Article pubs.acs.org/ac
White-Light-Exciting, Layer-by-Layer-Assembled ZnCdHgSe Quantum Dots/Polymerized Ionic Liquid Hybrid Film for Highly Sensitive Photoelectrochemical Immunosensing of Neuron Specific Enolase Xiangyang Yu,† Yanying Wang,†,‡ Xuemin Chen,† Kangbing Wu,‡ Danchao Chen,§ Ming Ma,§ Zhenjia Huang,† Wangze Wu,∥ and Chunya Li*,† †
Key Laboratory of Analytical Chemistry of the State Ethnic Affairs Commission, College of Chemistry and Materials Science, South-Central University for Nationalities, Wuhan 430074, China ‡ Key Laboratory for Large-Format Battery Materials and System, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China § Ningbo Entry−Exit Inspection and Quarantine Bureau of P.R.C., Ningbo 315012, China ∥ The Central Hospital of Wuhan, Wuhan 430014, China S Supporting Information *
ABSTRACT: ZnCdHgSe quantum dots (QDs) functionalized with N-acetyl-L-cysteine were synthesized and characterized. Through layer-by-layer assembling, the ZnCdHgSe QDs was integrated with a polymerized 1-decyl-3-[3-pyrrole-1-yl-propyl]imidazolium tetrafluoroborate (PDPIT) ionic liquid film modified indium tin oxide (ITO) electrode to fabricated a photoelectrochemical interface for the immobilization of rabbit antihuman neuron specific enolase (anti-NSE). After being treated with glutaraldehyde vapor and bovine serum albumin successively, an anti-NSE/ZnCdHgSe QDs/PDPIT/ITO sensing platform was established. Simplely using a white-light LED as an excitation source, the immunoassay of neuron specific enolase (NSE) was achieved through monitoring the photocurrent variation. The polymerized ionic liquid film was demonstrated to be an important element to enhance the photocurrent response of ZnCdHgSe QDs. The anti-NSE/ZnCdHgSe QDs/PDPIT/ITO based immunosensor presents excellent performances in neuron specific enolase determination. The photocurrent variation before and after being interacted with NSE exhibits a good linear relationship with the logarithm of its concentration (log cNSE) in the range from 1.0 pg mL−1 to 100 ng mL−1. The limit of detection of this immunosensor is able to reach 0.2 pg mL−1 (S/N = 3). The determination of NSE in clinical human sera was also demonstrated using anti-NSE/ZnCdHgSe QDs/PDPIT/ITO electrode. The results were found comparable with those obtained by using enzyme-linked immunosorbent assay method. n biomedical research and diagnosis fields, it is very important to detect disease-related biomarkers with high sensitivity, accuracy, and low-cost.1 Accurate determination of biomarkers related to malignant tumors is able to provide significant benefits to early diagnosis and disease surveillance.2 It is also helpful to evaluate the availability of therapy method through monitoring the concentration of specific biomarkers.3 NSE is known as a specific tumor marker for identifying some related diseases, such as small cell lung carcinoma4 and neuroblastoma.5 In the case of NSE examination, if the result of NSE is 5−12 ng mL−1 in serum and 100 ng mL−1) usually indicates high risk of these two diseases.7 Therefore, the NSE concentration is an indicator for diagnosing these two diseases and is also used to assess the patient’s
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© XXXX American Chemical Society
recovery progress. In addition, the rapid and accurate determination of NSE concentration shows great importance such as earlier prediction, simpler operation, and less cost than currently used clinical techniques.8,9 Hence, NSE determination is of great significance in terms of both clinic diagnosis and treatment for related cancers and diseases. In recent years, a lot of immunoassay methods have been constructed for NSE determination, such as radioimmunoassay, chemiluminescence immunoassay, ELISA, fluoroimmunoassay, and electrochemical immunoassay.10−15 Each of these analytical methods has its own advantage and limitation. Thus, it is highly desirable to fabricate a sensing platform to conveniently determine NSE Received: November 30, 2014 Accepted: March 19, 2015
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DOI: 10.1021/ac504456w Anal. Chem. XXXX, XXX, XXX−XXX
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QDs opened a new way for the design of photoelectrochemical sensors.
