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A Label-Free Photoelectrochemical Immunosensor Based on Water-Soluble CdS Quantum Dots Guang-Li Wang, Pei-Pei Yu, Jing-Juan Xu,* and Hong-Yuan Chen Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of China), School of Chemistry and Chemical Engineering, Nanjing UniVersity, Nanjing 210093, China ReceiVed: March 7, 2009; ReVised Manuscript ReceiVed: May 5, 2009
We demonstrate herein a newly developed lable-free photoelectrochemical immunosensor using a CdS quantum dots (QDs) multilayer film coupled with a biospecific interaction. The CdS QDs multilayer film was prepared by layer-by-layer assembling positively charged poly(dimethyldiallylammonium chloride) (PDDA) and thioglycolic acid (TGA)-capped water-soluble CdS QDs with negative charges on the surface of an indium-tin oxide (ITO) electrode. Ascorbic acid (AA) was exploited as an efficient and nontoxic electron donor for scavenging photogenerated holes under mild solution medium. The photoexcitation of CdS QDs modified electrode potentiostated at 0 V (vs. Ag/AgCl) in the presence of 0.1 M AA led to a stable anodic photocurrent. To perform the immunoassay, goat antimouse IgG was conjugated onto CdS QDs modified electrode by using the classic EDC coupling reactions between COOH groups on the surfaces of the TGA capped CdS QDs and NH2 groups of the antibody. The concentrations of mouse IgG were measured through the decrease in photocurrent intensity resulting from the increase in steric hindrances due to the formation of the immunocomplex. The synthetic conditions (different Cd/S ratio and different pH) of CdS QDs and the number of PDDA/CdS bilayers could influence the photoelectrochemical properties of CdS QDs modified electrodes used for immunosensor construction. Under the optimal conditions, a linear relationship between photocurrent decrease and mouse IgG concentration was obtained in the range of 10 pg/mL to 100 ng/mL with a detection limit of 8.0 pg/mL. This strategy opens a new perspective for the application of QDs, which might be of great significance for QDs in photoelectrochemical bioanalysis in the future. Introduction Immunosensors have gained increasing attention with the expectation of obtaining quick and sensitive immunological response for environmental monitoring, food safety, and clinical and medical diagnosis.1,2 The conventional immunoassay methods involve radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Despite the predominance of high sensitivity, all these methods involve labeling steps which complicate the analysis procedures. Moreover, RIA exposes laboratory workers to a significant safety hazard, and ELISA is tedious and time-consuming.3 So, the development of simple, sensitive, specific, and low cost immunoassays for quantitative analysis is a continuing challenge for the scientific community.4,5 Typically, the design of label-free affinity-based probing concepts is the objective of much current research. The main advantage of label-free methods is that they avoid the process of labeling antibody or antigen with markers, which makes the experimental process simple, low cost, and time saving. Different techniques such as piezoelectricity,6 surface plasmon resonance,7 electrochemistry,8,9 and electrochemiluminescence10 have been widely used in label-free immunoassays. The photoelectrochemical method is a newly developed and promising analytical method for biological assay.11 In the photoelectrochemical detection process, light is used to excite photoelectrochemically active species on the electrode and current is used as the detection signal, which is just the reverse process of electrochemiluminescence. Benefiting from the different form of energy for excitation and detection, its * To whom correspondence should be addressed. Phone/fax: +86-2583597294. E-mail:
[email protected].
