Novel Magnetic Fe3O4@CdSe Composite Quantum Dot-Based

Feb 9, 2012 - ... Jacinete L. Santos , Rodrigo V. Rodrigues , Luelc S. Costa , Diego Muraca , Kleber R. Pirota , Maria C. F. C. Felinto , Oscar L. Mal...
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Novel Magnetic Fe3O4@CdSe Composite Quantum Dot-Based Electrochemiluminescence Detection of Thrombin by a Multiple DNA Cycle Amplification Strategy Guifen Jie* and Jinxin Yuan State Key Laboratory Base of Eco-chemical Engineering, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China S Supporting Information *

ABSTRACT: A novel small magnetic electrochemiluminescent Fe3O4@CdSe composite quantum dot (QD) was facilely prepared and successfully applied to sensitive electrochemiluminescence (ECL) detection of thrombin by a multiple DNA cycle amplification strategy for the first time. The as-prepared composite QDs feature intense ECL, excellent magnetism, strong fluorescence, and favorable biocompatibility, which offers promising advantages for ECL biosensing. ECL of the composite QDs was efficiently quenched by gold nanoparticles (NPs). Taking advantages of the unique and attractive ECL and magnetic characteristics of the composite QDs, a novel DNA-amplified detection method based on ECL quenching was thus developed for a sensitive assay of thrombin. More importantly, the DNA devices by cleavage reaction were cycled multiple rounds, which greatly amplified the ECL signal and much improve the detection sensitivity. This flexible biosensing system exhibits not only high sensitivity and specificity but also excellent performance in real human serum assay. The present work opens a promising approach to develop magnetic quantum dot-based amplified ECL bioassays, which has wider potential application with more favorable analytical performances than other ECL reagent-based systems. Moreover, the composite QDs are suitable for long-term fluorescent cellular imaging, which also highlights the promising directions for further development of QD-based in vitro and in vivo imaging materials.

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resonance imaging, drug delivery, and cell separation.17,18 By combining magnetic nanocrystals and QDs together, magnetic quantum dots are possible to simultaneously show magnetic and optical properties and, therefore, have shown a promising future or revealed some novel applications in biomedical fields.19,20 Some researchers presented seeded growth methods for synthesizing magnetic fluorescent nanoarchitectures.21,22 However, so far, the resultant nanocomposite particles were usually larger (70−200 nm), which was rarely used as an optical probe for bioassays, and the used reagents such as polyelectrolytes were expensive.23,24 Therefore, the new small magnetic fluorescent QDs are highly desired in bioassays and biolabeling applications, they can not only be rapidly and conveniently separated in a biolabeling process but also be easily immobilized on the magnetic electrode, showing promising applications in bioassays. In our previous work, we fabricated a magnetic nanocomposite using QDs and polyelectrolyte for the ECL immunoassay,25 but the nanocomposite was very large (300−350 nm); no any amplification strategy was employed in the immunoassay. So far, the small

lectrochemiluminescence (ECL) has received considerable attention during the past several decades owing to its versatility, simplified optical setup, and good control. ECL detection offers inexpensive instrumentation, low background noise, high detection sensitivity, and a wide dynamic range.1−3 Recently, quantum dot (QD) ECL has attracted considerable interest in analysis.4,5 Compared with the conventional ECLemitting species such as luminol or Ru(bpy)32+, QD ECL possesses many advantages, such as neutral detection conditions and easy realizability in both cathodic and anodic processes with diverse coreactants, leading to multifarious biosensing strategies.6−8 The QD-based ECL analytical technique has been quickly developed in many fields,9,10 and the ECL biosensors were gradually reported.11−13 However, so far, there has been rare reports on applying QD ECL to DNA or cell assays.14−16 The main reason may be that the ECL signals of QDs by biolabeling are usually not high and that the separation of QDs from biomolecules is complicated, which limits the wide application of QD ECL in bioassays. Thus, it is significant to develop novel multifunctional QDs with highly intense ECL and excellent magnetic property, which has more promising applications in ECL biosensing. Magnetic nanocrystals exhibit superparamagnetism and have been widely used in biomedical fields, such as magnetic © 2012 American Chemical Society

