Technical Note pubs.acs.org/ac
Using Graphene-Based Plasmonic Nanocomposites to Quench Energy from Quantum Dots for Signal-On Photoelectrochemical Aptasensing Xianxiang Zeng, Shishi Ma, Jianchun Bao, Wenwen Tu,* and Zhihui Dai* Jiangsu Key Laboratory of Biofunctional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, P. R. China S Supporting Information *
ABSTRACT: On the basis of the absorption and emission spectra overlap, an enhanced resonance energy transfer caused by excition-plasmon resonance between reduced graphene oxide (RGO)-Au nanoparticles (AuNPs) and CdTe quantum dots (QDs) was obtained. With the synergy of AuNPs and RGO as a planelike energy acceptor, it resulted in the enhancement of energy transfer between excited CdTe QDs and RGO-AuNPs nanocomposites. Upon the novel sandwichlike structure formed via DNA hybridization, the exciton produced in CdTe QDs was annihilated. A damped photocurrent was obtained, which was acted as the background signal for the development of a universal photoelectrochemical (PEC) platform. With the use of carcinoembryonic antigen (CEA) as a model which bonded to its specific aptamer and destroyed the sandwichlike structure, the energy transfer efficiency was lowered, leading to PEC response augment. Thus a signal-on PEC aptasensor was constructed. Under 470 nm irradiation at −0.05 V, the PEC aptasensor for CEA determination exhibited a linear range from 0.001 to 2.0 ng mL−1 with a detection limit of 0.47 pg mL−1 at a signal-to-noise ratio of 3 and was satisfactory for clinical sample detection. Since different aptamers can specifically bind to different target molecules, the designed strategy has an expansive application for the construction of versatile PEC platforms.
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which is different from the mechanism of photoelectrochemisty. It detects the photocurrent signal. As a newly emerged but dynamically developing analysis technique, PEC determination has attracted substantial attention.14 It avoids the expensive equipment of optical techniques and high overpotential in electrochemistry or ECL measurements. By using light as the external stimulus at an appropriate wavelength, a selective PEC reaction can be achieved.8 Aqueous QDs and their assemblies have caught scientists’ eyes in different optoelectronic applications, including energy harvesting and photosensitive films15 because of their desirable features such as broad excitation, symmetric tunable emission spectra, photochemical stability, and binding compatibility with biomolecules.16 The ability of the QDs to
esonance energy transfer (RET) is a powerful technique for probing changes in the distance between energy donors and acceptors1,2 and is ideal for the sensitive detection of molecular binding events in response to interactions with a particular target molecule.3,4 Indeed, this phenomenon has been applied to biosensing contacting with various determination techniques, including fluorescence,5 surface plasma resonance (SPR),6 and electrochemiluminescence (ECL).7 However, so much effort was commonly put into steering the conduction band (CB) electrons to the electrode or solutionsolubilized electron acceptors to give rise to anodic or cathodic photocurrent, the related work focused on photoelectrochemical (PEC) biosensing based on RET was limited.4,8 On the basis of RET, previous work mainly focused on studying the underlying mechanisms of electron transfer between the quantum dots (QDs) and graphene9−11 or the biological assay using fluorescence.12,13 Fluorescence detects the optical signal produced from chemical reaction or energy donors, © 2013 American Chemical Society
Received: October 20, 2013 Accepted: November 20, 2013 Published: November 20, 2013 11720
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interact with adjacent nanoparticles or molecules gave rise to the RET-based assembly and nanoplasmonic devices as well as various sensors.5 Recently, graphene has been found to be a promising component in the development of RET biosensors due to its quenching capability toward various organic dyes17−19 and quantum dots.13,20 Besides, Au nanoparticles (AuNPs), with a high extinction coefficient and broad absorption spectrum in visible light that overlapped with the emission spectra of usual energy donors, played an important role in energy transfer systems.21,22 Therefore, the planelike nanocomposites of AuNPs and reduced graphene oxide (RGO) synthesized according to the previous report23 might improve the energy transfer efficiency.24,25 The residual oxygencontaining groups on the RGO surface were leveraged to stabilize AuNPs and made the RGO-AuNPs nanocomposites form well-dispersed aqueous suspension, which was beneficial for SH-probe conjugating with AuNPs.26 The planelike structure and good dispersion of RGO-AuNPs nanocomposites with high oligonucleotide densities were favorable for their applications in bioassays.27 In this work, a universal signal-on PEC platform based on RET between CdTe QDs and RGO-AuNPs nanocomposites was developed (Scheme 1). After an electrode modified with
Technical Note
EXPERIMENTAL SECTION
Materials and Reagents. Graphite (99.95%, 8000 mesh) was obtained from Aladdin Industrial Corporation. 