sensitivity could potentially match that of the electrochemiluminescence. In addition, the use of electronic readout makes the instrument simpler, cheaper, and easier to miniaturize than that of the optical detection methods. As a detection method, the photoelectrochemical method is also well-suited for rapid and high-throughput biological assay. Ruthenium tris(2,2′bipyridine) derivatives were the most often used materials in photoelectrochemical biosensors.12 Semiconductor nanocrystals, also known as quantum dots (QDs), have emerged as a significant new class of materials over the past decade. Due to the quantum confinement, QDs can exhibit size-dependent properties.13,14 As a result, QDs exhibit a wide range of electrical and optical properties and can be used for various applications such as light-emitting diodes, solar cells, lasers, and transistors. Especially, QDs-based bioassay has become one of the most exciting forefront fields in analytical chemistry. Extensive efforts have been made toward the development of QDs-based optical sensors including fluorescence and fluorescence resonance energy transfer-based sensors for detecting metal ions, small molecules, and biomolecules.15-20 Recently, electrochemiluminescence properties of QDs were also exploited for biosensor applications.21,22 While more and more research has been focusing on the exploration of QDs as optical sensors, little work was done on the photoelectrochemical properties of QDs and their potential applications as biosensors. On the basis of the reaction between photogenerated holes of illuminated CdS QDs and the substrate of acetylcholine esterase, the first example of a photoelectrochemical biosensor was developed for sensing of the enzyme inhibitors.23 Using triethanolamine as an electron donor, CdS
10.1021/jp902069s CCC: $40.75 2009 American Chemical Society Published on Web 06/02/2009
A Label-Free Photoelectrochemical Immunosensor QDs were used as labels for photoelectrochemical tyrosinase activity detection.24 In these detection systems, the photocurrent intensity of CdS QDs was small (about 10-9-10-8 A) and expensive, and sophisticated instrumentation (lock-in amplifier) was needed to detect the photocurrent. For photoelectrochemical applications of QDs in biosensing, the system that has improved photocurrent intensity will be desirable. In CdS-based photoelectrochemical cells, electron donors in strong alkaline solution are usually used to obtain high photocurrent intensity. However, the strong alkaline medium is improper for biosensing construction. So it is necessary to find an efficient electron donor in mild solution medium. In the experiment, ascorbic acid (AA) was exploited as an efficient and nontoxic electron donor for scavenging photogenerated holes of CdS QDs under mild solution medium. In AA solution, CdS showed much higher photocurrent intensity than that of the usually used electron donors. Thus, the first example of a label-free photoelectrochemical immunosensor with QDs was realized. The thioglycolic acid (TGA)-capped water-soluble CdS QDs were assembled on the electrode simply by layer-by-layer assembly. Goat antimouse IgG was conjugated onto CdS QDs modified electrode by using the classic EDC coupling reactions between COOH groups on the surfaces of the TGA-capped CdS QDs and NH2 groups of the antibody. The antigen concentratons were directly (label-free) measured through the decrease in photocurrent intensity resulting from the specific immunoreaction. Results indicated that the as-obtained lable-free immunosensor showed good sensitivity, selectivity, and stability. The established method could provide an approach for the assembly of QDs with other clinically important proteins, to further the design of novel QDs-based photoelectrochemical biosensors in the future. Experimental Section Reagents. CdCl2 · 2.5H2O was obtained from Shanghai Jinshan Tingxin Chemical Plant. Na2S · 9H2O was obtained from Shanghai Lingfeng Chemical Reagent Co. LTD (Shanghai, China). PDDA (20%, w/w in water, molecular weight ) 200 000-350 000) was obtained from Aldrich. 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) was purchased from Fluka. Bovine serum albumin (BSA) and Tween 20 were purchased from Amresco. N-Hydroxysuccinimide (NHS), thioglycolic acid (TGA), ascorbic acid (AA), triethanolamine (TEA), Na2SO3, and urea were obtained from Sinopharm Chemical Reagent Co., LTD (Shanghai, China). Mouse IgG, rabbit IgG, goat IgG, and goat antimouse IgG were purchased from Boster Bio-Technology Co. LTD (Wuhan, China). Mouse serum was obtained from ZhongBei LinGe Biotechnology LTD (Beijing, China). All other reagents were of analytical grade and were used as received. All aqueous solutions were prepared with doubly distilled water (18 MΩ cm-1), which was obtained from a Milli-Q water purification system. Dulbecco’s phosphate buffer (pH 7.4) containing 1.47 mM KH2PO4, 8.10 mM Na2HPO4, 2.67 mM KCl, and 138 mM NaCl was used for the preparation of the antibody and antigen solution, as well as the blocking solution and the washing solution for the immunoreactions. The washing buffer solution was Dulbecco’s phosphate buffer containing 0.05% Tween 20. The blocking buffer solution was Dulbecco’s phosphate buffer containing 3% (w/v) BSA. Apparatus. Photoelectrochemical measurements were performed with a homemade photoelectrochemical system. A 500 W Xe lamp equipped with monochromator was used as the irradiation source. The monochromatic illuminating light in-
