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Quenched sandwich-type photoelectrochemical aptasensor for protein detection based on exciton energy transfer
Talanta 198 (2019) 302–309
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Quenched sandwich-type photoelectrochemical aptasensor for protein detection based on exciton energy transfer
T
Yi Zhana, Jing Tangb, Di Huanga, Lina Zoua, , Baoxian Yea, ⁎
a b
⁎
College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, PR China Henan Provincial Institute of Cultural Relics and Archaeology, Zhengzhou 450001, PR China
ARTICLE INFO
ABSTRACT
Keywords: Photoelectrochemical aptasensor Energy transfer CdS:Mn AuNPs Thrombin
This work proposes a quenched photoelectrochemical sensing method for highly selective and sensitive detection of protein via Energy Transfer (ET) effect between the AuNPs and CdS:Mn quantum dots. This detection was performed on a sandwich-type aptamer sensing interface. Chitosan modified CdS:Mn/TiO2/ITO electrode was used to immobilize capture DNA (S1) via -CONH- bond. In the presence of target protein, AuNPs labeled DNA (AuNPs-S2) was further bonded to the protein to fabricate sandwich sensing platform, which forced the AuNPs away from the electrode surface. In this state, the photocurrent was greatly depressed, mainly due to two factors: (a) the ET effect produced by interparticle distance between CdS:Mn and AuNPs; (b) the steric hindrance of AuNPs-S2 partly obstructs the diffusion of the electron donor. The photocurrent decreased with the increasing concentration of the target protein. Using thrombin as a target, this sensitized method showed a detectable range of 0.1 pM to 8 nM and a detection limit of 30 fM. It possessed high selectivity and good stability for detection of thrombin. This method is extremely flexible and can be extended to varieties of protein targets.
1. Introduction Protein detection plays an important role in the disease prevention, health care, medical diagnosis and disease treatment [1,2]. Highly sensitive and selective analytical technology of special proteins is greatly desirable. Aptamer assay is one of the hot research areas for specific protein detection, which is developed on the basis of immunoassay. Compared with traditional recognition element antibodies, aptamers not only have high specificity comparable to antibodies, but also have features of easy synthesis, high stability, easy to label and nonimmunogenicity [3,4]. Therefore, various strategies of biosensors for protein detection based on aptamers have been developed, such as fluorescence [5], colorimetry [6], electrochemiluminescence [7], electrochemical method [8] and photoelectrochemical technique [9]. Among these biosensors, photoelectrochemical (PEC) analysis is a recently developed technology for rapid and sensitive bioassays, in which light excite photoactive materials to generate photocurrent. Due to the complete separation of the excitation signal (light) and the detection signal (current), PEC method perform a lower background signal and higher sensitivity compared to the other conventional methods. Thus PEC holds great promise and has attracted considerable attention in biological assays for its simplicity, low cost,simple sample preparation and potentially high sensitivity [10,11]. ⁎
For PEC bioanalysis, photoactive material with high photoelectric conversion efficiency plays a major role, meanwhile, the PEC sensing system with dramatically changed photocurrent is also necessary for highly sensitive detection. To achieve this aim, a sensitized structure, which is composed of large band gap photoactive material and one or more semiconductors with narrow band gap, is very effective [12]. TiO2 only absorb the ultraviolet light (< 387 nm) for the wide band gap of 3.2 eV, while the absorption range of narrow band gap quantum dots CdS (∼ 2.4 eV) extends to the wavelength of 550 nm [13,14]. Coupled TiO2 with Mn-doped CdS QDs to reduce the recombination of e--h+ pairs and increase the photocurrent intensity, for the doped Mn2+ ion could create a new band gap in the midst of CdS [15]. Therefore, coupling TiO2 with QDs would extend the absorption range and improve the photoelectric conversion efficiency. Moreover, Doping metal ions in quantum dots can further improve photoelectric conversion efficiency. After doping with optically active transition metal ions Mn2+, the electronic and photophysical properties of quantum dots vary greatly and can be used as photoelectrochemical enhancers [16]. Since Kamat et al. [17] first proposed Mn2+-doped CdS as a photoanode, it not only improves the electron transfer and photophysical properties of quantum dots, but also opens up new models for exploring new materials and methods. In view of the above report, by doping Mn2+, the intermediate region of the quantum dot produces an
Corresponding authors. E-mail addresses: [email protected] (L. Zou), [email protected] (B. Ye).
