Electrochemiluminescence Resonance Energy Transfer Between CdS

Jul 26, 2012 - Peng Wu , Xiandeng Hou , Jing-Juan Xu , and Hong-Yuan Chen ... Xiaojiao Du , Liming Dai , Ding Jiang , Henan Li , Nan Hao , Tianyan You...
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Electrochemiluminescence Resonance Energy Transfer Between CdS:Eu Nancrystals and Au Nanorods for Sensitive DNA Detection Hong Zhou, Yan-Yan Zhang, Jing Liu, Jing-Juan Xu,* and Hong-Yuan Chen State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China. ABSTRACT: This work provides a novel electrochemiluminescence resonance energy transfer (ECL-RET) system using CdS:Eu nanoscrytals (NCs) as an ECL donor and Au nanorods (Au NRs) as an ECL acceptor. CdS:Eu NC, prepared by doping 1.5% Eu3+ ions into CdS NCs, exhibits strong and stable cathodic ECL emissions in the presence of coreactant S2O82− ions with two ECL spectral bands at 450−550 nm from the host CdS and at 600−700 nm due to the energy transfer from host CdS to Eu3+ ions. Au nanorods (Au NRs) have two absorption peaks that are easily tuned to match well with the ECL emission spectrum of the CdS:Eu NCs film by adjusting the aspect ratio of the nanorods to get a highly effective ECL-RET. Here, we studied the spectrum, distance and shape dependence of the efficiency of ECL-RET between the NCs' ECL and different Au nanoparticles (Au NPs) on the basis of the stem-loop structure DNA with a 6-base-pair (bp) stem and a 12, 30, or 45 nucleotide (nct) loop. At the optimized conditions, the system could be used to ultrasensitivly and specifically detect target DNA, providing significant potential application in clinical analysis.

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INTRODUCTION Since the first work on the electrochemiluminescence (ECL) of silicon nanocrystals (NCs) was reported in 2002,1 semiconductor nanocrystals (S-NCs)-based ECL sensing strategies have been developed during the past decade.2−4 However, the S-NCs themselves have lower ECL signals comparing with conventional luminescent reagents, such as luminol or Ru(bpy)32+, which limits the application of S-NCs ECL in chemical analysis. Generally, metal ion dopants incorporated in semiconductors could improve the luminescence efficiency by creating anew electron energy level or perturbing the host energy level.5−7 Recently, we successfully demonstrated that Eu3+ ions could alter the surface of CdS NCs and create a new surface state− Eu3+ complex, resulting in a large enhancement in the ECL intensity,8 which may broaden S-NCs’ application in highly sensitive biosensors. Our recent studies investigated that ECL resonance energy transfer (ECL-RET) between a S-NCs film and nanoparticles (NPs) or Ru(bpy)32+ is an efficient strategy for designing sensitive biosensors.6,9−12 A key point in ECLRET is to find an effective ECL donor and a suitable acceptor, in which the donor’s ECL spectrum should be overlapped with the acceptor’s absorption spectrum. For Eu3+-doped CdS NCs, except for the electrochemluminescence (450−550 nm) from the host CdS, a red luminescence (620 nm) was also clearly observed due to the energy transfer from host CdS to Eu3+ ions,8 which increase the difficulty to find a suitable spectrumoverlapped acceptor. Compared with spherical Au nanoparticles (SAu NPs) we have used as an acceptor in ECLRET in our previous work,6,10,11 Au nanorods (Au NRs) display two distinct absorption bands, one at a shorter wavelength (∼520 nm) and the other at a longer wavelength © 2012 American Chemical Society