concentration with high sensitivity and selectivity, good stability, and high throughput. Photoelectrochemical (PEC) immunosensing has been progressed remarkably and paid more attention to as a method for biomolecules assay. Photoelectrochemical immunosensor commonly use light as an excitation source to produce photocurrent as a detection signal which can be measured by an electronic readout system. Combining with antibody/ antigen as the biosensing element, these type of immunosensors are facile to be fabricated and easily to be miniaturized. In addition, these methods are convenient and high-throughput for high selective and sensitive examination of biomarkers in clinical samples.16,17 To date, a variety of semiconductor materials, quantum dots and complexes have been used as photoelectrochemical sensing materials to design PEC sensors.18−26 Semiconductor nanocrystals show promising potential as near-infrared (NIR) emitting materials. They can be effectively excited to produce photocurrent by a light source with long wavelengths in the NIR region.27−30 Their photoelectrochemical properties are often highly dependent on their size and composition.31,32 These NIR nanocrystals are also considered as excellent sensing materials for biological analysis and bioimaging, because they can eliminate the interference from biosamples which mainly absorb or scatter visible light.33 Thus, to develop NIR nanocrystals and exploit their applications in bioassay are very attractive to many scientists and researchers. Ionic liquids (ILs) have been proved to possess many highly desirable properties34−37 and have got extensive applications in synthesis, separation and assay, photoelectric transformation, material preparation and biosensing.38−46 It has been demonstrated that the integration of ionic liquids and nanomaterials can produce some excellent features to improve their sensing performances. For instance, the electrochemical sensing of human IgG has proved a significantly enhanced effect of 4-amino-1-(3-mercapto-propyl)-pyridine hexafluorophosphate ionic liquid which was modified onto gold nanoparticles surface through covalent binding.47 1-Butyl-3methylimidazolium hexafluorophosphate modified on glassy carbon electrode surface can intensify the electrochemical luminescence intensity of CdTe QDs, and improve its sensing performance toward gossypol.48 Herein, a near-infrared water-soluble quaternary ZnCdHgSe QDs was synthesized. When ZnCdHgSe QDs are used as a sensing element to construct a photoelectrochemical platform, a commercial white-light LED is able to excite them to produce photocurrent with high efficiency. 1-Decyl-3-[3-pyrrole-1-ylpropyl]imidazolium tetrafluoroborate (DPIT) was electrochemically polymerized onto ITO electrode surface to construct a polymerized ionic liquid (PDPIT) interface for fixation of ZnCdHgSe QDs. Polymerized ionic liquid film has been proved to be an important element for enhancing photoelectrochemical response of ZnCdHgSe QDs. Anti-NSE was immobilized onto the ZnCdHgSe QDs/PDPIT/ITO interface to fabricate an immunosensor for neuron specific enolase. Through the combination of the merit characteristics of polymerized ionic liquid and ZnCdHgSe QDs, the sensing platform is able to improve the photocurrent response, selectivity and sensitivity toward NSE significantly. We also demonstrated that the established photoelectrochemical immunosensor can provide a practical approach for determination of NSE in clinical serum samples. In addition, the layerby-layer assembling of polymerized ionic liquid and ZnCdHgSe
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EXPERIMENTAL SECTION Materials. NAC was supplied by Shanghai Aladdin Reagent Inc. (Shanghai, China). CdCl2·2.5H2O, ZnCl2, and HgCl2 were purchased from Chengdu Chemical Reagent Plant (Chengdu, China). Selenium powder, NaBH4, and ascorbic acid were bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA), glutaraldehyde and Tween 20 were purchased from Amresco (Ohio, U.S.A.). NSE, rabbit antihuman neuron specific enolase (anti-NSE), prostate specific antigen (PSA), and α-fetoprotein (AFP) were bought from Beijing Biosynthesis Biotechnology Co., LTD (Beijing, China). Other chemicals were analytical grade and were used without any further purification. Human serum samples were obtained from the central hospital of Wuhan. Indium tin oxide (ITO) was obtained from China Southern Glass Holding Co., LTD (Shenzhen, China). Dulbecco’s phosphate buffer (pH 7.4) was prepared with 1.47 mmol L−1 KH2PO4, 8.10 mmol L−1 Na2HPO4, 2.67 mmol L−1 KCl, and 138 mmol L−1 NaCl. Dulbecco’s phosphate buffer containing 0.05% Tween 20 was used to wash electrode surface. Unspecific sites were blocked with Dulbecco’s