Received: December 9, 2011 Accepted: February 9, 2012 Published: February 9, 2012 2811

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magnetic electrochemiluminescent composite QDs with the particle size smaller than 20 nm and their amplified ECL bioassay applications have not been reported. Lately, various DNA-related processes were devised, and many of them were utilized in bioanalysis for signal amplification.26,27 DNA cycle amplification devices have now become powerful tools due to the specificity of molecular recognition capability and their robust physicochemical properties. In the DNA devices, movements were powered by DNAzymes, endonucleases, polymerases, and fuel oligonucleotides.28−30 DNA amplification devices were applied to ECL bioassy.31 Nevertheless, DNA was labeled by the pure QDs, which limited the ECL signal and stability; thus, graphene oxide (GO) has to be used to capture the labeled QDs; the complex procedures for QD ECL measurement would inevitably challenge the resultant stability and reproducebility. In addition, QD ECL quenching and enhancing by gold nanoparticles (NPs) have also found promising applications in biological studies.32,33 Wang et al. reported greatly enhanced ECL of CdS thin films by gold NPs for ultrasensitive detection of thrombin.32 In Zhou’s report, a dual-quenched ECL of CdS nanocrystals (NCs) film is achieved due to energy transfer from CdS NCs to both gold NPs and CdTe NPs, which makes the sensor exhibit relatively low background.33 However, the pure CdS NCs film as the ECL emitter in their works have some drawbacks, such as complicated purification procedures by ultracentrifugation and bad stability in ECL signal. The magnetic composite QDs have more advantages than the pure CdS NCs, they facilitate all the procedures such as more convenient and stable QD immobilization, rapid biological conjugation, separation and sensing target, etc., which is a significant advance in the application of ECL bioassays. Thrombin plays an essential role in some physiological and pathological processes, such as blood solidification, wound cicatrization, and inflammation. Therefore, specific recognition and quantitative detection of thrombin is extremely crucial in fundamental research as well as in clinical practice.34 Several lines of work reported ECL detection of thrombin using thrombin as binding agent of ECL emitter.35 Yet, in these reports, the ruthenium complex was usually employed whereas QDs were seldom exploited as ECL emitter to date.32,36 Enlighted by the advances of DNA amplification technique, coupled with the unique ECL and magnetic characteristics of the novel small composite QDs, we have developed a new amplified ECL strategy for sensitive thrombin assay based on ECL quenching of composite QDs by gold NPs. These new composite QDs display intense ECL, excellent magnetism, and strong fluorescence, which offers promising advantages for ECL biosensing as well as bioimaging. Benefiting from the magnetic property of the composite QDs, this approach can rapidly and conveniently be performed all the procedures such as QDs immobilization, biologic conjugation, separation, and sensing target. More specifically, this novel strategy is based on QD ECL quenching, allowing the QD film to be conveniently formed on the electrode with high ECL efficiency and stability, which avoided the shortcomings such as low ECL signal as well as complexity in biolabeling of QDs. Thus, this flexible ECL biosensing system exhibits not only high sensitivity and specificity but also excellent performance in real human serum assay. Furthermore, the present work opens a new approach to develop amplified ECL biosensing based on magnetic QDs, which has greater potential in bioassay applications.