that undergoes a bathochromic shift with increasing aspect ratio.13−16 Au NRs should be the better acceptor candidates of CdS:Eu NCs because their scattering and absorption bands can be tuned by adjusting the aspect ratio of the nanorods and their high extinction coefficient.17−19 The sequence-specific detection of DNA is of great importance for molecular diagnostics, genetic diseases, environmental monitoring, and early screening of cancers.20−24 Thus, sensitive, selective, and rapid methods for detecting specific DNA sequences are highly desirable. To fulfill these requirements, numerous DNA detection systems based on the hybridization between a DNA probe and its complementary target have been described. For example, to improve the sensitivity of DNA detection, polymerase chain reaction is often used to amplify the target DNA.25 However, this method generally suffers from low specificity, making it difficult to differentiate single-nucleotide polymorphisms (SNPs).26 Thus, a structured DNA probe, such as a molecular beacon (MB), has been used to recognize specific sequences.27,28 A molecular beacon is an oligonucleotide probe designed with selfcomplementarity at the 3′ and 5′ end, which forms a hairpinlike DNA stem−loop secondary structure in the absence of their target strand. The MB has been reported to exhibit high differentiation ability toward SNPs, which arise from the conformational constraint of the stem−loop structure. Therefore, the MB has been introduced for design of a novel DNA sensing strategy by combining with some signal amplification strategies.29,30 Received: May 24, 2012 Revised: July 16, 2012 Published: July 26, 2012 17773

dx.doi.org/10.1021/jp305076g | J. Phys. Chem. C 2012, 116, 17773−17780

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Table 1. DNA Sequence Used in This Worka

a

The underlined region of the molecular beacon identifies the stem sequence, and the mutant base is highlightened in the box.

for ECL detection, and 0.1 M Tris−HCl buffer containing 0.1 M NaCl (pH 7.4) was used for electrode stock solutions. All other reagents were of analytical grade and were used as received. Millipore ultrapure water (resistivity ≥18.2 MΩ cm) was used throughout the experiment. Labeled DNA oligonucleotides were ordered from Shenggong Bioengineering Ltd. (Shanghai, China), and their sequences are listed in Table 1. Synthesis of CdS:Eu NCs. The CdS:Eu NCs were prepared according to our previous work with some modification.8 Briefly, 112.5 μL of 0.08 M Eu(NO3)3 solution was added to 30 mL of aqueous solution containing Cd(NO3)2·4H2O (0.1683 g) under stirring and heated to 70 °C. Then a freshly prepared solution of Na2S·9H2O2 (0.7205 g) in 30 mL of ultrapure water was injected, and orange−yellow precipitates were obtained instantly. The reaction was held at 70 °C for 3 h with continuous refluxing. The final reaction precipitates were centrifuged and washed thoroughly with absolute ethanol three times, followed by washing with ultrapure water to get rid of any Eu3+ and other ions remaining outside the clusters. Then the resulting precipitate was ultrasonically dispersed into water for centrifugation to collect the upper yellow solution of CdS:Eu NCs. The final solution could be rather stable for 1 month when stored in a refrigerator at 4 °C. Preparation of CdS:Eu NCs Film. The GCE was polished in sequential order with 1.0, 0.3, and 0.05 μm alumina before the surface modification. Then the GCE was thoroughly rinsed with water; sonicated in ethanol and ultrapure water in turn; and finally, dried in air. The CdS:Eu NCs film was achieved by dropping 10 μL of the CdS:Eu NCs solution onto the pretreated surface of the GCE and evaporated in air at room temperature. Finally, the CdS:Eu NC-modified GCE was stored in 0.1 M NaCl + 0.1 M Tris−HCl buffer (pH 7.4) for characterization and further use. Synthesis and Modification of Au NRs. Au NRs were prepared in aqueous solutions using the silver ion-assisted seedmediated method.13,17 Specifically, the seed solution was first made by quickly injecting a freshly prepared ice-cold 0.01 M NaBH4 solution (0.6 mL) into an aqueous mixture consisting of 1% HAuCl4 (0.103 mL) and 0.1 M CTAB (10 mL), followed by vigorously stirring for 2 min. The resultant seed solution was kept at 30 °C for 2 h for the subsequent synthesis of Au NRs. The growth solution was prepared by reduction of 1% HAuCl4 (0.412 mL) in a solution containing 0.01 M AgNO3 and 0.1 M

Herein, we designed an ECL-RET system that combines the properties of a molecular beacon to sensitively and selectively detect DNA using CdS:Eu NCs as an ECL donor and Au nanoparticles as an ECL acceptor. In our study, a hairpin structure molecular beacon was used to improve selectivity and for efficient energy transfer between the NCs and Au NPs. Only after hybridization with complementary fragments did the hairpins open up and was the biotin forced away from the electrode surface and available for conjugation with the streptavidin−gold, resulting in an energy transfer. On the basis of the forementioned technique, we studied the spectrum-, distance-, and shape-dependent efficiency of ECLRET between the NCs and Au NPs. Finally, under the optimized conditions, this ECL-RET system was developed for ultrasensitive and specific detection of target DNA with the detection limit at the attomolar level. Therefore, this sensing ECL-RET platform may open up a new avenue for selective and ultrasensitive detection of DNA hybridization.

