescence enhancement upon its incubation with T0 over a period of 30 min. curve e) and thus makes no contribution to the fluorescence intensity of each CdS-involved sample measured. the specific hybridization between the dye-labeled DNA and its target disturbs the interaction between the dye-labeled ssDNA and CdSQD. 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. So. 3. respectively) plotted against logarithm of the concentration of T0. this species (PHIV) exhibit strong fluorescence emission in Tris-HCl buffer due to the presence of the fluorescein-based dye (Fig. (2) in the presence of target. In contrast. there are slight electrostatic repulsions between CdSQD and the negatively charged backbone of ssDNA.2 mV. These repulsions are offset by stronger coordination interaction between the nitrogen atom of DNA base and CdSQDs [37]. 3. demonstrating that the CdSQD can adsorb ssDNA and quench the dye effectively.

It is worthwhile mentioning that this sensing platform can differentiate complementary and mismatched sequences. respectively. 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. During the subsequent recovery process. the increase of CdS NPs in amount leads to increased quenching efficiency but decreased recovery efficiency. 5. two.4). fluorescence spectra of PHIV and CdSQDs. All measurements were done in Tris-HCl buffer in the presence of 5 mM Mg2+ (pH: 7. and T 3. All the above observations indicate that the CdSQD is superior to SWCNT and GO in detection speed [5. 15 and 20-μL CdSQDs sample in this system ([PHIV]=100 nM. In the presence of the target T0. the adsorption of targets on excessive CdSQDs will compete with the hybridization of target with ssDNA absorbed on CdSQD. The curve b shows the fluorescence recovery of PHIV-CdSQD by T0 as a function of time. however. as shown in Fig. The F/F0 value (where F0 and F are the fluorescence intensity without and with the presence of target. Inset: the corresponding fluorescence intensity histograms with error bar. 61%. Fig. T2.358 W. an optimal volume of 10 μL was chosen in our present study. and the emission was monitored at 517 nm. as well as of the PHIV-CdSQD complex with T0. In the absence of the target. Figure 5 shows the fluorescence responses of PHIVCdSQD complex toward perfect complementary target T0 and single. and three-base mismatched targets T1. and the emission was monitored at 517 nm Fig. the curve a exhibits a rapid reduction in the first 5 min and reaches equilibrium over a period of 10 min. e PHIV-CdSQD complex + 600 nM T3. The curve a shows the fluorescence quenching of PHIV by CdSQDs as a function of incubation time. leading to decreased hybridization and thus recovery efficiency. and T3 is about 66%. 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. It was found that the amount of CdSQDs has heavy influence on the fluorescence quenching and recovery. giving a higher quenching efficiency. b PHIV-CdSQD complex + 600 nM T0. 5 Fluorescence emission spectra of PHIV (100 nM) at different conditions: a PHIV-CdSQD complex. 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. 15 and 20-μL CdSQDs dispersion (0. and 49% of the value obtained upon addition of 600 nM of T0 into PHIV-CdSQD complex. Excitation was at 480 nm. indicating that the adsorption process of ssDNA on CdSQD is very fast. The inset in Fig. Excitation was at 480 nm. Excitation was at 480 nm. All measurements were done in 20 mM Tris–HCl buffer (pH 7. 5 mM KCl and 5 mM MgCl2) . 4. containing 100 mM NaCl. 5 presents the corresponding fluorescence intensity histograms. Obviously. 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 curve shows a fast increase in the first 3 min. and the best fluorescence response is obtained over a period of 15 min. d PHIV-CdSQD complex + 600 nM T2. 6 Fluorescence intensity histograms of PHIV + CdSQDs and PHIV + CdSQDs + T0 with the using of 0. All measurements were done in Tris-HCl buffer in the presence of 5 mM Mg2+ (pH: 7.4). 11.4. 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. 10. T2. c PHIV-CdSQD complex + 600 nM T1. [T0]=600 nM). respectively.3 mmol·L−1). Compared to the complementary target. 5. Lu et al. respectively) obtained upon addition of 600 nM of T1. Based on these observations. Fig.. 18].

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