Article

EXPERIMENTAL SECTION

Synthesis of Fe3O4@CdSe Composite QDs. A previously published procedure with slight modifications was used.37,38 Typically, ferric acetylacetonate (0.7063 g), 1,2-dodecanediol (2.023 g), octadecylamine (1.617 g), dibenzyl ether (20 mL), and oleic acid (2 mL) were placed in a 250 mL three-necked round-bottomed flask. The mixture was heated to 200 °C under N2 flow and kept at that temperature for 30 min. After the N2 gas was stopped, the mixture was further heated to 265 °C and proceeded for 30 min; then, the heating mantle was removed, and the mixture was cooled to room temperature. After ethanol was added to the reaction flask, the black precipitate was obtained. The final reaction precipitates were centrifugated and washed thoroughly with toluene and purified with ethanol. Finally, the Fe3O4 particles were isolated by centrifugation and dried in air. CdO (0.0460 g) and stearic acid (0.2940 g) were placed in a 250 mL round flask; the mixture was heated to 200 °C under N2 flow, and then, the clear reaction solution was cooled to room temperature. Fe3O4 nanoparticles (0.0165 g), trioctylphosphine oxide (TOPO, 2 g), and 1-octadecene (ODE, 2 g) were added to the above round flask; then, the mixture was heated to 280 °C and kept at that temperature for 10 min. After Se (0.16 g) was placed in a 5 mL beaker, trioctylphosphine (TOP, 3 mL) was added to the beaker to make Se resolve under ultrasonic irradiation. The resulting Se/TOP solution was quickly added to the above round flask, and the flask was heated for 5 min; then, the heating mantle was removed. After the reaction solution was cooled to room temperature, a certain amount of methanol was added to the solution to obtain the precipitate. Then, the precipitate was centrifugated and washed with toluene and methanol, and the resulting product was collected by a magnet and redispersed in toluene three times. Finally, the collected product was dispersed in toluene. Phase Transfer of Fe3O4@CdSe Composite QDs from the Organic to the Aqueous Phase. According to the literature,38 Fe3O4@CdSe composite QDs (∼50 mg) with desired sizes were dissolved in toluene (1 mL), and 2,3dimercaptosuccinic acid (DMSA) molecules (50 mg) were dissolved in dimethyl sulfoxide (DMSO, 1 mL). Then, the composite QDs and DMSA solutions were mixed. After isolation of black powders through centrifugation, the resulting nanocrystals were redispersed in 1 mL of water. ECL Detection of Thrombin by DNA Cycle Amplification Strategy. Thrombin aptamer and its complementary oligonucleotides 1 (s1) were previously hybridized in equal proportions (10 μL, 1.0 × 10−5 M) and incubated at 25 °C for 1 h. Then, 10 μL of this solution was added to 50 μL of thrombin solution at different concentrations and incubated at 37 °C for 2 h. Single-stranded target DNA s1 was released from the aptamer/DNA hybrids in the process. To form MB1-a1 capture probe, 20 μL of MB1 was washed three times with PBS buffer solution (pH 7.4); then, 20 μL of EDC (0.15 mol/L) was added and incubated for 30 min. At the same time, 40 μL of imidazol-HCl buffer (0.1 M) was added to 20 μL of DNA (a1, 10−5 mol/L) and incubated at 25 °C for 30 min. Then, these solutions were mixed and incubated at 25 °C for 12 h. MB2-hairpin DNA1 was conjugated as follows. Firstly, 20 μL of MB2 was washed three times with PBS buffer solution (pH 7.4); then, 20 μL of EDC (0.15 mol/L) was added and incubated for 30 min. At the same time, 40 μL of imidazol-HCl 2812