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EXPERIMENTAL SECTION Apparatus and Reagents. The electrochemical and ECL emission measurements were conducted on a MPI-A multifunctional electrochemical and chemiluminescent analytical system (Xi’An Remax Electronic Science & Technology Co. Ltd., Xi’An, China) at room temperature. The experiments were carried out with a conventional three-electrode system. The working electrode was a 3-mm-diameter glassy carbon electrode (GCE) modified with a CdS:Eu nanoscrytals film. Meanwhile, a Pt wire and SCE electrode served as the counter and reference electrodes, respectively. Transmission electron microscopy was performed with a JEOL model 2000 instrument operating at 200 kV accelerating voltage. The UV−vis absorption spectra were obtained on a Shimadzu UV3600 UV−vis−NIR photospectrometer (Shimadzu Co.). 6Mercapto-1-hexanol (MCH), cysteamine and streptavidin were obtained from Sigma-Aldrich (St. Louis, MO). Sodium sulfide (Na2S·9H2O), CTAB, NaBH4, and other routine chemicals were purchased from Nanjing Chemical Co. Ltd. Ascorbic acid, glutaraldehyde solution (25%), cadmium nitrate tetrahydrate (Cd(NO3)2·4H2O) and europium(III) oxide (Eu2O3) were supplied by Sinopharm Chemical Reagent Co. Ltd. Phosphate buffer solution (0.1 M; KH2PO4−K2HPO4−NaCl; PBS) containing 0.05 M K2S2O8 (pH 8.3) as a coreactant was used 17774

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Figure 1. (A) TEM picture of synthesized CdS:Eu NCs; (B) UV−vis spectrum (a) and ECL spectrum (b) of the CdS:Eu NCs; (C) ECL−potential curve and cyclic voltammogram (inset) of CdS:Eu NCs on the electrode; (D) ECL emission from GCE−CdS:Eu NC-modified GCE under a continuous cyclic potential scan for 10 cycles. ECL detection buffer: 0.1 M PBS (pH 8.3) containing 0.05 M S2O82−. Scan rate, 100 mV s−1.

Preparation of Au NRs−Streptavidin (Au NRs−SA) Bioconjugates. Briefly, 5 mL of amine-modified AuNRs solution (∼100 nM) was mixed with 1 mL of PBS (pH 7.4) containing 2% GA for about 1 h at room temperature. The resulting nanorods were then centrifuged at 7000 rpm for 10 min to remove the excess GA and resuspended in 5 mL PBS buffer (pH 7.4) containing 0.005 M CTAB. Then 100 μL of 1 mg mL−1 streptavidin was added to the mixture, which was incubated for 3 h at 37 °C. The functionalized nanorods were subsequently collected by centrifugation at 5000 rpm for 5 min, and the supernatant was removed. This step was repeated more than three times to remove free streptavidin. Finally, the obtained Au NRs−SA bioconjugates were redispersed in 1.0 mL of pH 7.4 PBS containing 3% BSA and stored at 4 °C. Preparation of Spherical Au Nanoparticles (SAu NPs) and SAu NPs−Streptavidin (SAu NPs−SA) Bioconjugates. SAu NPs were prepared according to the method reported previously with a minor modification.32 All glassware used in the preparation was thoroughly cleaned in aqua regia (3 parts concentrated HCl, 1 part concentrated HNO3), rinsed with distilled H2O, and oven-dried prior to use. HAuCl4 and trisodium citrate solutions were filtered through a 0.22-μm microporous membrane filter prior to use. Briefly, an aqueous solution of 0.01% HAuCl4 (100 mL) was boiled with vigorous stirring, and then an aqueous solution of 1% trisodium citrate (1.0 mL) was quickly added to the boiling solution. The color of the solution turned from gray yellow to deep red, indicating the formation of SAu NPs. The prepared SAu NPs were stored in brown glass bottles at 4 °C.