J. Phys. Chem. C, Vol. 113, No. 25, 2009 11143 tensity was about 400 µW/cm2, estimated with a radiometer (Photoelectric Instrument Factory of Beijing Normal University). Photocurrent was measured on a CHI 750a electrochemical workstation. CdS QDs modified ITO electrode with an area of 0.25 cm2 was employed as the working electrode. A Pt wire was used as the counter electrode and a saturated Ag/AgCl as the reference electrode. All the photocurrent measurements were performed at a constant potential of 0 V (vs saturated Ag/AgCl). A 0.1 M phosphate buffer solution (PBS, pH 7.0) containing 0.1 M AA was used as the supporting electrolyte for photocurrent measurements. The solution was deaerated by highly pure nitrogen for 15 min before photoelectrochemical experiments and then a N2 atmosphere was kept over the solution for the entire experimental process. UV-vis absorption spectra were obtained on a Shimadzu UV-3600 UV/vis spectrophotometer (Shimadzu corporation, Japan). High-resolution transmission electron microscopy (HRTEM) was carried out on a JEM-2100 transmission electron microscope (JEOL Co. LTD). Electrochemical impedance spectroscopy (EIS) was carried out on an Autolab potentiostat/galvanostat (PGSTAT30, Eco Chemie, B.V., Utrecht, The Netherlands) in 0.1 M KCl containing a redox probe of 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture at an open circuit potential of 216 mV with an applied voltage of 5 mV over a frequency range of 0.1 Hz-100 kHz. Synthesis of Water-Soluble CdS QDs. TGA-stabilized CdS QDs were synthesized by using a slightly modified procedure reported by Feng et al.25 Typically, 250 µL of TGA was added to 50 mL of 1.0 × 10-2 M CdCl2 aqueous solution, after that, 1.0 M NaOH was added to adjust the pH of the above solution to a desired value (7.0, 9.0, and 11.0). During the above process, N2 was bubbled throughout the solution to remove O2 for 30 min. Then, 5.0 mL of 0.1 M Na2S aqueous solution was injected into this solution to obtain TGA-capped water-soluble CdS QDs and the reaction mixture was refluxed under N2 atmosphere for 4 h. This procudure produced CdS QDs with a Cd to S (Cd/S) ratio of 1.0. When CdS QDs with Cd/S ratios of 2.0 and 1.6 were synthesized, 2.0 × 10-2 and 1.6 × 10-2 M CdCl2 solutions were used, respectively, while the amount of Na2S was unchanged. When CdS QDs with a Cd/S ratio of 0.83 was synthesized, 6 mL of 0.1 M Na2S was added, while the amount of CdCl2 remained unchanged. Due to the formation of precipitate, water-soluble CdS QDs with a smaller Cd/S ratio could not be obtained. All the finally obtained TGA-capped CdS QDs was diluted with the same volume of water and stored in a refrigerator at 4 °C for future use. Layer-by-Layer Assembly of CdS QDs on the ITO Electrode. The ITO slices (type N-STN-S1-10, China Southern Glass Holding Co., LTD, Shenzhen, China, ITO coating 180 ( 20 nm, sheet resistance 8.1 ( 0.6 Ω/square) were sonicated in acetone, NaOH (1 M) in 1:1 (v/v) ethanol/water, and water, respectively, for about 15 min each. The (PDDA/CdS)n multilayer film was grown by alternately dipping of the cleaned ITO slices into a solution of 2% PDDA containing 0.5 M NaCl and the as-obtained QDs solution for 10 min, respectively. The films were carefully washed with doubly distilled water after each dipping step. This process was repeated to obtain a desired number of bilayers (n). Immunosensor Construction. Conjugation of antibodies onto a CdS nanocrystals modified electrode was achieved by using the classic EDC coupling reactions between COOH groups on the surfaces of the TGA-capped CdS QDs and NH2 groups of the antibody. First, the CdS QDs modified electrode was activated by immersion in a solution containing 10 mg/mL of EDC and 20 mg/mL of NHS in distilled water for 50 min. Then
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SCHEME 1: Schematic Diagram of the Stepwise Immunosensor Fabrication Process
Figure 1. (A) The dependence of photocurrent of the PDDA/CdS film in different solutions: 0.1 M PBS (pH 7.0) alone; (b) 0.1 M PBS + 0.1 mol/L of Na2SO3; (c) 0.1 M PBS + 0.10 M TEA; and (d) 0.1 M PBS + 0.1 M AA. (B) The dependence of photocurrent of the PDDA/CdS film in 0.1 M PBS (pH 7.0) on the concentration of AA. The excitation wavelength was 390 nm. The light was turned on and off as indicated.