https://doi.org/10.1016/j.talanta.2019.02.019 Received 18 September 2018; Received in revised form 30 January 2019; Accepted 3 February 2019 Available online 06 February 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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electronic state that facilitates electron transfer, thereby Mn-doped CdS quantum dots can be used to sensitize TiO2 for further analysis. The changed photocurrent after biological recognition is the basis of PEC bioanalysis. Signal quenching strategy is one of the easy to use and sensitive methods, which can be realized via the consumption of electron donor or acceptor [18], steric hindrance effect [19], or energy transfer (ET) [20]. A classical and strong ET effect between CdS QDs and gold nanoparticles (AuNPs) for PEC biosensing application was firstly reported [21]. The quenched photocurrent caused by ET effect should meet two factors: (a) the emission spectrum of CdS QDs should overlap with the absorption spectrum of AuNPs, which would induce the surface plasmon resonance of the AuNPs; (b) there are should certain distance between the CdS QDs and AuNPs, making ET play a dominant role and further reducing the photocurrent significantly. The PEC bioanalysis based on ET effect have been reported in the detection of microRNA [22], carcinoembryonic antigen (CEA) [23], prostate specific antigen (PSA) [24]. High sensitivity and selectivity are the common characteristics of these systems. As a promising strategy, the research about ET-based PEC bioanalysis should be extended to various biological recognition systems. Herein, on the basis of the sensitization of CdS:Mn quantum dots on TiO2 and the ET effect between AuNPs and CdS:Mn QDs, we presented a sandwich-type PEC quenched assay for simple but high sensitive detection of protein, where thrombin (TB) was employed as the target. The PEC bioassay composed of the following steps (Scheme 1): coating TiO2 nanoparticles on the ITO surface and forming CdS:Mn/TiO2 hybrid structure; assembling capture DNA (S1) for the specific recognizing TB; on this biosensor surface, the TB could further induce the probe DNA functionalized AuNPs (AuNPs-S2) to from sandwich-type. In this state, a strong ET effect occurs under light illumination, combining with the
steric hindrance effect, the PEC current decreases dramatically then the concentration of target TB should be detected. The prepared biosensor was successfully applied for the determination of TB in real samples. To the best of our knowledge, this is the first report of ET-based PEC aptasensor for TB detection. Because common sandwich-type recognition mode was applied in this strategy, it can be extended to other protein detection easily and flexibly. 2. Experiment section 2.1. Materials and experiment measurements ITO slices (type JH52, ITO coating 30 ± 5 nm, sheet resistance ≤ 10 Ω/square) was purchased from Zhuhai Kaivo Optoelectronic Technology Co., Ltd. (China). Methanol, cadmium nitrate (Cd (NO3)2·4H2O), sodium sulfide (Na2S·9H2O) and manganese acetate (Mn (Ac)2·4H2O) were purchased from Tianjin Chemical Reagent Co., Ltd. (China). TiO2 powder (P25) was purchased from the Degussa Co. (Germany). Bovine serum albumin (BSA,98%; EMD Chemicals Inc.) and tris (2-carboxyethyl)-phosphine (TCEP) were obtained from SigmaAldrich. Ascorbic acid (AA) was purchased from Sinopharm Chemical Reagent Co., Ltd. (China). All other reagents were of analytical grade and used as received. All aqueous solutions were prepared with deionized water (DI water, 18 MΩ/cm), which was obtained from a Milli-Q water purification system. 0.01 M phosphate buffer solution (PBS, pH=7.5) was prepared with K2HPO4·3H2O, NaH2PO4·2H2O and KCl, being employed for washing buffer solution and detecting bottom liquid which contained 0.1 M AA. The oligonucleotides [25] with following sequences (5′ to 3′) were from Sangon Biotechnology Inc. (Shanghai, China):
Scheme 1. The fabrication of PEC biosensor. 303
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Thrombin aptamer(S1): 5′-GGT TGG TGT GGT TGG-(CH2)6-NH2-3′; Thrombin aptamer(S2): 5′-GGT TGG TGT GGT TGG-(CH2)6-SH-3′. A PEAC200A photochemical reaction apparatus (Tianjin Aida Hengsheng technology development Co.，LTD) was used as the irradiation source, The photocurrent was recorded on a RST5210F electrochemical workstation (Zhengzhou Shiruisi Instrument Technology Co., Ltd) through a three-electrode system with a 0.25 cm2 ITO as working electrode, a Pt wire as counter electrode, and a saturated Ag/ AgCl electrode as the reference electrode. Scanning electron microscopy was conducted using (SEM, JEOL JSM-6700, Japan) equipped with an energy-dispersive spectroscopy (EDS, Oxford Inca Energy 400, UK). UV–Vis spectra was obtained from a Lambda 35 UV–Vis spectrometer (Pgeneral General Instrument, Beijing, China). Electrochemical impedance spectroscopy (EIS) was performed on an RST5210F electrochemical workstation (Zhengzhou Shiruisi Instrument Technology Co., Ltd) via a three-electrode system in 0.1 M KCl solution containing 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture as a redox probe and recorded in the frequency range of 0.01 Hz to 100 kHz with an amplitude of 50 mV.