CTAB (20 mL). The length−diameter ratio of Au NRs was controlled by the amounts of added Ag+. To obtain Au NRs640, Au NRs-680, Au NRs-710, and Au NRs-750, 0.05, 0.10, 0.20, and 0.25 mL of AgNO3 was added, respectively. After gentle mixing of the solution, 0.16 mL of ascorbic acid solution (0.1 M) was added, and the obtained growth solution changed from dark yellow to colorless. Finally, the CTAB-stabilized seed solution (0.048 mL) was rapidly injected. The resultant solution was gently mixed for 10 s and left undisturbed 20 h in a 30 °C water bath to initiate growth to yield Au NRs. Excess CTAB was removed by centrifuging twice at 10 000 rpm, the supernatant was discarded, and the particles were redispersed in pure water. The amine-terminated Au NRs were obtained according to the methods reported previously with a slight modification.17,31 Briefly, 0.5 mL of 20 mM cysteamine was added into 5 mL of the Au NRs solution under vigorous stirring. The mixture was sonicated for 0.5 h at 50 °C, which was further heated in a 50 °C water bath for 3 h for the modification of both tips and sides of Au NRs. The resultant Au NRs were then collected by centrifugation twice at 7000 rpm for 15 min to remove excess cysteamine and desorbed CTAB. The purified amine-modified Au NRs were dispersed in a 0.005 M CTAB solution to yield a final concentration of 100 nM. The concentrations were estimated by the Lambert−Beer Law and the report of Murphy.13 The remaining amine groups of the Au NRs could be subsequently used to attach protein molecules (streptavidin) with the glutaraldehyde (GA) protocol to construct multifunctional nanorod bioprobes. 17775

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Figure 2. TEM picture of synthesized SAu NPs (A), SEM images of the Au NRs with four different aspect ratios (B, C, D and E, respectively), and the relative normalized plasmon absorption spectra of the sample (F).

contact with 0.1 M PBS (pH 8.3) containing 0.05 M K2S2O8 and scanned from 0 to −1.55 V. The voltage of the PMT was set at −500 V in the process of detection. ECL signals related to the target DNA concentrations could be measured. The data of three independent measurements are presented with an error margin of 1 standard deviation.

Streptavidin-coated SAu NPs were prepared according to a modified literature procedure.28 The conjugation process was carried out as follows: 100 μL of 1 mg mL−1 streptavidin was added to 5 mL of pH-adjusted (pH 6.4, adjusted by 0.1 M K2CO3) SAu NPs suspension, followed by incubation at room temperature for 30 min. The conjugate of SAu NPs−SA was centrifuged at 13 000 rpm for 30 min, and the red precipitates were dispersed with 0.01 M PBS solution containing 3% BSA and stored at 4 °C. Fabrication of ECL-RET Biosensor. The CdS:Eu NCmodified GCE was immersed in 60 μL of 0.1 M NaCl + 5 mM MgCl2 + 0.1 M Tris−HCl buffer (pH 7.4) containing 0.2 μM stem−loop structure probes and incubated overnight at 4 °C to immobilize the probe on the surface of the electrode. Subsequently, the electrode was rinsed with 0.1 M Tris−HCl buffer (pH 7.4) to remove the unbound probes on the surface of the electrode. Finally, the resulting thiol-capped molecularbeacon-modified electrode was immersed in 1 mM MCH for 2 h to remove the nonspecifically adsorbed DNA and force the probe DNA to adopt an upright surface orientation that favors DNA hybridization. The electrode surface was rinsed with 0.1 M NaCl−Tris−HCl buffer (pH 7.4) after each step to remove nonspecifically adsorbed species. ECL Measurements. The resulting electrode was immerged in 100 μL solution consisting of 0.1 M NaCl−Tris− HCl hybridization buffer and 5 mM MgCl2 (pH 7.4). A series of target DNA at different concentrations were then added to the mixture solution and the reaction was incubated at 37 °C for 60 min. Subsequently, the electrode was washed thoroughly with 0.1 M NaCl−Tris−HCl buffer (pH 7.4) to remove unhybridized oligonucleotide and then incubated with 60 μL Au NRs−SA bioconjugates at 37 °C for 40 min. The sensor was finally washed again with 0.1 M NaCl−Tris−HCl buffer (pH 7.4) three times to remove the unbound Au NRs−SA bioconjugates, followed by the measurement of ECL. ECL detection was accomplished with the electrodes in each step in