1-Ethyl-3[3-(dimethylamino)-propyl] carbodiimide (EDC), N-hydroxysuccinimide (NHS), tris(2-carboxyethyl)phosphine (TCEP), bovine serum albumin (BSA), and thrombin were obtained from Sigma-Aldrich. Cadmium acetate [Cd(CH3COO)2· 2H2O] and mercaptoacetic acid (MPA) (97%) were purchased from Alfa Aesar China Ltd. Carcinoembryonic antigen (CEA) was purchased from Beijing Keybiotech Company, Ltd. (China). Chloroauric acid (HAuCl4·4H2O) was obtained from Shanghai Reagent Company (Shanghai, China). Ascorbic acid (AA) was obtained from Sinopharm Chemical Reagent Company, Ltd. (China). Other chemicals were of analytical reagent grade. Ultrapure water obtained from a Millipore water purification system (≥18 MΩ cm, Millipore) was used in all assays. The washing solution was tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffered saline (10 mmol L−1, pH 7.4, CNaCl = 0.1 mol L−1). The oligonucleotides were purchased from Sangon Biological Engineering Technology & Company Ltd. (Shanghai, China) and purified using high-performance−liquid chromatography. Their sequences were CEA aptamer, 5′-ATACCAGCTTATTCAATT-3′; NH2-probe, 5′-NH2-AAAAAATTGAATA-3′; and SH-probe, 5′-AGCTGGTATAAAA-SH-3′. Apparatus. Transmission electron microscope (TEM) images were taken using a Hitachi H-7650 type transmission electron microscope at an accelerating voltage of 80 kV (Hitachi, Japan). Ultraviolet−visible (UV−vis) absorption spectra were obtained on Cary 60 spectrophotometer (Agilent). Photoluminescence (PL) spectra were recorded on LS 50B (Perkin-Elmer). The PEC measurements were performed with a Zahner PEC workstation (Zahner, Germany). All experiments were carried out at room temperature using a conventional three-electrode system with a modified indium tin oxide (ITO) electrode as a working electrode, a platinum wire as an auxiliary electrode, and a Ag/AgCl electrode as a reference electrode. The geometrical area of the working electrode was 1.0 ± 0.1 cm2. All of the photocurrent measurements were carried out under 470 nm irradiation at a constant potential of −0.05 V in Tris-HCl-buffered saline (0.1 mol L−1, pH 7.4) containing AA (0.01 mol L−1), which was deaerated with high purity nitrogen for 15 min before PEC experiments and then kept over a N2 atmosphere for the entire experimental process. Electrochemical impedance spectroscopy (EIS) was carried out at open circuit potential with an Autolab potentiostat/galvanostat PGSTAT302N (Metrohm, Netherland) and controlled by Nova 1.8 with a three-electrode system, in KCl solution (0.1 mol L−1) containing a K3Fe(CN)6/ K4Fe(CN)6 (5.0 mmol L−1) (1:1) mixture as a redox probe from 0.1 Hz to 100 kHz with a signal amplitude of 10 mV. Preparation of RGO-AuNPs Nanocomposites and RGO-AuNPs-SH-probe. MPA-CdTe QDs and AuNPs were synthesized using modified procedures reported previously (Supporting Information).34,35 Graphene oxide (GO) was synthesized by the modified Hummers’ method (Supporting Information).36 After the GO was reduced (Supporting Information), RGO-AuNPs nanocomposites were prepared, according to the previous report (Supporting Information).23 The RGO-AuNPs nanocomposites were resuspended in washing solution (1 mL) containing SH-probe DNA (100 nmol L−1), which was activated with TCEP (10 mmol L−1),
Scheme 1. Schematic Illustration of the Signal-On PEC Aptasensing Platform Based on RET
mercaptoacetic acid wrapped CdTe (MPA-CdTe) QDs, under visible light (470 nm) excitation, a cathodic photocurrent was obtained at negative bias potential (−0.05 V). Notably, for the negatively charged electric field of the electrode surface in PEC measurements, it owned two merits: reorient the negatively charged oligonucleotide perpendicular to the electrode surface due to the static repulsion force28−30 and less interference for the electron donors to scavenge the photogenerated holes.31 Through activation, monoethanolamine (MEA) blocking and DNA hybridization, the carcinoembryonic antigen (CEA) aptamer and RGO-AuNPs-SH-probe assembled on the electrode in sequence. CEA bonded to its aptamer and destroyed the sandwichlike structure accompanied with declined quenching efficiency of the exciton produced in QDs,32 resulting in the interruption of energy transfer between excited CdTe QDs and RGO-AuNPs nanocomposites and the augment of PEC signal. On the basis of the RET and using the aptamer as a recognition module,33 a signal-on PEC aptasensor for CEA detection was proposed. 11721
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and stirred for 12 h at room temperature. To obtain the optimum PEC response, here the amount of RGO-AuNPs nanocomposites was excessive, leading to full coverage of SHprobe on the RGO-AuNPs nanocomposites. Excess amount of RGO-AuNPs nanocomposites was removed by centrifugation with 10000 rpm at 4 °C for 30 min, and then the supernatant containing RGO-AuNPs-SH-probe was decanted and stored at 4 °C prior to the hybridization assay. Preparation of PEC Aptasensor. After activating by the classic EDC/NHS coupling reactions for 60 min at room temperature, the NH2-probe was conjugated to the MPA-CdTe QDs modified electrode through the reaction between the COOH and NH2 groups. Then they were rinsed with washing solution carefully. Twenty microliters NH2-probe (100 nmol L−1) was dropped onto the surface of the electrode and incubated at 4 °C overnight. After blocking with 10 mmol L−1 MEA at 4 °C for 2 h, the hybridization reaction was carried out by dropping 20 μL prepared solution of CEA aptamer (100 nmol L−1) and RGO-AuNPs-SH-probe (100 nmol L−1) on the NH2-probe-modified ITO electrode at 37 °C for 1 h incubation in the presence of 20 mmol L−1 MgCl2, in sequence. Between each step, the electrode was washed thoroughly with washing solution to remove the unhybridized oligonucleotides. PEC Measurements. To carry out the PEC measurements, the aptasensor was first incubated with 20 μL CEA solutions of different concentrations at 37 °C for 60 min, followed by rinsing with washing solution, thoroughly. Afterward, the aptasensor was inserted in Tris-HCl buffered saline (0.1 mol L−1, pH 7.4), containing 10 mmol L−1 AA to record the PEC response at an applied potential of −0.05 V with 470 nm irradiation for bioassay under N2 atmosphere.
Figure 1. (A) PEC responses of CdTe QDs modified ITO electrode. (B) PEC responses of (a) ITO/CdTe, (b) ITO/CdTe/NH2-probe, (c) ITO/CdTe/NH2-probe/aptamer, (d) ITO/CdTe/NH2-probe/ aptamer/AuNPs-SH-probe, (e) ITO/CdTe/NH2-probe/aptamer/ RGO-AuNPs-SH-probe, and (f) after the introduction of CEA (0.1 ng mL−1). (C) PL spectra of (a) CdTe/NH2-probe/aptamer, (b) CdTe/NH 2 -probe/aptamer/AuNPs-SH-probe, (c) CdTe/NH 2 probe/aptamer/RGO-AuNPs-SH-probe, and after introducing (d) 0.1 and (e) 0.05 ng mL−1 CEA. (D) EIS spectra of (a) bare ITO electrode, (b) ITO/CdTe, (c) ITO/CdTe/NH2-probe, (d) ITO/ CdTe/NH2-probe/aptamer, (e) ITO/CdTe/NH2-probe/aptamer/ RGO-AuNPs-SH-probe, and (f) after the introduction of CEA (0.1 ng mL−1).