BSA solution was dropped on the electrode for 1 h to block the unmodified sites. After being washed with 10 mM PBS (pH = 7.4), the PEC biosensor was obtained and stored at 4 °C before use.
2.2. Preparation of AuNPs-S2
SEM was utilized to characterize the surface topographies of TiO2 and CdS:Mn/TiO2, Fig. 1A and B exhibit the typical SEM images of TiO2 and CdS:Mn/TiO2, respectively. As shown in Fig. 1A, scattered TiO2 nanoparticles with a average diameter of 21 nm have been successfully fabricated on the ITO. From Fig. 1B, after CdS:Mn deposited, a great deal of small particles were distributed on the TiO2 nanoparticles. For further investigating the situation of CdS:Mn coating on the TiO2 nanoparticles, the corresponding EDX spectra were obtained. As demonstrated by Fig. 1C, Ti and O existed, indicating that TiO2 has been successfully loaded on ITO electrode. From Fig. 1D, the existence of Cd, Mn and S showed that CdS:Mn has been assembled on TiO2/ITO. In Fig. 2, curve b shows that the synthetic AuNPs has the maximum absorption peak at 520 nm, which have aquasi-spherical structure with a diameter of about 12 nm (insert in Fig. 2). The PL spectrum of CdS:Mn (curve a) has a peak at 524 nm. Obviously the PL spectrum of CdS:Mn overlap with the UV–vis absorption of Au NPs, which is essential for the strong ET effect.
2.5. Measurement procedure A volume of 10 μL different concentration of target thrombin solution was dropped on the PEC biosensor surface for 1 h incubation, followed by washing with PBS buffer (pH = 7.4). At last, 10 μL AuNPsS2 was fabricated on the electrode through an immune binding. Finally, the PEC detection was performed at room temperature in 4 mL of PBS buffer (10 mM, pH = 7.4), which contained 0.1 M AA as a sacrificial electron donor. White light, with a spectral range of 400–700 nm, was utilized as excitation light and was switched on and off at the interval of 10 s. The applied potential was selected as 0.0 V. 3. Results and discussion 3.1. Characterization of TiO2, CdS:Mn/TiO2, AuNPs and CdS:Mn QDs
Brifely, A typical method for synthesizing AuNPs [26] was to add 2.5 mL 1% weight ratio sodium citrate solution into 100 mL of HAuCl4 (0.01% weight ratio), stirring the mixture until it turned to orange-red. After cooling down to room temperature, the AuNPs solution was obtained and stored at 4 °C for later use. Then, 10 μL of S2 (100 μM) was slowly added into the mixture which contained 500 μL of above prepared AuNPs solution, 10 μL of tris (2-carboxyethyl)- phosphine hydrochloride (TCEP; 10 mM), and 100 μL of NaCl (0.5 M). After stirred in the dark for 16 h, the above obtained solution was centrifuged under 14000 rpm for 15 min, after the suspension suspended, the red-oil precipitation was dispersed in 1 mL 10 mM PBS contained 0.1 M NaCl, the AuNPs-S2 was obtained and stored at 4 °C for later use. 2.3. Preparation of modified electrode Prior to use, The ITO electrode was sequentially ultrasonic with acetone, de-ionized water, ethyl alcohol and de-ionized water for 15 min respectively, then dried with N2 for later use. 10 mg of TiO2 powder was ultrasonically dispersed in 10 mL de-ionized water, and then 20 μL of this homogeneous suspension (1.0 mg/mL) was dropped onto a piece of ITO slice with a fixed area of 0.25 cm2. After dried in air, the film was sintered at 450 °C for 30 min to obtain a stable TiO2/ITO electrode. The deposition of CdS on the TiO2/ITO electrode was performed with a successive ionic layer adsorption and reaction (SILAR) method [27]. Briefly, the TiO2/ITO electrode was first dipped into the mixture methanol solution which contained 0.1 M Cd(NO3)2 and 0.08 M Mn(Ac)2 for 1 min and rinsed with methanol, Subsequently dipping into 0.1 M Na2S methanol/water mixture (1:1, v/v) for 1 min, after each soaked in solution, the electrode was carefully washed with methanol. This SILAR cycle was repeated five times to acquire the CdS:Mn/TiO2/ ITO electrode.