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RESULTS AND DISCUSSION Characterization of the CdS:Eu NCs and CdS:Eu NCs Film. Recently, we have found that the Eu3+-doped CdS nanocrystals (CdS:Eu NCs) showed stronger and more stable ECL signals compared with the pure CdS NCs due to the fact that Eu3+ ions could alter the surface of CdS NCs and create a new surface state−Eu3+ complex. We also found the maximum of Eu3+ ion−surface crystal lattice complex formation was at the 1.5% doping level, which means that the 1.5% Eu3+-doped CdS NCs exhibited the biggest ECL intensity.8 So the CdS:Eu NCs used here were synthesized by doping 1.5% Eu3+ ions into CdS NCs, and the average size of the prepared CdS:Eu NCs was about 6 ± 1 nm, as demonstrated by transmission electron microscopy (TEM, Figure 1A). The diameter of 6.18 nm was further calculated according to the UV−vis spectrum, which exhibited an absorption maximum of CdS:Eu NCs at ∼470 nm (Figure 1B, curve a), and the empirical equations,33 which was consistent with the TEM results. Figure 1C shows an ECL−potential curve and cyclic voltammogram (inset) of a CdS:Eu NCs film on the electrode. In our design, GCE was modified by drop-coating of 10 μL of CdS:Eu NCs used as ECL emitter. As the electrode potential became sufficiently negative, the CdS:Eu NC was reduced to CdS:Eu−•, and the coreactant S2O82− was reduced to SO4−•. Thus, a cathodic peak occurred in the cyclic voltammogram (inset of Figure 1C). SO4−• could react with CdS:Eu−• to obtain an excited state (CdS:Eu*). This state emitted light in the aqueous solution to produce an ECL signal (Figure 1C). The ECL emission spectrum of the CdS:Eu film showed two 17776

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Scheme 1. Schematic Representation of Proposed ECL-RET Platform

Figure 3. (A) Cyclic ECL on potential curves in various cases. (a) GCE−CdS:Eu NCs/MB; (b) GCE−CdS:Eu NCs/MB/Au NRs; (c) GCE− CdS:Eu NCs/MB/tDNA(1 × 10−11 M); (d) GCE−CdS:Eu NCs/MB/tDNA (1 × 10−14 M)/Au NRs; (e) GCE−CdS:Eu NCs/MB/tDNA (1 × 10−11 M)/Au NRs. ECL detection buffer: 0.1 M PBS (pH 8.3) containing 0.05 M S2O82−. Scan rate, 100 mV s−1. (B) EIS of the proposed ECL-RET platform: (a) bare GCE; (b) GCE−CdS:Eu NCs; (c) GCE−CdS:Eu NCs/MB; (d) GCE−CdS:Eu NCs/MB/tDNA(1 × 10−11 M); (e) GCE− CdS:Eu NCs/MB/tDNA (1 × 10−11 M)/Au NRs. EIS are measured in 0.1 M KCl containing 5 mM [Fe(CN)6]3−/[Fe(CN)6]4−.

increasing aspect ratio. Compared with organic dyes, Au NPs should be a better acceptor candidate in the resonance energy transfer (RET) system because of their tuned absorption bands, unique optical properties, better biocompatibility, and high extinction coefficient. Figure 2A shows the TEM images of SAu NPs. According to the TEM observation, the average size of the SAu NPs was about 20 ± 1 nm. The surface plasmon resonance (SPR) absorption peak of SAu NPs at about 520 nm was characterized by the UV−vis spectrum (Figure 2F, curve a). Au NRs with four different aspect ratios were synthesized by tuning the amounts of added Ag+, which were characterized by SEM (Figure 2B−E) and UV−vis spectrum (Figure 2F, curves b−e). The Au NRs with four different aspect ratios were named for their longitudinal peak emission wavelengths of Au NRs640, Au NRs-680, Au NRs-710, and Au NRs-750, respectively. Construction and Characterization of the ECL-RET Platform. As illustrated in Scheme 1, we employed a molecular beacon probe dually labeled with a thiol at its 5′ end and a biotin at its 3′ end, respectively. This probe was immobilized on a GCE modified with CdS:Eu NCs. In the absence of target DNA, the immobilized MB probe was in a “closed” state, which kept the streptavidin−Au NPs off the biotin due to the large steric effect. Upon contact with the target DNA, the hairpin structure MB was damaged, and the biotin group was

spectral bands (Figure 1B, curve b): one was from 450 to 550 nm, which belonged to the host CdS; the other was from 600 to 700 nm, which was due to the energy transfer from host CdS to Eu3+ ions.8 The ECL mechanism was listed as follows:34,35 CdS: Eu + e− → (CdS: Eu)−•