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AuNPs, the RGO-AuNPs nanocomposites could serve as a more efficient energy acceptor. Once the sandwichlike structure formed, RET caused excitons depletion between CdTe QDs and RGO-AuNPs nanocomposites, which restricted the separation of the electron and hole. As a result, it suppressed the cathodic photocurrent generation1 and offered a low PEC background signal for the further detection of CEA. While the target, CEA was introduced, the sandwichlike structure was destroyed.32 Hence, the suppression of RGO-AuNPs nanocomposites weakened, leading to an enhanced photocurrent (curve f). What’s more, the exciton-plasma interaction (EPI) might be another interaction between AuNPs and CdTe QDs, which could greatly attenuate and even completely damp the photocurrent of CdTe QDs,41−44 as the plasma absorption spectrum of AuNPs was overlapped with the emission spectrum of CdTe QDs, which was vital to initiate the efficient EPI. The energy-transfer efficiency between energy donor and acceptor is distance dependent. After the hybridization, the face-to-face distance between RGO-AuNPs nanocomposites and CdTe QDs was about 10 nm, which was sufficient for RET45,46 and EPI,43 as verified by fluorescence (concentrations of all the probe and aptamer were 100 nmol L−1) (Figure 1C). As seen, the most intensive fluorescence was shown in the CdTe/NH2-probe/aptamer (curve a). The fluorescence quenched after the introduction of AuNPs-SH-probe (curve b) and then quenched more when the AuNPs-SH-probe was displaced by the RGO-AuNPs-SH-probe (curve c). This might be that, in contrast to the AuNPs-SH-probe, the planelike RGO-AuNPs-SH-probe could provide a more efficient energy acceptor, strengthening the energy transfer efficiency between excited CdTe QDs and RGO-AuNPs nanocomposites.25,47 Thus it would inhibit the photocurrent generation.1 It was
RESULTS AND DISCUSSION PEC Mechanism and Characterizing the Fabrication Process of PEC Aptasensor. Characterizations of CdTe QDs, AuNPs, RGO, and RGO-AuNPs nanocomposites and RGO-AuNPs-SH-probe were provided in Figures S-1−S-5 of the Supporting Information. Upon light irradiation, the photoexcited CdTe QDs underwent electron−hole pair (excitions) generation, which then generated the electron and energy transfer process37 between the valence band (VB) and conduction band (CB)38 and could be classified into two forms: electrical signal enhancement or inhibition. At the negatively applied bias, the holes generated in VB of CdTe QDs were scavenged through electron donors. Simultaneously, together with proton (H+) in Tris-HCl-buffered saline, AA served H+ source for H2 production (Scheme 1). This process consumed electrons from the CB of the CdTe QDs.39,40 In this way, a periodically cathodic photocurrent generated under irradiation by the light turned on or off (Figure 1A). As shown in Figure 1B, the cathodic photocurrent responses to light were instantaneous, followed by a slight decay (curve a). A likely rationale for the initial decay was filling of the traps, which led to an inherent loss in photocurrent.1 With the modification processing, the photocurrent decreased accordingly (curves b− e). Compared with the photocurrent of the electrode modified by AuNPs-SH-probe (curve d), the photocurrent of the electrode modified by RGO-AuNPs-SH-probe declined more (curve e). The main reason for the large signal decay should be ascribed to energy transfer rather than steric hindrance or the photoinduced electron transfer from CdTe QDs to AuNPs.41 Since the absorption and emission spectra of CdTe QDs overlapped well with the plasmonic resonance spectrum of 11722
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reasonable to expect a stronger synergy between AuNPs and 2D nanomaterials. However, after the introduction of 0.1 and 0.05 ng mL−1 CEA, fluorescence recovered (curves d and e), which could be attributed to the partial destruction of the sandwichlike structure. Meanwhile, it verified the energy transfer was caused by the RGO-AuNPs-SH-probe rather than collisional quenching.42 The decay and recovery of fluorescence triggered by distance variation between energy donor and acceptor, critically attested to the effectiveness of this RET method in biomolecules determination. The activity of the RGO-AuNPs-SH-probe nanocomposites also compared with the AuNPs-SH-probe as well as the simple physical mixtures of the AuNPs-SH-probe with RGO (Figure S-6 of the Supporting Information). The quenching efficiency was in the sequence of the RGO-AuNPs-SH-probe (curve d) > the simple physical mixtures of AuNPs-SH-probe with RGO (curve c) > AuNPs-SH-probe (curve b), which further verified that the RGO-AuNPs-SH-probe was more suitable than the other two for the system. As shown in Figure 1D, the stepwise assembly process of the PEC aptasensor was investigated by the EIS. Compared with the impedance spectrum of bare ITO electrode (curve a), after the MPA-CdTe QDs were coated onto ITO, the electrontransfer resistance (Ret) increased significantly (curve b), which was attributed to the electronegative carboxyl groups of MPACdTe QDs repelling the transfer of the negatively charged probe [Fe(CN)63−/4−]. This suggested that MPA-CdTe QDs were deposited on the ITO electrode. Subsequently, after EDC/NHS activation, NH2-probe covalently bonded to the MPA-CdTe QDs films through amine and carboxyl groups, leading to an further increase of Ret (curve c), which was probably due to the poor conductivity of single strand DNA. After MEA blocking and subsequent stepwise incubation of aptamer and RGO-AuNPs-SH-probe, the Ret augmented gradually on account of the steric hindrance (curves d and e), indicating the successful hybridization of aptamer with the NH2-probe and the RGO-AuNPs-SH-probe, respectively, in sequence. The stepwise increase of electron transfer resistance verified the successful fabrication of the PEC aptasensor (curves a−e). However, after the introduction of CEA, the Ret diminished (curves f), which could be attributed to the partial destroy of the sandwichlike structure, weakening the steric hindrance and accelerating the electron transfer between the redox probe and electrode surface. Correspondingly, the resulting photocurrent responses to the different concentrations of CEA could provide a quantitative readout signal. PEC Aptasensing. Under optimal conditions (Figures S-7− S-9 of the Supporting Information), after corroborating the successful assembling of the PEC aptasensor and an efficient RET occurring between CdTe QDs and RGO-AuNPs nanocomposites, the system was applied to PEC determination of CEA as a model, with AA concentration of 0.01 mol L−1 and applied bias potential of −0.05 V under irradiation of 470 nm. The photocurrent responses measured by varying the CEA concentrations (C) were revealed in Figure 2A. The photocurrents increased with increasing concentrations of the CEA. The calibration plot was constructed by plotting ΔI (ΔI = I − I0, where I was the photocurrent of CEA solutions with different concentrations and I0 was the back ground photocurrent) against CEA concentrations in a linear range from 0.001 to 2.0 ng mL−1 (Figure 2B). The correlation coefficient was 0.992, and the detection limit was estimated to be 0.47 pg mL−1 at a signal-to-noise ratio of 3. Although the commonly
Figure 2. (A) PEC responses of the aptasensor to (a−j) 0, 0.001, 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2.0, and 2.2 ng mL−1 CEA. Inset: magnified responses from 0 to 0.01 ng mL−1. (B) Linear calibration curve. Inset: magnified responses from 0.001 to 0.1 ng mL −1 for CEA determination.