phosphate buffer containing 1% (w/v) BSA. Apparatus. FTIR spectra were conducted on Nicolet NEXUS-470 FTIR spectrometer (Thermo Nicolet, USA). Fluorescence spectrum was measured on PerkinElmer LS-55 luminescence spectrometer (PerkinElmer, USA). UV−vis analysis was performed on PE Lambda Bio 35 (PerkinElmer, USA). NMR was carried out with Avance 400 MHz NMR Spectrometer (Bruker, Switzerland). FEI Tecnai G2 20S-TWIN instrument (FEI Company, Netherlands) which was operated at an acceleration voltage of 200 kV was employed for transmission electron microscopic (TEM) images. X-ray photoelectron spectroscopy (Thermo Electron Corp., USA) was used to analysis the composition of ZnCdHgSe QDs. Scanning electron microscopic images were obtained on a Sirion 200 microscope (FEI) with the electron beam voltage of 3.0 kV and working distance of 4.6 mm. A white-light LED lamp (5W) was applied to excite the ZnCdHgSe QDs. The light intensity was about 160 mW cm−2 measured with a radiometer which is supplied by Photoelectric Instrument Factory of Beijing Normal University (Beijing, China). All electrochemical measurements were performed with CHI 660E electrochemical workstation (Chen Hua Corp., Shanghai, China) using a three-electrode system including an ITO working electrode (0.28 cm2), a Pt wire counter electrode and a saturated calomel reference electrode (SCE). All the photocurrent measurements were performed at a constant potential of 0 V (vs. SCE). Photocurrents were measured in a 0.1 mol L−1 phosphate buffer solution (pH 7.0) containing 0.2 mol L−1 ascorbic acid. In electrochemical impedance spectroscopic measurements, the frequency range was varied from 100 kHz to 100 mHz, the initial potential was the average value of the redox peak potentials, and the amplitude was 5 mV. Neuron specific enolase concentration in clinical serum samples were measured with Olympus AU5421 automatic biochemical analyzer (Olympus Optical Co., Ltd., Japan). ELISA has been achieved in terms of the instructions of a commercial kit (Leadman Group Co., Ltd., China). Synthesis of ZnCdHgSe QDs. ZnCdHgSe QDs were synthesized using an aqueous phase method: NaHSe solution B
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Figure 1. Characterizations of ZnCdHgSe QDs with UV−vis (a) and fluorescence emission spectroscopy (b), FTIR spectroscopy (d), transmission electron microscopy (e), X-ray photoelectron spectroscopy (f), and X-ray diffraction (g). Curve c is the FTIR spectrum of NAC.
(D2O) δ 8.396 (1H, d), 7.37 (2H, d), 6.69(2H, d), 6.08 (2H, d), 4.08(2H, t), 4.03(2H, t), 3.98(2H, t), 3.03(2H, t), 2.32 (2H, t), 2.00 (2H, t), 1.135 (12H, t), 1.17 (3H, t); m/z = 316.278. Preparation of ZnCdHgSe QDs/PDPIT/ITO Electrode. ITO slices were washed with 15 min sonications in acetone, NaOH (1 mol L−1) in ethanol/water (v/v, 1:1) and water, respectively. After cleaning, the ITO slices were dried at room temperature and covered with a plastic film with a round hole whose area is 0.28 cm2. Electrochemical polymerization was carried out in a 0.01 mol L−1 sodium tetrafluoroborate solution containing 0.04 mmol L−1 DPIT with the potential step from 0.5 to 1.3 V. Step time for 0.5 and 1.3 V was set as 2 and 5 s, respectively. After thoroughly washed with ultrapure water, a polymerized ionic liquid film electrode (PDPIT/ITO) was fabricated. Then, the PDPIT/ITO electrode was dipped into a ZnCdHgSe QDs solution for 10 min. After each step for the assembling of ZnCdHgSe QDs onto PDPIT film surface, the electrode was gently washed with ultrapure water. A desired layer number (n) of ZnCdHgSe QDs/PDPIT film can be obtained by repeating the above process accordingly. Fabrication of NSE Immunosensors. Twenty microliters of anti-NSE (5.0 μg mL−1) was drop coated onto the ZnCdHgSe QDs/PDPIT/ITO electrode surface. After incubation overnight at 4 °C, the electrode was washed with a buffer solution. Then, the electrode was immersed in glutaraldehyde vapor for 5 min. The covalent immobilization of anti-NSE onto ZnCdHgSe QDs/PDPIT/ITO surface can be achieved via the cross-linking reaction between aldehyde groups and amino groups. The electrode was then incubated in bovine serum albumin solution to block all unspecific sites to produce a NSE immunosensor which was denoted as anti-NSE/ZnCdHgSe QDs/PDPIT/ITO electrode. During specific recognition process, 20 μL of NSE solution with different concentration was added onto antiNSE/ZnCdHgSe QDs/PDPIT/ITO electrode surface, and was incubated at 35 °C. After incubation for 30 min, the