(1)

S2 O82 − + e− → SO4 2 − + SO4 −•

(2)

(CdS: Eu)−• + SO4 −• → (CdS: Eu)* + SO4 2 −

(3)

(CdS: Eu)* → CdS: Eu + hν

(4)

The ECL signal−time curve under continuous potential scanning for 10 cycles was shown in Figure 1D. The stable, high ECL signals suggested that the CdS:Eu NCs film is an excellent platform to construct ECL-based biosensors. Characterization of the Au NPs. Au nanoparticles exhibit strong plasmon bands, depending on their geometric shape and size. For example, spherical Au nanoparticles display a strong absorption band around 520 nm due to the excitation of plasmons by incident light and almost independent of their size. Au nanorods display two distinct absorption bands: one at a shorter wavelength (∼520 nm) and the other at a longer wavelength, which undergoes a bathochromic shift with 17777

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Figure 4. (A) ECL-RET efficiency (IE) in the presence of Au NRs-640, Au NRs-680, Au NRs-710, Au NRs-750, SAu NPs, and a blank, respectively. The concentration of the target DNA is 0.1 pM or 0.1 nM, and the MB with a 6-bp stem and a 12-nct loop is used as the probe DNA. (B) A comparison of ECL responses with different separation distances between the CdS:Eu NCs film and Au NRs-640 or SAu NPs. The concentration of the target DNA is 0.1 pM or 0.1 nM, and the ECL is measured in 0.1 M PBS (pH 8.3) containing 0.05 M S2O82−. The scan rate is 100 mV s−1.

“activated” by a force away from the surface of the electrode, which made it available for conjugating with the streptavidin− Au NPs. Then ECL-RET occurred by the conjugation between CdS:Eu NCs and Au NPs in the presence of the target DNA. ECL signals at each immobilization step were recorded to characterize the fabrication process of the ECL-RET platform. As shown in Figure 3A, the GCE−CdS:Eu NCs/MB showed a strong, stable cathodic ECL emission in the presence of coreactant S2O82− ions when the potential of electrodes becomes sufficiently negative (curve a). In the absence of target DNA, the immobilized stem−loop structure (with a 6-bp stem and a 12-nct loop) probe was in a “closed” state, and there was no significant change in ECL intensity due to the steric effect (curve b), which shielded biotin from being approached by the large-sized streptavidin−Au NRs bioconjugate. Upon contact with the target DNA but without streptavidin−Au NRs, there was also no significant change in the ECL intensity (curve c). The ECL peak height decreased by 41% and 84% (curves d and e) in the presence of the target DNA (1 × 10−14 M and 1 × 10 −11 M) and streptavidin−Au NRs. Combining that information with the lower resistance of Au NRs (Figure 3B, curve e), we deduced that the ECL emission of the NCs could be quenched by the proximal Au NRs via ECL resonance energy transfer, which was due to the fact that the Au NRs could efficiently absorb ECL emission from a CdS:Eu NCs film followed by nonradiative dissipation. The fabrication process of the ECL-RET platform was also confirmed by electrochemical impedance spectroscopy (EIS, Figure 3B). The impedance spectrum includes a semicircle portion and a linear portion. The semicircle diameter at higher frequencies corresponds to the electron−transfer resistance (Ret), and the linear part at lower frequencies corresponds to the diffusion process. It was observed that the EIS of the bare electrode displayed an almost straight line (curve a), which was characteristic of a diffusion process. When CdS:Eu NCs were assembled on the electrode surface, the Ret increased obviously (curve b), which indicated that the CdS:Eu NCs were immobilized on the electrode surface and decreased the electron-transfer efficiency. The addition of the molecular beacon layer resulted in a larger electron-transfer resistance (curve c), mainly due to the large stereospecific blockade of the stem−loop structure, and the electrostatic repulsion between negative charges of the DNA backbone and Fe(CN)6 3−/4−, then inhibiting the interfacial charge transfer. On hybridization