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buffer (0.1 M) was added to 20 μL of hairpin DNA (10−5 mol/ L) and incubated at 25 °C for 30 min. Then, these solutions were mixed and incubated at 25 °C for 12 h. The solutions of S1, MB1-a1, and MB2-hairpin DNA mentioned above were mixed, and then, two kinds of endonuclease were added according to the instruction. The mixture was incubated at 37 °C for 2 h. To form the quencher probe gold NPs-hairpin DNA2, 1 mL of gold colloid was first centrifuged at 10 000 rpm for 10 min, following removal of the supernatant; the precipitate was then redispersed in 100 μL of doubly distilled water. 50 μL of 1.0 × 10−5 M thiol-modified hairpin DNA2 was added to 100 μL of the purified gold colloid. After shaking gently for 24 h at room temperature, the gold NPs-hairpin DNA2 conjugates were “aged” in the solution (0.3 M NaCl, 10 mM Tris-acetate, pH 8.2) for another 48 h. To remove excess thiol-DNA, the solution was centrifuged at 15 000 rpm for 30 min. The oily precipitate was then washed with 10 mM phosphate buffer two times, recentrifuged, redispersed in 0.3 M PBS, and stored in a refrigerator (4 °C). For ECL detection, the magnetic Au electrodes were polished with alumina slurries (1, 0.3, 0.05 μm), sonicated in deionized water, and dried with nitrogen gas. After 7 μL of the magnetic QDs solution was dropped to the electrode surface and dried naturally at the room temperature, 20 μL of gold NPs-hairpin DNA2 solution was dropped to the electrode surface and reacted at 37 °C for 12 h. Unconjugated gold NPshairpin DNA2 was removed by washing the electrodes with doubly distilled water. Then, the electrode was immersed in the above mixture (S1, MB1-a1, MB2-hairpin DNA together with the two kinds of endonuclease), and the next endonuclease (Nb.) was added according to the instruction, which was incubated at 37 °C for 2 h, and then washed twice for ECL measurement. The ECL emission was detected with a model MPI-A electrochemiluminescence analyzer using a three-electrode system at room temperature. The electrodes were a modified magnetic Au disk working electrode (4 mm diameter), a saturated calomel reference electrode, and a Pt counter electrode. The modified electrodes above were in contact with 0.1 M PBS (pH 7.4) containing 0.05 M K2S2O8 and 0.1 M KCl and scanned from 0 to −1.5 V. The spectral width of the photomultiplier tube (PMT) was 200−800 nm, and the voltage of the PMT was −800 V in the detection process. ECL signals related to the thrombin concentrations were measured. Fluorescent Microscopy Imaging. After the composite QDs were activated by EDC (0.1 M) and NHS (0.025 M) for 30 min, the aptamer with amine groups was added to the composite QDs, and the mixture was incubated at 37 °C for 12 h with gentle shaking and then magnetically separated, washed twice, and resuspended in buffer. Subsequently, for cell imaging, the QDs/aptamer was incubated with 5000 cells/ 200 μL of PBS at room temperature for 50 min and then magnetically separated, washed twice, and resuspended in buffer. For confocal microscopy imaging, a 10 μL sample solution was deposited onto a microscope slide and covered with a standard microscope slide. The composite QDs and cancer cells were observed with HCX PL APO 40/0.85 objective; a 488 nm laser beam (Argon ion laser) was employed in this experiment.

Article

RESULTS AND DISCUSSION Preparation and Characterization of the Magnetic Electrochemiluminescent Fe 3 O 4 @CdSe Composite Quantum Dots. The small magnetic Fe3O4@CdSe composite QDs were prepared via two steps. The central core was Fe3O4 nanoparticle; it was first prepared through high temperature thermal decomposition of acetylacetonate in the presence of dibenzyl ether, oleic acid, octadecylamine, and 1,2-dodecanediol. Figure 1A shows the transmission electron microscopy

Figure 1. Representative TEM images of (A) Fe3O4 nanoparticles and (B) Fe3O4@CdSe composite quantum dots.

(TEM) image of the Fe3O4 nanoparticle; the average diameter was about 6−7 nm. Then, the CdSe shell was formed using TOPO, ODE, and Se. The plausible formation mechanism may be as follows.39 One is that Fe3O4/CdSe heterostructures formed with the process of heterogeneous nucleation in the initial stage. In the latter case, the Fe3O4 and CdSe nanoparticles seemed to form ‘‘bilayer structure’’ due to hydrophobic·hydrophobic interactions. As shown in Figure 1B, the as-prepared Fe3O4@CdSe nanoparticle has an average diameter of about 9−10 nm, and the composite QDs exhibit unique magnetism and high quality optical properties. Figure 2 shows the magnetic properties of the Fe3O4@CdSe QDs and Fe3O4 NPs (inset) measured at the temperature of

Figure 2. Magnetic hysteresis loops of the Fe3O4@CdSe composite QDs and Fe3O4 NPs (inset) measured at 300 K.