3.2. PEC behavior of CdS:Mn/TiO2/ITO electrode TiO2 have a wide energy band gap (∼ 3.2 eV), which absorbs the ultraviolet light (< 387 nm), while CdS has a narrow energy band gap (∼ 2.4 eV) with the absorption range extending to the wavelength of 550 nm. Therefore, combining CdS with TiO2 to form a hybrid structure can evidently extend the absorption range, improving the utilization of light energy. Meanwhile, the doped Mn2+ in CdS can effectively extend the life of carriers, delay the recombination of electrons with holes, which will improve the photoelectric conversion efficiency. The photocurrent response of ITO, TiO2/ITO, CdS/TiO2/ITO and CdS:Mn/TiO2/ ITO were showed in Fig. 3, the photocurrent of CdS:Mn/TiO2/ITO was the highest. Eventually, CdS:Mn/TiO2/ITO was used as the working electrode for its excellent PEC performance in this work. 3.3. Characterization of the PEC biosensor
2.4. Fabrication of the PEC aptasensor
As is shown in Fig. 4, the preparation of PEC biosensor was characterized with electrochemical impedance spectroscopy (EIS) in 0.1 M KCl containing 5 mM K3Fe(CN)6 and K4Fe(CN)6. Compared with the CdS:Mn/TiO2/ITO electrode (curve a), the chitosan assembled CdS:Mn/ TiO2/ITO electrode (curve b) showed a much larger electron transfer impedance Ret (curve b) due to the insulativity of chitosan. After incubation of S1, Ret increased (c) a lot for negatively charged oligonucleotide repelling the [Fe(CN)6]3−/4−. After the electrode surface was incubated with BSA (curve d), target thrombin (curve e), and AuNPs-S2 (curve f) gradually, the Ret increased step and step, implying the successful hybridization of target thrombin with S1 and AuNPs-S2.
First of all, the CdS:Mn/TiO2/ITO electrode electrode was treated with 20 μL of 2% acetic acid solution containing 0.1 mg/m L CS to acquire a -COOH modified interface and dried at 60 °C. Then 10 μL of 2.5% GLD was covered onto the electrode surface and remained for 1 h at room temperature. After rinsed with de-ionized water, a volume of 10 μL 1 μM capture DNA S1 solution was dropped directly onto the CdS:Mn/TiO2/ITO electrode and incubated for 1 h in the dark to form a self-assembled monolayer (SAM) via -CONH- chemical bond. The excess capture-DNA was removed via a room temperature de-ionized water rinse (∼ 20 s). For avoiding the nonspecific adsorption, 10 μL of 1% 304
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Fig. 1. SEM image of (A) TiO2/ITO and (B) CdS:Mn/TiO2/ITO; EDS image of (C) TiO2/ITO and (D) CdS:Mn/TiO2/ITO.
Fig. 4. EIS of (a) CdS:Mn/TiO2/ITO, (b) CS assembly, (c) Glutarldehyde and S1 immobilization, (d) BSA immobilization, (e) incubated with 10 μL of 8 nM thrombin and (f) incubated with AuNPs-S2.
Fig. 2. Fluorescence spectrum of CdS:Mn (curve a) and UV–vis spectrum of AuNPs-S2 (curve b), insert: TEM image of AuNPs.
Fig. 5. Photocurret responses of (a) ITO/TiO2, (b) CdS:Mn/TiO2/ITO, (c) CS assembly, (d) glutarldehyde and S1 immobilization, (e) BSA immobilization, (f) incubation of 10 μL of 8 nM thrombin, (g) incubation of S2 and (h) the immobilization of AuNPs-S2.