required diagnostic level for normal CEA concentration is 5 ng mL−1,6 the real serum samples did not need pretreatment, except for an appropriate dilution before the test. The linear range (0.001 to 2.0 ng mL−1) was much wider than 1.0−60 ng mL−1 by the chemiluminescence method48 and 1.0−100 ng mL−1 by the ECL method.49 The detection limit of 0.47 pg mL−1 was lower than 20 ng mL−1 by SPR immunoassay6 and 4.4 pg mL−1 by the ECL immunoassay.50 In addition, the detection potential was closer to the physiological potential than −0.84 V by the electrochemical method51 and −1.25 V by the ECL method,7 leading to less interference. Thus the PEC aptasensor at a low-overpotential exhibited wide linear response range and high sensitivity in the determination of CEA. Moreover, the results of specificity, reproducibility, and stability of the PEC aptasensor showed good precision, acceptable fabrication reproducibility, and good long-term stability (see the Supporting Information). In addition, the selectivity of the PEC aptasensor illustrated only CEA; excluding other test samples could cause an obvious difference of the photocurrent response, verifying an excellent specificity of the PEC aptasensor to CEA detection (Figure S-10 of the Supporting Information). Results from real serum sample measurements validate the feasibility of the proposed strategy (Table S-1 of the Supporting Information).
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CONCLUSION A universal signal-on PEC platform based on RET between CdTe QDs and RGO-AuNPs nanocomposites was developed. The plasma absorption spectrum of AuNPs overlapped with the emission spectrum of CdTe QDs, which initiated the efficient EPI. The synergy of plasmonic nanoparticles and planelike structure of RGO improved RET efficiency and decreased the background signal. On the basis of RET and the aptamer as a recognition module, with the use of CEA as a model, a sandwichlike structure PEC aptasensor prepared via DNA hybridization was constructed. Under the optimal conditions, the PEC aptasensor exhibited good analytical performance, such as wide linear response range, low detection limit, low overpotential and good reproducibility, stability, and specificity. The proposed PEC aptasensor was successfully applied to clinical sample assays, showing acceptable accuracy. The RET between QDs and RGO-AuNPs nanocomposites provided a novel avenue for preparation of PEC devices in analytical fields. Since different aptamers can specifically bind to different target molecules, the designed strategy has an expansive and promising perspective of application in other plasmonic nanomaterials for the construction of versatile PEC platforms. 11723
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Technical Note
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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 Authors
*E-mail: [email protected]. *E-mail: [email protected]. Tel/Fax: +86-25-85891051. 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 for the project (Grants 21175069 and 21205061) and the Foundation of the Jiangsu Education Committee (Grant 11KJA150003). We appreciate the financial support from the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Program for Jiangsu Collaborative Innovation Center of Biomedical Functional Materials.