300 K. It can be seen that both the composite QDs and Fe3O4 NPs show ferromagnetic behavior. The saturation magnetization (Ms) values of Fe3O4 NPs and Fe3O4@CdSe QDs are about 51.1 and 1.29 emu/g, respectively, which are sufficient to allow them to be separated easily from the solution with the help of external magnet force. The decrease in magnetic 2813

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saturation for Fe3O4@CdSe QDs can be explained by taking into account the diamagnetic contribution of the thick CdSe layer surrounding the magnetic Fe3O4 cores.40 Figure 3 presents the photoluminescence (PL) spectra of the composite Fe3O4@CdSe (curve a) and the pure CdSe QDs

also observed (B), confirming the promising fluorescence imaging ability of the composite QDs in targeting cells assays. ECL of the Fe3O4@CdSe Composite Quantum Dots. Figure 4 shows an ECL−potential curve and cyclic voltammo-

Figure 3. Photoluminescence (PL) of (a) Fe3O4@CdSe composite QDs and (b) CdSe QDs.

Figure 4. ECL−potential curve and cyclic voltammogram (inset) of the Fe3O4@CdSe composite quantum dots on the electrode.

(curve b). Both the QDs were synthesized under the same conditions, including [Cd]/[Se] molar ratio and reaction time. The PL emission peak of the composite QDs was at 611 nm, and the PL intensity was very high. By comparison, there is no obvious difference in PL intensity between the Fe3O4@CdSe and pure CdSe QDs, but the peak position of the Fe3O4@CdSe is slightly blue-shifted compared to CdSe QDs. According the classical nucleation theory,41 the nucleation activation energy for heterogeneous nucleation is lower than that for homogeneous nucleation. The participation of the Fe3O4 seed would increase the nucleation rate, and the growth rate for CdSe domains in Fe3O4@CdSe would be decelerated; thus, a blue-shift on PL spectra was observed. In addition, the PL quantum yields (QY) of the Fe3O4@ CdSe QDs was estimated using Rohodamine 6G as a PL reference by following equation:42

gram (CV, inset) of the Fe3O4@CdSe composite quantum dots on the electrode. There is one cathodic peak at −0.95 V in the CV response, corresponding to the reduction of S2O82−. One ECL peak was observed at −1.376 V in the ECL−potential curve, resulting from the reaction of the composite QDs with S2O82−. QDs immobilized on the electrode were reduced to nanocrystalline species (QD−·), while the coreactant S2O82− was reduced to strong oxidant SO4−·. Then, SO4−· reacted with QD−· to form the excited state (QD*) that emits light. The possible ECL mechanisms are as follows:44

QYx = QYr(A r L x n x 2 /Ax L rnr 2)

QD + e− → QD−·

(1)

S2O82 − + e− → SO4 2 − + SO4−·

(2)

QD−· + SO4−· → QD* + SO4 2 −

(3)

QD* → QD + hν

(4)

The ECL signal from the composite QDs was very high, suggesting that the composite QDs exhibit excellent ECL properties and have promising potential in ECL biosensing. Recently, the QD-based ECL energy transfer systems were reported and applied to bioassays.32 Herein, ECL of the Fe3O4@CdSe composite QDs was efficiently quenched by gold NPs, which was used to the thrombin detection by multiple DNA cycle amplification strategy. Mechanism for Thrombin Detection by Multiple DNA Cycle Amplification Strategy Based on ECL Quenching of Fe3O4@CdSe QDs. A novel strategy for thrombin assay using multiple DNA cycle devices for amplifying signal based on ECL quenching of the magnetic composite QDs was successfully designed. Scheme 1 shows the fabrication principle of the strategy. The DNA duplex contained an aptamer and its complementary oligonucleotides (s1). In the presence of the target thrombin, the double-stranded aptamer/DNA (s1) dehybridized by the recognition of thrombin to the aptamer; then, the DNA (s1) detached from aptamer. In addition, DNA a1* previously hybridized with DNA a1 on MB-1, when 18-