with the target sequence, a relatively large increased Ret is observed (curve d). It is expected that the stem of the MB is opened in the presence of target DNA and forms a duplex structure which becomes more dense and crowded, therefore impeding the electron transfer more than the stem−loop structure of MB. After further conjugating with the streptavidin−Au NRs, the Ret obviously decreased (curve e), which implied that the conductive Au NRs accelerated the electron transfer. These results were consistent with the fact that the electrode was fabricated as expected. Influencing Factors on the ECL-RET Efficiency. Obviously, the key to improving the detection sensitivity of the ECL-RET biosensor is to enhance the ECL-RET efficiency, which is highly dependent on the spectral overlap between the donor’s emission and the acceptor’s absorption, the distance between the donor and acceptor molecules, and the extinction coefficient of the acceptor.36,37 The ECL-RET efficiencies are estimated by the value of the quenching efficiency, defined as IE = 1 − I/I0, where I and I0 are the ECL intensities in the presence and absence of target DNA, respectively. Herein, we used the MB (with a 6-bp stem and a 12-nct loop) as the probe DNA, then in the presence of streptavidin−Au NRs (Au NRs640, Au NRs-680, Au NRs-710, and Au NRs-750) or streptavidin−SAu NPs, the ECL quenching efficiency was 57%, 48%, 37%, 33%, or 28% for 0.1 pM target DNA, respectively, and 90%, 79%, 65%, 60%, or 41% for 0.1 nM target DNA, respectively (Figure 4A). Obviously, the Au NRs640 was the better acceptor due to their high quenching efficiency. Such high quenching efficiency is attributed to the complete overlap between the ECL emission spectrum of the CdS:Eu film and the absorption bands of the Au NRs-640 (Figure 1B, curve b and Figure 2F, curve b), which indicates that the greater the spectrum overlap between donor and acceptor, the greater the energy transfer efficiency in the ECLRET system. Compared with Au NRs, SAu NPs showed lower energy transfer efficiency in ECL-RET system. On one hand, the only one absorption band of SAu NPs makes a smaller spectral overlap between the CdS:Eu NCs emission and SAu NPs absorption (Figure 1B, curve b and Figure 2F, curve a). On the other hand, Au NRs have a larger surface area and lower curvature, which can increase the quenching sites and improve the quenching efficiency; furthermore, the extinction coefficient of Au NRs is higher than that of SAu NPs. 17778

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Figure 5. (A) ECL signal responses for detection of different concentrations of target DNA. The concentrations of the target DNA: (a) 0, (b) 10 aM, (c) 100 aM, (d) 1 fM, (e) 10 fM, (f) 100 fM, (g) 1 pM, and (h) 10 pM. Inset: linear relationship between ECL-RET efficiency (IE) and the logarithm of target DNA concentration, three measurements for each point. (B) ECL-RET efficiency (IE) for 1 fM of different DNA sequences: (a) complementary sequence (target DNA), (b) single-base-mismatched sequence, (c) noncomplementary sequence, and (d) blank. Three measurements for each point.

Sensitivity, Specificity and Reproducibility of the Proposed ECL-RET Platform. On the basis of the obtained results, we used a CdS:Eu NCs film labeled with 12-nct loop MB and Au NRs-640 as the ECL-RET biosensor for target DNA detection. As shown in Figure 5A, the intensity of ECL was very sensitive to the change of the target DNA concentration and decreased with the increase in concentration of the target DNA, ranging from 10 aM to 10 pM, and the ECL quenching efficiency (IE) was found to be logarithmically related to the concentration of target DNA in the range from 10 aM to 10 pM (R = 0.997, shown as the inset in Figure 5A). The detectable concentration range of 6 orders of magnitude was relatively wide, and the detection limit was experimentally found to be 10 aM, which was much lower than 0.19 fM for ECL detection of DNA based on the luminol-functionalized gold nanoparticles39 and was comparable to those obtained from an enzyme-based electrochemical DNA sensor.40,41 This high sensitivity reflected the highly effective ECL-RET between the CdS:Eu NCs film and Au NRs. In this ECL-RET biosensor, the selectivity was investigated with the same concentration of complete complementary target DNA sequences (T1), the one-base-mismatched DNA sequences (T2), the noncDNA sequences (T3), and a blank (without DNA), respectively. As shown in Figure 5B, in the presence of T1, the ECL quenching efficiency (IE) showed a significant increase compared with that of the blank test (a). This is attributed to the hybridization between the T1 and the linker strand, leading to efficient ECL-RET between the CdS:Eu NCs film and Au NRs-640. Although the IE increase was observed by adding 1.0 fM T2 (b), it was not comparable with that caused by the T1 (a), and the T3 showed little response (c). These results indicate that the developed approach exhibits an excellent specificity to distinguish the single-base mismatched DNA, which benefits from using the stem−loop DNA probe as the sensing component. The reproducibility of the proposed ECL-RET biosensor was investigated with interassay. The interassay variation coefficient was 8.53%, which was evaluated from the response to 1.0 fM target DNA at eight different electrodes independently. Thus, this fact demonstrated the proposed ECL-RET biosensor possessed acceptable reproducibility.