SCHEME 2: Photocurrent Generation Mechanism of CdS QDs
the activated electrode was thoroughly rinsed with water and dried. Following this step, 25 µL of goat antimouse IgG (0.5 mg/mL) was dropped on the electrode surface and the electrode was incubated at 4 °C overnight. After incubation, the electrode was rinsed with the washing buffer solution. This electrode was then blocked with 25 µL of blocking buffer solution for 1 h at room temperature and washed with the washing buffer solution thoroughly. Finally, 25 µL of analyte (mouse IgG) solution with different concentrations were added and the mixture was incubated at 37 °C for 1 h followed by washing with the washing buffer solution four times. A detailed description of the immunosensor process is illustrated in Scheme 1. The analytical application of the immunosensor was evaluated by determining the recoveries of mouse IgG in a mouse serum spiked with mouse IgG (0.4, 1, 5, 10, and 50 ng/mL). The mouse serum was diluted 1:50 000 (v/v) for determining the recoveries of 10 and 50 ng/mL of mouse IgG, and 1:1 000 000 (v/v) for determining the recoveries of 0.4, 1, and 5 ng/mL of mouse IgG. Results and Discussion Photoelectrochemical Property of the PDDA/CdS Film. CdS is one of the most widely studied nanocrystalline semiconductor as photoanode in photoelectrochemical (PEC) cells due to its excellent photoelectrochemical propeties. The photocurrent generation mechanism of CdS QDs is shown in Scheme 2. When the material absorbs photons with energies higher than that of its band gap, electrons are excited from the (occupied) valence band to the (empty) conduction band, forming the electron-hole pairs. Once the process happened, the electron-hole pairs would recombine or the charges would be transferred. The electron transfers to the ITO electrode and
generates photocurrent because the energy level of the conduction band of ITO is lower than that of CdS.26,27 The hole transfers to the surface of the semicondutor material and can be captured by an electron donor (or hole scavenger) present in solution. For CdS nanoparticles, there exits a corrosion process (lattice dissolution) under illumination: 2h+ + CdS f Cd2+ + S, if there is no electron donor for scavenging of the holes.28 When an efficient electron donor is in the solution, the photodissolution reaction and the electron-hole recombination are inhibited and the photocurrent intensity can be enhanced. Polysulfide (Na2S and S),29 Na2SO3,30 and triethanolamine (TEA)31 are widely used as electron donors in strong alkaline solution (around pH 12) for the CdS-based photoelectrochemical cells for obtaining high and stable photocurrent. However, a mild solution medium is strongly required when QDs are used for biosensing.23,24 As is well-known, ascorbic acid (AA) is a powerful antioxidant used widely in the food industry32 and for disease treatment33 because it is in general regarded to be safe and healthy. The formal potential of the redox couple of AA is -0.185 V (vs. SCE).34 So, AA can be easily oxidized by the holes (E0 ) 1.38 V)27 generated by illuminated CdS nanoparticles. Here, we investigated the photoelectrochemical behaviors of PDDA/CdS film under different electron donors’ (such as Na2SO3, TEA, and AA) solutions (shown in Figure 1A). All the photocurrents in these solutions containing electron donors were much higher than that in PBS medium alone. It should be especially pointed out that the photocurrent in PBS solution containing AA was greatly higher than that in other cases (containing Na2SO3 and TEA), which indicated that AA was an excellent hole scavenger in such a mild medium. Thus, AA was selected as an efficient and nontoxic electron donor for the following experiments. The intensity of the photocurrent increased with the increased concentrations of added electron donor AA and reached a maximum for 0.1 M AA, then a weak decrease was observed for