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REFERENCES
(1) Ruland, A.; Schulz-Drost, C.; Sgobba, V.; Guldi, D. M. Adv. Mater. 2011, 23, 4573−4577. (2) Lunz, M.; Gerard, V. A.; Gun′ko, Y. K.; Lesnyak, V.; Gaponik, N.; Susha, A. S.; Rogach, A. L.; Bradley, A. L. Nano Lett. 2011, 11, 3341− 3345. (3) Freeman, R.; Liu, X. Q.; Willner, I. J. Am. Chem. Soc. 2011, 133, 11597−11604. (4) Liu, X. Q.; Niazov-Elkan, A.; Wang, F.; Willner, I. Nano Lett. 2013, 13, 219−225. (5) Bigall, N. C.; Paraka, W. J.; Dorfs, D. Nano Today 2012, 7, 282− 296. (6) Hu, W. H.; Liu, Y. S.; Lu, Z. S.; Li, C. M. Adv. Funct. Mater. 2010, 20, 3497−3503. (7) Shan, Y.; Xu, J. J.; Chen, H. Y. Chem. Commun. 2010, 46, 5079− 5081. (8) Tu, W. W.; Wang, W. J.; Lei, J. P.; Deng, S. Y.; Ju, H. X. Chem. Commun. 2012, 48, 6535−6537. (9) Kaniyankandy, S.; Rawalekar, S.; Ghosh, H. N. J. Phys. Chem. C 2012, 116, 16271−16275. (10) Zhang, D. Y.; Gan, L.; Cao, Y.; Wang, Q.; Qi, L. M.; Guo, X. F. Adv. Mater. 2012, 24, 2715−2720. (11) Lightcap, I. V.; Kamat, P. V. J. Am. Chem. Soc. 2012, 134, 7109− 7116. (12) Liu, M.; Zhao, H. M.; Quan, X.; Chen, S.; Fan, X. F. Chem. Commun. 2010, 46, 7909−7911. (13) Dong, H. F.; Gao, W. C.; Yan, F.; Ji, H. X.; Ju, H. X. Anal. Chem. 2010, 82, 5511−5517. (14) Gill, R.; Zayats, M.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 7602−7625. (15) Lesnyak, V.; Gaponik, N.; Eychmüller, A. Chem. Soc. Rev. 2013, 42, 2905−2929. (16) Hildebrandt, N. ACS Nano 2011, 5, 5286−5290. (17) Lu, C. H.; Yang, H. H.; Zhu, C. L.; Chen, X.; Chen, G. N. Angew. Chem., Int. Ed. 2009, 48, 4785−4787. (18) He, S. J.; Song, B.; Li, D.; Zhu, C. F.; Qi, W. P.; Wen, Y. Q.; Wang, L. L.; Song, S. P.; Fang, H. P.; Fan, C. H. Adv. Funct. Mater. 2010, 20, 453−459. (19) Chang, H. X.; Tang, L. H.; Wang, Y.; Jiang, J. H.; Li, J. H. Anal. Chem. 2010, 82, 2341−2346. (20) Zhang, C. L.; Xu, J.; Zhang, S. M.; Ji, X. H.; He, Z. K. Chem. Eur. J. 2012, 18, 8296−8300. (21) Oh, E.; Hong, M. Y.; Lee, D.; Nam, S. H.; Yoon, H. C.; Kim, H. S. J. Am. Chem. Soc. 2005, 127, 3270−3271. 11724
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Using Graphene-Based Plasmonic Nanocomposites to Quench Energy from Quantum Dots for Signal-On Photoelectrochemical Aptasensing Xianxiang Zeng, Shishi Ma, Jianchun Bao, Wenwen Tu,* and Zhihui Dai* Jiangsu Key Laboratory of Biofunctional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, P. R. China S Supporting Information *
ABSTRACT: On the basis of the absorption and emission spectra overlap, an enhanced resonance energy transfer caused by excition-plasmon resonance between reduced graphene oxide (RGO)-Au nanoparticles (AuNPs) and CdTe quantum dots (QDs) was obtained. With the synergy of AuNPs and RGO as a planelike energy acceptor, it resulted in the enhancement of energy transfer between excited CdTe QDs and RGO-AuNPs nanocomposites. Upon the novel sandwichlike structure formed via DNA hybridization, the exciton produced in CdTe QDs was annihilated. A damped photocurrent was obtained, which was acted as the background signal for the development of a universal photoelectrochemical (PEC) platform. With the use of carcinoembryonic antigen (CEA) as a model which bonded to its specific aptamer and destroyed the sandwichlike structure, the energy transfer efficiency was lowered, leading to PEC response augment. Thus a signal-on PEC aptasensor was constructed. Under 470 nm irradiation at −0.05 V, the PEC aptasensor for CEA determination exhibited a linear range from 0.001 to 2.0 ng mL−1 with a detection limit of 0.47 pg mL−1 at a signal-to-noise ratio of 3 and was satisfactory for clinical sample detection. Since different aptamers can specifically bind to different target molecules, the designed strategy has an expansive application for the construction of versatile PEC platforms.
R
which is different from the mechanism of photoelectrochemisty. It detects the photocurrent signal. As a newly emerged but dynamically developing analysis technique, PEC determination has attracted substantial attention.14 It avoids the expensive equipment of optical techniques and high overpotential in electrochemistry or ECL measurements. By using light as the external stimulus at an appropriate wavelength, a selective PEC reaction can be achieved.8 Aqueous QDs and their assemblies have caught scientists’ eyes in different optoelectronic applications, including energy harvesting and photosensitive films15 because of their desirable features such as broad excitation, symmetric tunable emission spectra, photochemical stability, and binding compatibility with biomolecules.16 The ability of the QDs to
esonance energy transfer (RET) is a powerful technique for probing changes in the distance between energy donors and acceptors1,2 and is ideal for the sensitive detection of molecular binding events in response to interactions with a particular target molecule.3,4 Indeed, this phenomenon has been applied to biosensing contacting with various determination techniques, including fluorescence,5 surface plasma resonance (SPR),6 and electrochemiluminescence (ECL).7 However, so much effort was commonly put into steering the conduction band (CB) electrons to the electrode or solutionsolubilized electron acceptors to give rise to anodic or cathodic photocurrent, the related work focused on photoelectrochemical (PEC) biosensing based on RET was limited.4,8 On the basis of RET, previous work mainly focused on studying the underlying mechanisms of electron transfer between the quantum dots (QDs) and graphene9−11 or the biological assay using fluorescence.12,13 Fluorescence detects the optical signal produced from chemical reaction or energy donors, © 2013 American Chemical Society
Received: October 20, 2013 Accepted: November 20, 2013 Published: November 20, 2013 11720
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interact with adjacent nanoparticles or molecules gave rise to the RET-based assembly and nanoplasmonic devices as well as various sensors.5 Recently, graphene has been found to be a promising component in the development of RET biosensors due to its quenching capability toward various organic dyes17−19 and quantum dots.13,20 Besides, Au nanoparticles (AuNPs), with a high extinction coefficient and broad absorption spectrum in visible light that overlapped with the emission spectra of usual energy donors, played an important role in energy transfer systems.21,22 Therefore, the planelike nanocomposites of AuNPs and reduced graphene oxide (RGO) synthesized according to the previous report23 might improve the energy transfer efficiency.24,25 The residual oxygencontaining groups on the RGO surface were leveraged to stabilize AuNPs and made the RGO-AuNPs nanocomposites form well-dispersed aqueous suspension, which was beneficial for SH-probe conjugating with AuNPs.26 The planelike structure and good dispersion of RGO-AuNPs nanocomposites with high oligonucleotide densities were favorable for their applications in bioassays.27 In this work, a universal signal-on PEC platform based on RET between CdTe QDs and RGO-AuNPs nanocomposites was developed (Scheme 1). After an electrode modified with