Here, QYx and QYr are the quantum yields of QDs sample and Rohodamine 6G, respectively. The quantum efficiency of Rohodamine 6G in ethanol is 95% from the literature. Ax and Ar are the optical densities at excitation wavelength (around 0.05). nx and nr are the refractive index of solvents: nethanol is 1.359 and nwater is 1.333 at room temperature. Lx and Lr are PL integrate intensities of the composite QDs and Rohodamine 6G excited at 488 nm. The PL QY of the Fe3O4@CdSe QDs was thus estimated to be 20.6%, which is higher than the reported magnetic luminescent nanocomposites (3.2%, 13− 18%,),43 indicating that the Fe3O4@CdSe QDs possess high quality luminescent properties and have promising optical applications. The fluorescence imaging of the Fe3O4@CdSe composite QDs for the application of cell assays was investigated. As shown in Figure S1, Supporting Information, the Fe3O4@CdSe composite QDs exhibited highly intense fluorescence (A). When the Ramos cells were labeled with the composite QDs via cell aptamers, obvious fluorescent imaging on the cell was 2814

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Scheme 1. Schematic Representation for the Strategy of Thrombin Detection by a Multiple DNA Cycle Amplification Strategy Based on ECL Quenching of the Magnetic Fe3O4@CdSe Composite QDs

technique was obtained, which was directly detected on the magnetic electrode. ECL Detection of Thrombin by Multiple DNA Cycle Amplification Strategy. The strategy feasibility for thrombin detection based on ECL quenching of the composite QDs was investigated. As shown in Figure 5, very low background ECL signal was observed on the bare electrode (curve a), while the Fe3O4@CdSe composite QD modified electrode displayed high ECL signal (curve b). When the gold NPs as ECL quencher were close to the composite QDs on electrode, the ECL signal obviously decreased (curve c), indicating that the gold NPs efficiently quenched the QD ECL. In the presence of target thrombin together with the mixture containing both DNA probe and the endonuclease, the ECL signal was obviously enhanced (curve d), indicating that thrombin was detected by the amplification strategy based on ECL quenching of the composite QDs. Figure 6A shows the ECL signal responses obtained upon different concentrations of target thrombin. In the absence of thrombin, the ECL signal was quite low (curve a), indicating that little unspecific binding occurs. However, in the presence of target thrombin by amplification strategy, the ECL peak signal gradually increased with increasing thrombin concentrations (curve b to j), indicating that the thrombin concentration was quantitatively measured by the ECL signal.

base DNA s1 enters into the system, s1 binds to a1 and displaces a1* from MB-1 owing to the competition hybridization. Subsequently, the formation of double-stranded (s1/ a1) recognition region of Nt. AlwI will lead to scission of strand a1. Target s1 and the fragment t1 spontaneously dissociate from MB-1 because of instability of hybridization structure. Then, single-stranded DNA s1 can hybridize to other strands a1 to continue the strand-scission self-cycle (the first cycle working mode), which will result in the massive release of a1* and t1 from MB-1. t1 can then trigger a series of the abovementioned reactions in MB-2 under the effect of Nb. BbvCI and release a number of s1 fragments, which can then be recycled to the headstream. Meanwhile, the gold NPs as the ECL quencher were linked to the thiol-modified hairpin DNA2 by Au−S bond. After the Fe3O4@CdSe composite QDs were immobilized on the magnetic Au electrode and used as the ECL emitters, the gold NPs-hairpin DNA2 as the quenching probe was conjugated to the composite QDs through covalent interactions between the carboxyl groups of QDs and amino groups of DNA2. In the presence of a1*, a series of the scission reactions of the hairpin strand were then triggered under the effect of Nb. BbvCI and released a number of DNA fragments with quencher. Because the above DNA devices were cycled for multiple rounds, hundreds of DNA fragments with quencher were released. After the quencher was washed off the electrode, much enhanced ECL signal by the multiple amplification 2815

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Figure 7. Selectivity of the strategy for thrombin detection by comparing it with different interfering proteins. The concentration of all interfering proteins was 0.1 nM, and thrombin concentration was 0.01 nM.