was prepared by adding selenium powder into a cooled NaBH4 solution in a molar ratio of 2:1. Fifteen milliliters of 0.005 mol L−1 ZnCl2, 1 mL of 0.005 mol L−1 CdCl2, 5 mL of 0.005 mol L −1 HgCl 2 , and 5 mL of 0.05 mol L −1 NAC were homogeneously mixed in a three-necked flask, and were deaerated by bubbling nitrogen. The reaction solution was diluted to 50 mL with ultrapure water. Then, the pH of the solution was adjusted to 11.1 by adding 1.0 mol L−1 of NaOH solution dropwisely. Zn, Cd, Hg, and NAC was approximately loaded at the molar ratio of 15:1:5:50. Under vigorous stirring condition, the freshly prepared NaHSe solution was instantly injected into the homogeneously mixed solution and kept on stirring for 4 h at 80 °C to promote the growth of the quaternary ZnCdHgSe QDs. After cooling to room temperature, ZnCdHgSe QDs was purified using centrifugation performed on a Millipore ultrafiltration centrifuge tube at 10000 rpm for 15 min. Synthesis of 1-Decyl-3-[3-pyrrole-1-yl-propyl]imidazolium Tetrafluoroborate. Under nitrogen atmosphere, N-decyl imidazole and 1-(3-bromopropyl) pyrrole were dissolved into toluene. With a continue stirring, the reaction temperature was maintained at 80 °C for 24 h. Then, the reaction was terminated by cooling to room temperature. The product was extracted with ultrapure water and then the collected aqueous phase was washed with diethyl ether. The aqueous layer was evaporated under vacuum to produce 3-decyl-1-(3-pyrrol-1-ylpropyl)imidazolium bromide as a pale yellow oil. Subsequently, 3-decyl-1-(3-pyrrol-1-yl-propyl)imidazolium bromide was reacted with sodium tetrafluoroborate to exchange anions. After filtering and removing the solvent, the residue was thoroughly washed using ethyl acetate, filtered, and evaporated under reduced pressure to afford 3-decyl-1-(3-pyrrol-1-yl-propyl)imidazolium tetrafluoroborate (DPIT) ionic liquid. The produced ionic liquid was characterized with 1H NMR and HPLC-MS. The results are shown in Supporting Information as Figures S1 and S2. Some data are selected as follows: 1H NMR C
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Analytical Chemistry immunosystem was washed with a buffer solution for three times, and then was stored at 4 °C. Supporting Information Scheme S1 illustrated a typical process for the fabrication of an anti-NSE/ZnCdHgSe QDs/PDPIT film based immunosensor.
Figure 1f shows the composition of ZnCdHgSe QDs, which was investigated by X-ray photoelectron spectroscopy. The peaks located at 284.6, 531.88, 399.25, and 161.07 eV are attributed to C1s, O1s, N1s, and S2p levels, respectively. Meanwhile, Zn2p, Cd3d, Hg4f, and Se3d levels are also detected at 1021.44, 404.37, 100.52, and 53.39 eV. The presence of the above-mentioned elements belong to NAC and ZnCdHgSe QDs means the successful integration of them. The crystal characteristic of ZnCdHgSe QDs was investigated by X-ray diffraction. The XRD pattern obtained from ZnCdHgSe QDs shows some broad peaks and is presented in Figure 1g. The 2θ values of these peaks are measured to be 23.98°, 43.44°, and 51.57°, respectively. These peaks are the planes corresponding to (111), (220), and (311) planes of ZnSe. The average diameter for the ZnCdHgSe QDs is calculated to be 2.64 nm from the (111) peak width using the Scherrer-equation. The result is in good agreement with the value, 2.34 ± 0.21 nm, estimated from TEM image. Layer-by-layer assembled polyelectrolytes and nanoparticles with opposite charges provides a promising route to prepare hybrid materials.49 This method was reasonably employed in assembling ZnCdHgSe QDs/PDPIT film whose thickness can be easily modulated by adjusting the assembled layer number. DPIT was electrochemically deposited onto the ITO electrode surface to produce a polymerized ionic liquid film, which would offer a solid interface with positive charges. Consequently, negatively charged ZnCdHgSe QDs can be efficiently assembled onto the PDPIT/ITO surface with the help of an anion-exchange process based on the electrostatic interaction. The effect of electrostatic interaction on the structure of ZnCdHgSe QDs/PDPIT was investigated using scanning electron microscope. As shown in Supporting Information Figure S4, a significant difference was observed in the morphologic image of the cross section of the ZnCdHgSe QDs/PDPIT/ITO electrode fabricated using ZnCdHgSe QDs capped with NAC which was transformed from a negatively charged molecule to an uncharged neutral molecule by adjusting the pH from 9.6 to 3.0. Compared with a single layer of ZnCdHgSe QDs/PDPIT film, multilayer film is more effective in improving the photocurrent response due to the fact that a significant amount of ZnCdHgSe QDs can be loaded onto the sensing interface. Supporting Information Figure S5a displays photocurrent responses of ZnCdHgSe QDs/PDPIT hybrid films independently modified on ITO electrode surface from one layer to eight layers. When