Technical Note
EXPERIMENTAL SECTION
Materials and Reagents. Graphite (99.95%, 8000 mesh) was obtained from Aladdin Industrial Corporation. 1-Ethyl-3[3-(dimethylamino)-propyl] carbodiimide (EDC), N-hydroxysuccinimide (NHS), tris(2-carboxyethyl)phosphine (TCEP), bovine serum albumin (BSA), and thrombin were obtained from Sigma-Aldrich. Cadmium acetate [Cd(CH3COO)2· 2H2O] and mercaptoacetic acid (MPA) (97%) were purchased from Alfa Aesar China Ltd. Carcinoembryonic antigen (CEA) was purchased from Beijing Keybiotech Company, Ltd. (China). Chloroauric acid (HAuCl4·4H2O) was obtained from Shanghai Reagent Company (Shanghai, China). Ascorbic acid (AA) was obtained from Sinopharm Chemical Reagent Company, Ltd. (China). Other chemicals were of analytical reagent grade. Ultrapure water obtained from a Millipore water purification system (≥18 MΩ cm, Millipore) was used in all assays. The washing solution was tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffered saline (10 mmol L−1, pH 7.4, CNaCl = 0.1 mol L−1). The oligonucleotides were purchased from Sangon Biological Engineering Technology & Company Ltd. (Shanghai, China) and purified using high-performance−liquid chromatography. Their sequences were CEA aptamer, 5′-ATACCAGCTTATTCAATT-3′; NH2-probe, 5′-NH2-AAAAAATTGAATA-3′; and SH-probe, 5′-AGCTGGTATAAAA-SH-3′. Apparatus. Transmission electron microscope (TEM) images were taken using a Hitachi H-7650 type transmission electron microscope at an accelerating voltage of 80 kV (Hitachi, Japan). Ultraviolet−visible (UV−vis) absorption spectra were obtained on Cary 60 spectrophotometer (Agilent). Photoluminescence (PL) spectra were recorded on LS 50B (Perkin-Elmer). The PEC measurements were performed with a Zahner PEC workstation (Zahner, Germany). All experiments were carried out at room temperature using a conventional three-electrode system with a modified indium tin oxide (ITO) electrode as a working electrode, a platinum wire as an auxiliary electrode, and a Ag/AgCl electrode as a reference electrode. The geometrical area of the working electrode was 1.0 ± 0.1 cm2. All of the photocurrent measurements were carried out under 470 nm irradiation at a constant potential of −0.05 V in Tris-HCl-buffered saline (0.1 mol L−1, pH 7.4) containing AA (0.01 mol L−1), which was deaerated with high purity nitrogen for 15 min before PEC experiments and then kept over a N2 atmosphere for the entire experimental process. Electrochemical impedance spectroscopy (EIS) was carried out at open circuit potential with an Autolab potentiostat/galvanostat PGSTAT302N (Metrohm, Netherland) and controlled by Nova 1.8 with a three-electrode system, in KCl solution (0.1 mol L−1) containing a K3Fe(CN)6/ K4Fe(CN)6 (5.0 mmol L−1) (1:1) mixture as a redox probe from 0.1 Hz to 100 kHz with a signal amplitude of 10 mV. Preparation of RGO-AuNPs Nanocomposites and RGO-AuNPs-SH-probe. MPA-CdTe QDs and AuNPs were synthesized using modified procedures reported previously (Supporting Information).34,35 Graphene oxide (GO) was synthesized by the modified Hummers’ method (Supporting Information).36 After the GO was reduced (Supporting Information), RGO-AuNPs nanocomposites were prepared, according to the previous report (Supporting Information).23 The RGO-AuNPs nanocomposites were resuspended in washing solution (1 mL) containing SH-probe DNA (100 nmol L−1), which was activated with TCEP (10 mmol L−1),
Scheme 1. Schematic Illustration of the Signal-On PEC Aptasensing Platform Based on RET
mercaptoacetic acid wrapped CdTe (MPA-CdTe) QDs, under visible light (470 nm) excitation, a cathodic photocurrent was obtained at negative bias potential (−0.05 V). Notably, for the negatively charged electric field of the electrode surface in PEC measurements, it owned two merits: reorient the negatively charged oligonucleotide perpendicular to the electrode surface due to the static repulsion force28−30 and less interference for the electron donors to scavenge the photogenerated holes.31 Through activation, monoethanolamine (MEA) blocking and DNA hybridization, the carcinoembryonic antigen (CEA) aptamer and RGO-AuNPs-SH-probe assembled on the electrode in sequence. CEA bonded to its aptamer and destroyed the sandwichlike structure accompanied with declined quenching efficiency of the exciton produced in QDs,32 resulting in the interruption of energy transfer between excited CdTe QDs and RGO-AuNPs nanocomposites and the augment of PEC signal. On the basis of the RET and using the aptamer as a recognition module,33 a signal-on PEC aptasensor for CEA detection was proposed. 11721
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and stirred for 12 h at room temperature. To obtain the optimum PEC response, here the amount of RGO-AuNPs nanocomposites was excessive, leading to full coverage of SHprobe on the RGO-AuNPs nanocomposites. Excess amount of RGO-AuNPs nanocomposites was removed by centrifugation with 10000 rpm at 4 °C for 30 min, and then the supernatant containing RGO-AuNPs-SH-probe was decanted and stored at 4 °C prior to the hybridization assay. Preparation of PEC Aptasensor. After activating by the classic EDC/NHS coupling reactions for 60 min at room temperature, the NH2-probe was conjugated to the MPA-CdTe QDs modified electrode through the reaction between the COOH and NH2 groups. Then they were rinsed with washing solution carefully. Twenty microliters NH2-probe (100 nmol L−1) was dropped onto the surface of the electrode and incubated at 4 °C overnight. After blocking with 10 mmol L−1 MEA at 4 °C for 2 h, the hybridization reaction was carried out by dropping 20 μL prepared solution of CEA aptamer (100 nmol L−1) and RGO-AuNPs-SH-probe (100 nmol L−1) on the NH2-probe-modified ITO electrode at 37 °C for 1 h incubation in the presence of 20 mmol L−1 MgCl2, in sequence. Between each step, the electrode was washed thoroughly with washing solution to remove the unhybridized oligonucleotides. PEC Measurements. To carry out the PEC measurements, the aptasensor was first incubated with 20 μL CEA solutions of different concentrations at 37 °C for 60 min, followed by rinsing with washing solution, thoroughly. Afterward, the aptasensor was inserted in Tris-HCl buffered saline (0.1 mol L−1, pH 7.4), containing 10 mmol L−1 AA to record the PEC response at an applied potential of −0.05 V with 470 nm irradiation for bioassay under N2 atmosphere.