Figure 5. ECL−time curves for (a) bare electrode, (b) the Fe3O4@ CdSe composite QD modified electrode, (c) ECL quenching by gold NPs, (d) in the presence of target thrombin after ECL quenching by gold NPs, 0.1 M PBS (pH 7.4) containing 0.1 M KCl and 0.05 M K2S2O8.

investigated by detecting thrombin in a human real serum sample. As no coagulation factors were contained in healthy human serum sample, no thrombin in the serum sample was detected. Thus, we spiked thrombin into 10-fold-diluted serum samples to examine the applicability of this ECL method in real serum sample. The serum samples were spiked with 5.0, 10.0, 100.0, and 50.0 pM and then measured by our ECL method. The recoveries were obtained by comparing the measured amounts to that of added human thrombin, which varied from 90.0 to 104.0% (Table S2, Supporting Information). These results suggest that the ECL method may be competent for monitoring thrombin samples.

Figure 6B displays the relationship between ΔI and thrombin concentrations (1.0 pM to 100 nM). The ΔI was found to be logarithmically related to the thrombin concentrations in the range from 1.0 pM to 5.0 nM (R = 0.994) with a detection limit of 0.12 pM at 3σ. (inset in Figure 6B), which is comparable to the previously reported detection of thrombin.45 A series of five duplicate measurements of 0.1 nM were used for estimating the precision, and the relative standard deviation (RSD) was 5.1%, showing good reproducibility of the ECL method. Specificity of the ECL-Based Strategy for Thrombin Detection. To assess the specificity of the ECL-based strategy for thrombin detection, the influences of some other proteins such as bovine serum albumin (BSA), IgG, and lysozyme were examined under the same experimental conditions. Figure 7 shows that none of these proteins caused obvious ECL alteration even with a concentration as high as 0.1 nM, while only 0.01 nM of thrombin resulted in significant ECL enhancement, indicating those proteins did not interfere with the thrombin assay. The results demonstrated that the proposed amplification strategy exhibited good specificity for thrombin assay. Real Sample Analysis of Thrombin. The practical applicability of the proposed ECL-based strategy was

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CONCLUSIONS In summary, we have prepared a novel small multifunctional composite QD with intense ECL, excellent magnetism, and strong fluorescence and developed a DNA-amplified method for sensitive thrombin assay based on ECL quenching of the composite QDs by gold NPs. Several advantages of our method have been demonstrated: (1) The unique magnetic property of the composite QDs facilitates all the procedures such as convenient QD immobilization, rapid biological separation, and sensing target, etc., which allows rapid thrombin detection. (2) This novel strategy is based on QD ECL quenching, allowing QD film to be conveniently formed on the electrode with high

Figure 6. (A) ECL signal responses for detection of different concentrations of thrombin. The concentrations of thrombin: (a) 0 nM; (b) 0.001 nM; (c) 0.01 nM; (d) 0.05 nM; (e) 0.1 nM; (f) 1.0 nM; (g) 5.0 nM; (h) 10.0 nM. (B) Relationship between ΔI and thrombin concentration, three measurements for each point. Inset: The logarithmic calibration curve for thrombin detection. 2816

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Analytical Chemistry

Article

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ECL efficiency and stability, which enables the method to have high sensitivity and good reproducibility. (3) The DNA devices were cycled multiple rounds by cleavage reaction, which greatly amplified the ECL signal and improved the detection sensitivity. (4) This strategy provides a new and invaluable flexible ECL biosensing system which exhibits not only high sensitivity and specificity but also excellent performance in real human serum assay. (5) The present work opens a promising new approach to magnetic QD-based amplified ECL bioassays, which has extensive potential in analytical applications. (6) The composite QDs are suitable for long-term fluorescent cellular imaging, which also highlights the promising direction for development of QD-based imaging nanomaterials.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-532-84022750. Fax: +86-532-84022750. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 21175078), the Natural Science Foundation of Shandong province (No. ZR2010BZ003), and the State Key Laboratory of Analytical Chemistry for Life Science SKLACLS11**(SKLACLS1109). This work was also supported by the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT).

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REFERENCES

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dx.doi.org/10.1021/ac203261x | Anal. Chem. 2012, 84, 2811−2817