the layer number (n) was varied from one to four, the corresponding photocurrent increased accordingly, suggesting that the photoelectrochemical response is dependent on the amounts of ZnCdHgSe QDs. Nevertheless, if the ZnCdHgSe QDs/PDPIT film continues to increase over four layers, an adverse effect occurs, namely that the corresponding photocurrent decreases. The photocurrent intensity of the ZnCdHgSe QDs/PDPIT multilayer would be influenced by two factors. On the one hand, more amount of photoelectrochemically active material was loaded onto the sensing interface with the ZnCdHgSe QDs/PDPIT layer increasing, generating more holes and hence, being scavenged by ascorbic acid to enhance its photocurrent response. On the other hand, ascorbic acid is difficult to diffuse to the inner portion of a thicker ZnCdHgSe QDs/PDPIT film to scavenge the oxidant, holes, to generate photocurrent. As a result, the photocurrent of the developed ZnCdHgSe QDs/PDPIT/ITO sensing platform decreases reversely. This observation is consisted with the
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RESULTS AND DISCUSSION ZnCdHgSe QDs were characterized using UV−vis spectroscopy in the wavelength range from 380 to 800 nm. The spectrum, shown as curve a in Figure 1, reveals that ZnCdHgSe QDs have a strong absorption in the wavelength range. It implies that the ZnCdHgSe QDs can be excited by a visible-light source, and thus it is suitable to be employed as a substrate for photoelectrochemistry. As expected, a commercial white-light LED was demonstrate to be an effective light source to excite ZnCdHgSe QDs modified on PDPIT/ITO interface to produce photocurrent response, suggesting that a convenient and cheap way to fabricate a photoelectrochemical sensor is feasible. The photocurrent response of a ZnCdSe QDs/ PDPIT/ITO electrode was also studied to investigate the effect of Hg. From Supporting Information Figure S3a, it was found that photocurrent decreased significantly in the absence of Hg. The result indicates that the photoelectric transformation efficiency of ZnCdHgSe QDs is distinctly higher than that of ZnCdSe QDs. A typical fluorescence emission spectrum of ZnCdHgSe QDs at an excitation wavelength of 470 nm was presented in Figure 1b. The maximum emission wavelength is observed at 690 nm. It is especially noted that the fluorescence may also be noticeable when the wavelength is longer than 800 nm, although it did not show in the figure because of the limitation of our instruments. The fluorescence emission spectrum of ZnCdSe QDs was also displayed in Supporting Information Figure S3b. The maximum emission wavelength appears at 540 nm which is obviously shorter than that of ZnCdHgSe QDs. FTIR spectroscopy was carried out to characterize NAC (c) and the NAC capped ZnCdHgSe QDs (d) and was illustrated in Figure 1. From curve c, the characteristic absorption signal of S−H stretching vibration was observed at 2547.48 cm−1. An infrared absorption peak appeared at 1717.16 cm−1 originates from the asymmetric stretching of carboxyl groups. Furthermore, the spectral features of the C−H stretching vibration belong to methyl and methylene in NAC are found at 2964.85, 2900.71, and 2808.78 cm−1. These characteristic C−H bond absorption signals can also be observed obviously in the ZnCdHgSe QDs. An exceptional example was observed during the characterization. It is obvious to note that the S−H absorption peak at 2547.48 cm−1 in NAC is not observed in the spectrum of ZnCdHgSe QDs. This could be explained by the possible chemical reactions occurred between the metal ions and sulfur during the preparation process. By the comparison of FTIR spectra, it is reasonable to conclude that ZnCdHgSe QDs have been successfully functionalized with NAC. Transmission electron microscopy (TEM) is a powerful tool for morphologic analysis of nanomaterials, thus was used to characterize the morphology of ZnCdHgSe QDs. As shown in Figure 1e, TEM image reveals that the shape of ZnCdHgSe QDs is mainly spherical. With the diameter of 2−3 nm, the particle size distribution of ZnCdHgSe QDs turns out to be homogeneous. After being stored in a refrigerator for 6 months, no observable change was found in color and dispersity of the ZnCdHgSe QDs sample in solution, meaning that ZnCdHgSe QDs possesses well stability and dispersity. D
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Figure 2. Electrochemical impendence spectroscopy (A) and photocurrent response (B) of ITO electrode (a); ZnCdHgSe QDs/PDPIT/ITO electrode before (b) and after (c) anti-NSE immobilization; blocking with BSA (d); and being incubated in NSE solution at the concentration of 5.0 ng mL−1 (e). Inset is the electrical equivalent circuit.