The distance between energy donor and acceptor is also important for the efficiency of energy transfer. By varying the DNA lengths, the separation distance between CdS:Eu NCs film and Au NPs can be systematically varied. Herein, the MBs with a 6-bp stem and a 12-nct, 30-nct, and 45-nct loop were used to investigate the distance dependence of the ECL-RET efficiency. In the presence of target DNA and streptavidin−Au NRs-640 or streptavidin−SAu NPs, the distances between the CdS:Eu NCs film and gold were about 8, 14, and 19 nm for the structure-damaged MBs with 12-nct, 30-nct, and 45-nct loops, respectively. For Au NRs-640, the ECL quenching efficiency was 57%, 44%, and 30%, respectively, or 90%, 67%, and 55%, respectively, in the presence of 0.1 pM or 0.1 nM target DNA, which means that the higher quenching efficiency was obtained with the separation distance being about 8 nm (Figure 4B). This is presumably due to the energy transfer efficiency inversely linked to separation distance of the donor and acceptor.38 For SAu NPs, on the other hand, an ECL enhancement with different extent was observed because the CdS:Eu NCs film− SAu NP separation was about 19 nm (1.4- and 1.7-fold enhancement) or 14 nm (1.2- and 1.4-fold enhancement) in the presence of 0.1 pM and 0.1 nM target DNA, respectively (Figure 4B). The reason for the ECL enhancement is that the ECL emission from the CdS:Eu NCs film could induce surface plasmon resonances (SPR) of SAu NPs, and the induced SPR in turn enhances the ECL response of CdS:Eu NCs film at a large separation, which was consistent with our previous work.6 The ECL enhancement efficiency between the CdS:Eu NCs film and SAu NPs was much lower than that between the CdS NCs film and SAu NPs and similar to that between the CdS:Mn NCs film and SAu NPs reported in our previous work.10 The reason is that both CdS:Eu and CdS:Mn NCs act as diluted magnetic semiconductors, and the extra magnetic field of the doping of magnetic Eu3+ and Mn2+ ions in the CdS NCs has negative influence on the ECL excited SPR in SAu NPs.11 As for Au NRs-640, there was no ECL enhancement at large separation because of the disorder assembly of Au NRs640, which makes some parts of the Au NRs close to the CdS:Eu NCs film, and combined with the high extinction coefficient of the Au NRs, the absorption dominates over scattering, resulting in the observed quenching. 17779

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CONCLUSION We have demonstrated an efficient ECL-RET system between CdS:Eu NCs film and Au NRs for highly sensitive and excellently specific detection of the target DNA. The CdS:Eu NCs film on the electrode showed a strong and stable cathodic ECL emission in the presence of coreactant S2O82− ions, which was suitable for application in the ECL detection. The absorption spectrum of Au NRs with high extinction coefficient could be easily tuned to match well with the ECL emission spectrum of the CdS:Eu NCs film by adjusting the aspect ratio of the nanorods to get the highly effective ECL-RET. Under the optimized conditions, this ECL-RET system was developed for ultrasensitive and specific detection of target DNA with a detection limit at the attomolar level. Moreover, this study provides a pathway toward the construction of a sensitive ECLRET assay for homogeneous DNA detection.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the 973 Program (2012CB932600), the National Natural Science Foundation (Nos. 21025522 and 21135003), and the National Natural Science Funds for Creative Research Groups (21121091) of China.

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