Figure 1. (A) PEC responses of CdTe QDs modified ITO electrode. (B) PEC responses of (a) ITO/CdTe, (b) ITO/CdTe/NH2-probe, (c) ITO/CdTe/NH2-probe/aptamer, (d) ITO/CdTe/NH2-probe/ aptamer/AuNPs-SH-probe, (e) ITO/CdTe/NH2-probe/aptamer/ RGO-AuNPs-SH-probe, and (f) after the introduction of CEA (0.1 ng mL−1). (C) PL spectra of (a) CdTe/NH2-probe/aptamer, (b) CdTe/NH 2 -probe/aptamer/AuNPs-SH-probe, (c) CdTe/NH 2 probe/aptamer/RGO-AuNPs-SH-probe, and after introducing (d) 0.1 and (e) 0.05 ng mL−1 CEA. (D) EIS spectra of (a) bare ITO electrode, (b) ITO/CdTe, (c) ITO/CdTe/NH2-probe, (d) ITO/ CdTe/NH2-probe/aptamer, (e) ITO/CdTe/NH2-probe/aptamer/ RGO-AuNPs-SH-probe, and (f) after the introduction of CEA (0.1 ng mL−1).
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AuNPs, the RGO-AuNPs nanocomposites could serve as a more efficient energy acceptor. Once the sandwichlike structure formed, RET caused excitons depletion between CdTe QDs and RGO-AuNPs nanocomposites, which restricted the separation of the electron and hole. As a result, it suppressed the cathodic photocurrent generation1 and offered a low PEC background signal for the further detection of CEA. While the target, CEA was introduced, the sandwichlike structure was destroyed.32 Hence, the suppression of RGO-AuNPs nanocomposites weakened, leading to an enhanced photocurrent (curve f). What’s more, the exciton-plasma interaction (EPI) might be another interaction between AuNPs and CdTe QDs, which could greatly attenuate and even completely damp the photocurrent of CdTe QDs,41−44 as the plasma absorption spectrum of AuNPs was overlapped with the emission spectrum of CdTe QDs, which was vital to initiate the efficient EPI. The energy-transfer efficiency between energy donor and acceptor is distance dependent. After the hybridization, the face-to-face distance between RGO-AuNPs nanocomposites and CdTe QDs was about 10 nm, which was sufficient for RET45,46 and EPI,43 as verified by fluorescence (concentrations of all the probe and aptamer were 100 nmol L−1) (Figure 1C). As seen, the most intensive fluorescence was shown in the CdTe/NH2-probe/aptamer (curve a). The fluorescence quenched after the introduction of AuNPs-SH-probe (curve b) and then quenched more when the AuNPs-SH-probe was displaced by the RGO-AuNPs-SH-probe (curve c). This might be that, in contrast to the AuNPs-SH-probe, the planelike RGO-AuNPs-SH-probe could provide a more efficient energy acceptor, strengthening the energy transfer efficiency between excited CdTe QDs and RGO-AuNPs nanocomposites.25,47 Thus it would inhibit the photocurrent generation.1 It was
RESULTS AND DISCUSSION PEC Mechanism and Characterizing the Fabrication Process of PEC Aptasensor. Characterizations of CdTe QDs, AuNPs, RGO, and RGO-AuNPs nanocomposites and RGO-AuNPs-SH-probe were provided in Figures S-1−S-5 of the Supporting Information. Upon light irradiation, the photoexcited CdTe QDs underwent electron−hole pair (excitions) generation, which then generated the electron and energy transfer process37 between the valence band (VB) and conduction band (CB)38 and could be classified into two forms: electrical signal enhancement or inhibition. At the negatively applied bias, the holes generated in VB of CdTe QDs were scavenged through electron donors. Simultaneously, together with proton (H+) in Tris-HCl-buffered saline, AA served H+ source for H2 production (Scheme 1). This process consumed electrons from the CB of the CdTe QDs.39,40 In this way, a periodically cathodic photocurrent generated under irradiation by the light turned on or off (Figure 1A). As shown in Figure 1B, the cathodic photocurrent responses to light were instantaneous, followed by a slight decay (curve a). A likely rationale for the initial decay was filling of the traps, which led to an inherent loss in photocurrent.1 With the modification processing, the photocurrent decreased accordingly (curves b− e). Compared with the photocurrent of the electrode modified by AuNPs-SH-probe (curve d), the photocurrent of the electrode modified by RGO-AuNPs-SH-probe declined more (curve e). The main reason for the large signal decay should be ascribed to energy transfer rather than steric hindrance or the photoinduced electron transfer from CdTe QDs to AuNPs.41 Since the absorption and emission spectra of CdTe QDs overlapped well with the plasmonic resonance spectrum of 11722
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reasonable to expect a stronger synergy between AuNPs and 2D nanomaterials. However, after the introduction of 0.1 and 0.05 ng mL−1 CEA, fluorescence recovered (curves d and e), which could be attributed to the partial destruction of the sandwichlike structure. Meanwhile, it verified the energy transfer was caused by the RGO-AuNPs-SH-probe rather than collisional quenching.42 The decay and recovery of fluorescence triggered by distance variation between energy donor and acceptor, critically attested to the effectiveness of this RET method in biomolecules determination. The activity of the RGO-AuNPs-SH-probe nanocomposites also compared with the AuNPs-SH-probe as well as the simple physical mixtures of the AuNPs-SH-probe with RGO (Figure S-6 of the Supporting Information). The quenching efficiency was in the sequence of the RGO-AuNPs-SH-probe (curve d) > the simple physical mixtures of AuNPs-SH-probe with RGO (curve c) > AuNPs-SH-probe (curve b), which further verified that the RGO-AuNPs-SH-probe was more suitable than the other two for the system. As shown in Figure 1D, the stepwise assembly process of the PEC aptasensor was investigated by the EIS. Compared with the impedance spectrum of bare ITO electrode (curve a), after the MPA-CdTe QDs were coated onto ITO, the electrontransfer resistance (Ret) increased significantly (curve b), which was attributed to the electronegative carboxyl