fabricated by modifying the ZnCdHgSe QDs/PDPIT/ITO interface with anti-NSE. The immunoreaction between antiNSE/ZnCdHgSe QDs/PDPIT/ITO and NSE forms an immunocomplex to block the transport of ascorbic acid to the ZnCdHgSe QD surface to react with holes. In the event of this process, more electrons and holes may be recombined, thus leading to a decline in the photocurrent accordingly. The photocurrent variation caused by the specific immunoreaction is directly correlated with the NSE concentration, and thus it can be used as an indicator for NSE determination. A mechanism for photocurrent generation was proposed in Supporting Information Scheme S2. The charge-transfer resistance (Rct) obtained from electrochemical impedance spectroscopy reflects the restricted diffusion of K3[Fe(CN)6]/K4[Fe(CN)6] probes through a multilayer film, and directly relates to its permeability. As shown in Supporting Information Figure S6a, the Rct value calculated from the Nyquist plot of K3[Fe(CN)6]/K4[Fe(CN)6] at the unmodified ITO electrode is about 90.9 Ω. After the layer-by-layer modification with ZnCdHgSe QDs and PDPIT film, as shown in Supporting Information Figure S6b− g, the Rct progressively increased to 134.9, 333.2, 619, 396, 1017, and 1514 Ω, respectively. The increasing in the charge transfer resistance of K3Fe(CN)6/K4Fe(CN)6 would be attributed to the hindrance effects of a nonconductive long carbon chain in the polymerized ionic liquid and the ZnCdHgSe QDs which possess lower conductivity because of the intrinsic property of a semiconductor. Each step for the fabrication of anti-NSE/ZnCdHgSe QDs/ PDPIT/ITO based immunosensor was also characterized using electrochemical impedance spectroscopy. Figure 2A exhibits the Nyquist plots of K3Fe(CN)6/K4Fe(CN)6 at different electrodes fabricated with a step-by-step procedure. The inserted picture is an equivalent electrical circuit used to simulate the data for Nyquist plots. Where, Rs, Rct, Zw, and Cdl represent the resistance of electrolyte solution, charge transfer resistance, Warburg impedance and the double-layer capacitance, respectively. From Nyquist plots, we can obviously see that, for a bare ITO, the charge transfer resistance which was calculated to be 90.9 Ω is very small (curve a). After the antiNSE was covalently immobilized onto ZnCdHgSe/PDPIT/ ITO electrode surface, Rct significantly increased from 396 Ω (curve b) to 696 Ω (curve c). When nonspecific sites were deactivated by BSA, Rct increased further to 1926 Ω (curve d). The increase in charge transfer resistance may come from the nonconductive characteristics of BSA which would retard the
previous reports that ions and small molecules can permeate polyelectrolyte films, however, the transport process is retarded by increasing the polyelectrolyte film thickness.50 The balance between the above-mentioned factors can be easily modulated by varying the film thickness. As far as a thinner film is concerned, when the layer number is less than 4, the first one is the predominated factor, and the photocurrent intensity is positively correlated to the ZnCdHgSe QDs/PDPIT film thickness directly. Conversely, with further increasing the layer number, the ZnCdHgSe QDs/PDPIT film becomes thicker and produce diffusion barrier for ascorbic acid, and thus causing a decrease in the photocurrent intensity. In addition, as the layer number of ZnCdHgSe QDs increasing, the negative charge of the electrode surfaces also increases, and leading to the increase of the repulsion toward negatively charged ascorbic acid. Consequently, the photocurrent response of ZnCdHgSe QDs/ PDPIT/ITO decreases. The enhanced effect of polymerized ionic liquid was also demonstrated by comparing the photocurrent responses of ZnCdHgSe QDs/PDPIT/ITO electrode (Supporting Information Figure S5b) and ZnCdHgSe QDs/ITO electrode (Supporting Information Figure S5c). Using the polymerized ionic liquid film as the mediate, the photocurrent response increases significantly. The response improvement is originated from the large amounts of ZnCdHgSe QDs assembled onto the PDPIT/ITO electrode surface because of the electrostatic interaction and the large surface area. In the photoelectrochemical process, those photons possess higher energy than the band gap of ZnCdHgSe QDs are absorbed partially. Lowenergy electrons in the valence band accept energy from these high-energy photons and are effectively excited to the conduction band. As a result of the excitation, many electron−hole pairs will be produced immediately. Subsequently, the electrons will either eventually recombine with the holes or pass through the ITO electrode surface to generate photocurrent. If electron donors were added into, they can scavenge holes and reduce the electron−hole recombination, leading to photocurrent enhancement.51 In addition, the scavenging of holes can inhibit the photo corrosion of semiconductor modified photoelectrodes.52 Using ascorbic acid as an electron donor, ZnCdHgSe QDs/PDPIT/ITO interface produces a stable anodic photocurrent, which can be observed from Supporting Information Figure S5 obviously. The high photoelectrical activity makes ZnCdHgSe QDs/ PDPIT/ITO a good substrate for photoelectrochemical biosensor. In our experiments, a NSE immunosensor was E
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Figure 3. Scanning electron microscopic images of the bare ITO (a), PDPIT/ITO (b), ZnCdHgSe QDs/PDPIT/ITO (c), and anti-NSE/ ZnCdHgSe QDs/PDPIT/ITO (d) electrode surface.