much higher concentrations of AA (see Figure 1B). As a hole scavenger, AA in lower concentrations would result in a fall in electrical output because fewer reducing agent molecules were available for electron donation to photogenerated holes. While a much higher concentration of AA would also result in the increase of the absorbance of AA in solution, as a consequence, the intensity of the irradiation arriving at the electrode surface decreased and the efficiency of excited QDs would decrease. Due to the lack of systematic investigation on the photoelectrochemical properties of water-soluble CdS QDs, we investigated the effects of synthetic conditions such as reaction pH and mole ratio of Cd/S on the properties of the synthesized
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Figure 3. (A) UV-vis absorption spectra of alternating deposition of (PDDA/CdS)n films (n ) 1-5); the insert image represents the absorption spectrum of CdS dispersion in aqueous solution. (B) The absorbance of the multilayer films measured at 350 nm vs. the number of bilayers (n).
Figure 2. The photocurrent action spectra of CdS QDs synthesized at pH (A) 7.0, (B) 9.0, and (C) 11.0. At each reaction pH, CdS QDs was synthesized with various ratios of Cd/S: (9) 2.0, (b) 1.6, (2) 1.0, and (1) 0.83.
products in detail. The synthesized CdS QDs under different conditions were assembled on PDDA modified ITO electrodes for photocurrent measurements. For CdS QDs synthesized with a fixed Cd/S ratio, the reaction pH prior to Na2S addition could influence the photoelectrochemical properties of the final products. At higher pH, the surface trap states of the CdS QDs, which can act as recombination center of the electron-hole pairs, would be removed.35 As a result, the CdS QDs which had fewer surface states would have larger photocurrent. This has been proven by our experiments. With increasing reaction pH, the photocurrent at the corresponding CdS QDs modified electrode increased (shown in Figure 2A-C). The photocurrents at the CdS QDs modified electrode which were synthesized at pH 11.0 were much higher than those obtained at pH 7.0 and 9.0 at the same Cd/S ratio. At the same reaction pH, the CdS QDs obtained with different Cd/S ratio also had different photoelectrochemical properties. The photocurrent increased with the decrease of the Cd/S ratio (from 2.0 to 0.83) (Figure 2), which indicated that CdS QDs synthesized with excess Cd2+ have relatively lower photocurrent intensity, although these QDs have been demonstrated to have high photoluminescence quantum yield. Here, a large Cd/S ratio could provide a large amount of Cd2+ sites on the particle surface; adsorption of OHand TGA onto free Cd2+ sites might lead to the formation of a shell-like structure on its surface and the shell-like structure could build a potential wall for confinement of photogenerated holes.36 Due to the confinement, the photogenerated holes could not react with AA in solution and as a result, the photocurrent intensity decreased. The fact that excess Cd2+ could decrease the photocurrents of CdS synthesized by other methods was also observed previously.37,38 From Figure 2, we can see that CdS QDs synthesized with a Cd/S mole ratio of 1.0 and 0.83 at pH 11.0 have identical and highest photocurrent intensity.
However, the photocurrent of the CdS QDs obtained at a Cd/S ratio of 0.83 decreased when at storage, while the product obtained at a Cd/S ratio of 1.0 was rather stable for storage at 4 °C for more than one month. Finally, we chose the product synthesized at a Cd/S mole ratio of 1.0 (pH 11.0) for assembling the multilayer film for biosensor applications. All the photocurrent action spectra in Figure 2 showed that the maximum photocurrent intensity is around 380-390 nm. Longer wavelength (>390 nm) went against the absorbance of CdS QDs, which would result in a decrease of photocurrent. At shorter wavelength (