groups of MPACdTe QDs repelling the transfer of the negatively charged probe [Fe(CN)63−/4−]. This suggested that MPA-CdTe QDs were deposited on the ITO electrode. Subsequently, after EDC/NHS activation, NH2-probe covalently bonded to the MPA-CdTe QDs films through amine and carboxyl groups, leading to an further increase of Ret (curve c), which was probably due to the poor conductivity of single strand DNA. After MEA blocking and subsequent stepwise incubation of aptamer and RGO-AuNPs-SH-probe, the Ret augmented gradually on account of the steric hindrance (curves d and e), indicating the successful hybridization of aptamer with the NH2-probe and the RGO-AuNPs-SH-probe, respectively, in sequence. The stepwise increase of electron transfer resistance verified the successful fabrication of the PEC aptasensor (curves a−e). However, after the introduction of CEA, the Ret diminished (curves f), which could be attributed to the partial destroy of the sandwichlike structure, weakening the steric hindrance and accelerating the electron transfer between the redox probe and electrode surface. Correspondingly, the resulting photocurrent responses to the different concentrations of CEA could provide a quantitative readout signal. PEC Aptasensing. Under optimal conditions (Figures S-7− S-9 of the Supporting Information), after corroborating the successful assembling of the PEC aptasensor and an efficient RET occurring between CdTe QDs and RGO-AuNPs nanocomposites, the system was applied to PEC determination of CEA as a model, with AA concentration of 0.01 mol L−1 and applied bias potential of −0.05 V under irradiation of 470 nm. The photocurrent responses measured by varying the CEA concentrations (C) were revealed in Figure 2A. The photocurrents increased with increasing concentrations of the CEA. The calibration plot was constructed by plotting ΔI (ΔI = I − I0, where I was the photocurrent of CEA solutions with different concentrations and I0 was the back ground photocurrent) against CEA concentrations in a linear range from 0.001 to 2.0 ng mL−1 (Figure 2B). The correlation coefficient was 0.992, and the detection limit was estimated to be 0.47 pg mL−1 at a signal-to-noise ratio of 3. Although the commonly
Figure 2. (A) PEC responses of the aptasensor to (a−j) 0, 0.001, 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2.0, and 2.2 ng mL−1 CEA. Inset: magnified responses from 0 to 0.01 ng mL−1. (B) Linear calibration curve. Inset: magnified responses from 0.001 to 0.1 ng mL −1 for CEA determination.
required diagnostic level for normal CEA concentration is 5 ng mL−1,6 the real serum samples did not need pretreatment, except for an appropriate dilution before the test. The linear range (0.001 to 2.0 ng mL−1) was much wider than 1.0−60 ng mL−1 by the chemiluminescence method48 and 1.0−100 ng mL−1 by the ECL method.49 The detection limit of 0.47 pg mL−1 was lower than 20 ng mL−1 by SPR immunoassay6 and 4.4 pg mL−1 by the ECL immunoassay.50 In addition, the detection potential was closer to the physiological potential than −0.84 V by the electrochemical method51 and −1.25 V by the ECL method,7 leading to less interference. Thus the PEC aptasensor at a low-overpotential exhibited wide linear response range and high sensitivity in the determination of CEA. Moreover, the results of specificity, reproducibility, and stability of the PEC aptasensor showed good precision, acceptable fabrication reproducibility, and good long-term stability (see the Supporting Information). In addition, the selectivity of the PEC aptasensor illustrated only CEA; excluding other test samples could cause an obvious difference of the photocurrent response, verifying an excellent specificity of the PEC aptasensor to CEA detection (Figure S-10 of the Supporting Information). Results from real serum sample measurements validate the feasibility of the proposed strategy (Table S-1 of the Supporting Information).
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CONCLUSION A universal signal-on PEC platform based on RET between CdTe QDs and RGO-AuNPs nanocomposites was developed. The plasma absorption spectrum of AuNPs overlapped with the emission spectrum of CdTe QDs, which initiated the efficient EPI. The synergy of plasmonic nanoparticles and planelike structure of RGO improved RET efficiency and decreased the background signal. On the basis of RET and the aptamer as a recognition module, with the use of CEA as a model, a sandwichlike structure PEC aptasensor prepared via DNA hybridization was constructed. Under the optimal conditions, the PEC aptasensor exhibited good analytical performance, such as wide linear response range, low detection limit, low overpotential and good reproducibility, stability, and specificity. The proposed PEC aptasensor was successfully applied to clinical sample assays, showing acceptable accuracy. The RET between QDs and RGO-AuNPs nanocomposites provided a novel avenue for preparation of PEC devices in analytical fields. Since different aptamers can specifically bind to different target molecules, the designed strategy has an expansive and promising perspective of application in other plasmonic nanomaterials for the construction of versatile PEC platforms. 11723
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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 Authors
*E-mail: [email protected]. *E-mail: [email protected]. Tel/Fax: +86-25-85891051. 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 for the project (Grants 21175069 and 21205061) and the Foundation of the Jiangsu Education Committee (Grant 11KJA150003). We appreciate the financial support from the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Program for Jiangsu Collaborative Innovation Center of Biomedical Functional Materials.
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