photocurrent response of NSE (5.0 ng mL−1) with the asprepared immunosensor and the incubation temperature was illustrated in Supporting Information Figure S7a. The maximum value of the photocurrent variation was obtained at 35 °C. Therefore, the optimum incubation temperature for photoelectrochemical determination of NSE was set at 35 °C. Supporting Information Figure S7b depicts the relationship between the photocurrent response of 5.0 ng mL−1 NSE at the anti-NSE/ZnCdHgSe QDs/PDPIT/ITO electrode and the incubation time. When the incubation time prolonged from 10 to 30 min, the photocurrent variation for the specific recognization of NSE increased gradually, and then nearly tends to be a steady response, indicating an equilibrium state for the immunosensing system. So that, being incubated in NSE solution for 30 min was employed for immunoassay. The topography of the electrode surface at each fabrication step was characterized with scanning electron microscope. It could be found from Figure 3a that the indium tin oxide nanoclusters were homogeneously distributed on the unmodified ITO electrode surface. When DPIT ionic liquid was polymerized onto ITO electrode surface, from Figure 3b, on which a polymer film with some pores was found homogeneously covered. After assembling of ZnCdHgSe QDs, some particles in nanometer were observed obviously and aggregated together to form some nanobundles and nanoclusters (Figure 3c). The results confirm the successful decoration of ZnCdHgSe QDs onto the PDPIT/ITO surface. After anti-NSE was subsequently assembled, as shown in Figure 3d, not only ZnCdHgSe QDs were embedded by anti-NSE proteins, but also the formation of a network structure was clearly found because of cross-linking reactions between aldehyde groups and amino groups. The morphologic differ-
electron transfer and the block effect on mass transport of K3Fe(CN)6/K4Fe(CN)6 probes. Similarly, it was found that Rct has been changed to be 3208 Ω (curve e) after the specific recognition reaction between anti-NSE and NSE. To monitor this fabrication process, the photocurrent response of ZnCdHgSe QDs/PDPIT/ITO electrode in an ascorbic acid solution is another important index. Figure 2B indicates that the photocurrent intensity gradually decreased after anti-NSE and BSA were immobilized onto the ZnCdHgSe QDs/PDPIT/ITO electrode surface. Proteins assembled onto the ZnCdHgSe QDs/PDPIT/ITO electrode surface would obstruct the diffusion of ascorbic acid to ZnCdHgSe QDs, thus leading to retard its reaction with photogenerated holes and also weakening the photocurrent intensity. When NSE specifically bound to anti-NSE, the photocurrent response decreased significantly (curve e). In the light of the photocurrent variation in the immunoreaction process, a photoelectrochemical immunosensor can be successfully developed for NSE determination. Control experiment turns out that there is no observable change in photocurrent intensity. To verify that, an identical immunosensor was incubated into a phosphate buffer solution without NSE, and determined according the method mentioned above. The result reveals that the relative deviation for photocurrent response of the immunosensor before and after incubation is only 0.82% (n = 5). Consequently, it is reasonable to deduce that the immunoreaction between the anti-NSE immobilized on ZnCdHgSe QDs/PDPIT/ITO surface and NSE in bulk solution was the main reason for the reduction of photocurrent. In the process of immunoreaction, the incubation time and temperature are two important factors which influence the sensing properties toward NSE. The relationship between the F
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Analytical Chemistry
Figure 4. Typical photocurrent response (a) and calibration curve (b) for NSE determination at the anti-NSE/ZnCdHgSe QDs/PDPIT/ITO electrode based immunosensor. (Error bars are standard deviation for three independent determinations.)
turning on and off the white-light LED more than 20 times. As presented in Supporting Information Figure S10, only a negligible variation in the photocurrent response was found in the studied time range, meaning an excellent stability. The stability of the anti-NSE/ZnCdHgSe QDs/PDPIT/ITO electrode was also examined by measuring the photocurrent response of 1.0 ng mL−1 NSE. After being stored at 4 °C for more than 2 weeks, only 4.7% changes deviated from its original response was found in the photocurrent response. This result demonstrates that the anti-NSE/ZnCdHgSe QDs/ PDPIT/ITO electrode can be stably maintained for a long period of time. The reproducibility was evaluated by determining 1.0 ng mL−1 of NSE solution using five immunosensors, which were respectively fabricated with the same procedure. The relative standard deviation for photocurrent responses was calculated to be 5.8%, from which a good reproducibility was demonstrated. The practicability of this photoelectrochemical immunosensor was illustrated by the determination of NSE levels in clinical human sera. These samples used for determination were prepared by diluting the clinical human sera using phosphate buffer solution in the proportion of 1:10000.The accuracy of the as-prepared immunosensor for the determination of NSE in these samples was also verified by ELISA method. All results summarized in Supporting Information Table S2 were the original concentrations of NSE in the serum samples calculated from the determined values. The results from the immunosensor and ELISA method were assessed by a t test method. All calculated t values, among them the maximum is 2.55, are less than the standard value (t0.05,4 = 2.78, the confidence coefficient is 95.0%), indicating that there is no statistical difference between the immunosensor and ELISA method. The results also demonstrate that the anti-NSE/ZnCdHgSe QDs/PDPIT/ ITO based immunosensor can be used to assay NSE in real samples with high accuracy and selectivity.
ences coming from these interfaces suggest the successful fabrication of anti-NSE/ZnCdHgSe QDs/PDPIT/ITO sensing platform step by step. On the basis of the photocurrent variation, which is directly correlated to the NSE concentration, a sensitive and selective method can be developed for NSE analysis. Figure 4a presents a typical photocurrent response of the immunosensor after being incubated in a NSE solution at different concentrations. Photocurrent responses decreased accordingly with the increase of NSE concentrations. From Figure 4b, the percentage of the photocurrent decrement, which can be defined as ΔI/I0 = (I0 − I)/I0, exhibits a linear relation with the logarithm of NSE concentration (logcNSE) in the range of 1.00 pg mL−1 to 100.00 ng mL−1. Where, I0 and I are the photocurrents of the antiNSE/ZnCdHgSe QDs/PDPIT/ITO electrode based immunosensor prior to (I0) and after (I) being incubated in NSE solution at the evaluated concentration. The detection limit for NSE determination on the anti-NSE/ZnCdHgSe QDs/ PDPIT/ITO electrode is 0.20 pg mL−1 (S/N = 3). Supporting Information Table S1 presents the comparison of the analytical characteristics of this immunosensor with other methods developed for NSE determination. This immunosensor exhibits not only a lower detection limit but also a wider linear range than others. Furthermore, we also illustrate that the proposed photoelectrochemical strategy is able to realize high selectivity, and this immunosensor is also a promising platform for the NSE level evaluation in the early diagnosis, the treatment process, and disease surveillance. Evaluation of the specificity was achieved by surveying the photocurrent responses of the anti-NSE/ZnCdHgSe QDs/ PDPIT/ITO electrodes after being incubated in a NSE solution at the concentration of 0.1 ng mL−1 and a mixed solution containing 0.1 ng mL−1 of NSE and 5.0 ng mL−1 of PSA, AFP and human IgG. Similarly, NSE in a real clinical serum sample was also tested using this immunosensor to examine its selectivity. The photocurrent responses are shown in Supporting Information Figures S8 and S9, respectively. By comparing the photocurrent responses, only a negligible difference can be observed for the determination of the NSE solution and the mixed solution (SD < 5.0%, n = 3). The results indicated that the immunosensor that based on anti-NSE/ ZnCdHgSe QDs/PDPIT/ITO electrode possesses acceptable selectivity and was not interfered by the nonspecific adsorption. The stability of the photocurrent response of the ZnCdHgSe QDs/PDPIT/ITO electrode was studied through the manipulation of the photoelectrochemical excitation process by
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CONCLUSIONS
A promising photoelectrochemical immunosensing platform for highly sensitive determination of NSE was established based on ZnCdHgSe QDs and polymerized ionic liquid. The polymerized ionic liquid can remarkably improve the photocurrent intensity of ZnCdHgSe QDs, which was excited by a commercial white-light LED. To illustrate the application of the anti-NSE/ZnCdHgSe QDs/PDPIT/ITO electrode, immunoassay of NSE was designed and was demonstrated effectively. Compared to those previously reported, this immunosensor for G
DOI: 10.1021/ac504456w Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry NSE not only has a detection limit as low as 0.2 pg mL−1 but also gives a wider linear range (1.0 pg mL−1 to 100 ng mL−1). NSE level in the human body fluid is commonly over the span of 5 ng mL−1 to >100 ng mL−1. A wider linear range is thus very significant for direct detection of NSE in clinical samples, and also improves the potential application in the early stage diagnosis and long-term prognosis. This work is able to promote the further developments on photoelectrochemical immunosensor combining with quantum dots and ionic liquid and also extends its potential applications in biological or clinical targeted analysis.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information is 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
*E-mail:
[email protected]. Tel: +86 27 67842752. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial supports from National Natural Science Foundation of China (No.21275166) and China Scholarship Council (No. 201307780006).
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