Fig. 3. Photocurret responses of (a) ITO, (b) TiO2/ITO, (c) CdS/TiO2/ITO and (d) CdS:Mn/TiO2/ITO.
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Scheme 2. Energy transfer mechanism of the operating PEC system in 0.1 M AA electrolyte.
Fig. 6. Photocurrent response of (A) the CdS:Mn/TiO2/ITO electrode with different TiO2 concentration, the SILAR of CdS:Mn is 5, (B) the different SILAR of CdS:Mn, C(TiO2) = 1.0 mg/mL, (C) the different incubation time of 10 μL 1 μM S1 and (D) the different incubation time of 10 μL 8 nM thrombin.
3.4. Feasibility of the PEC biosensor
recombination. As compared to the CdS:Mn/TiO2/ITO electrode, the immobilization of CS (curve c) and capture DNA S1 (curve d) caused gradually decrease of photocurrent intensity which shown the successful fabrication of DNA S1 using CS as cross linker, due to the poor conductivity and steric hindrance. After the incubation with BSA (curve e) and target thrombin (curve f), the photocurrent intensity decreased due to the insulativity of protein. In order to research with the PEC mechanism of this biosensor, S2 (curve g) and AuNPs-S2 (curve h) were further incubated respectively, it was found that the photocurrent
The validation of the designed strategy was further performed via photocurrent characterization of the PEC biosensing process. As shown in Fig. 5, the TiO2/ITO electrode displays a small photocurrent intensity (curve a), because TiO2 could only absorb UV light so that the photoelectric conversion efficiency is low. After deposition of CdS:Mn, the photocurrent intensity (curve b) had a sharp rise, revealing that CdS:Mn could promote charge separation and depress the electron-hole 306
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Fig. 7. (A) Photocurrent responses of thrombin at 10–13,10–12,10–11,10−10,10−9,10−8.3 M respectively. (B) Calibration curve for thrombin. (C) The stability of the developed biosensor. (D) Selectivity of the sensor incubated with CRP, ALP and Mb.
Table 1 Comparison of different methods for the determination of thrombin. Method
Platform
Fluorescence
Aptamer, Aptamer, Aptamer, Aptamer, Aptamer, Aptamer, Aptamer, Aptamer,
colorimetric sensor Electrochemical Aptasensor Photoelectrochemical Sensing
DNAzyme powered DNA motor Protein-induced gold nanoparticle aggregation cationic polythiophene derivative Co-based metal-organic frameworks rolling circle amplification Au nanoparticles modified nanoporous BiVO4 CdS:Mn/TiO2/ITO
intensity of assembling with AuNPs-S2 (curve h) showed a larger decrease than S2 (curve g). Therefore, the PEC mechanism was shown in Scheme 2: AuNPs-S2 bonded on target thrombin will force the AuNPs away from the electrode surface. In this state, strong quenching of photocurrent intensity will be realized for the energy transfer effect between AuNPs and CdS:Mn. All of these results suggest the successful fabrication of the PEC aptasensor as designed.
Linear range
Detection limit
Ref.
10 pM~10 nM 0.25 pM~25 nM 10 nM~5 μM 0.01–1 nM 1 pM to 30 nM 0.1 pM~10 nM 1 pM~10 nM 0.1 pM~ 8 nM
4 pM 8.9 pM 7.5 nM 4 pM 0.32 pM 35 fM 0.5 pM 30 fM
[28] [29] [30] [31] [32] [33] [34] This work
In the low concentration range of TiO2 (0.3–1.0 mg/mL), more amount of CdS:Mn could be absorbed on the surface of TiO2, which benefit for photocurrent intensity increase. However, In the high concentration range of TiO2 (1.0–2.0 mg/mL), excessive TiO2 hinder the electron transfer, which leads to the gradual decrease of the photocurrent intensity. Therefore, a 1.0 mg/mL TiO2 suspension was used to make the electrodes. The deposition of CdS:Mn can be adjusted by the period of SILAR. Similarly, the photocurrent of the CdS:Mn/TiO2/ITO electrode was investigated with different CdS:Mn deposition period at 1.0 mg/mL TiO2. which reached the maximum at 5 cycles (Fig. 6B). For best signal amplification, 5 SILAR cycles of CdS:Mn deposition and 1.0 mg/mL TiO2 were chosen to make the electrodes. Fig. 6C and Fig. 6D showed the optimization incubation time for S1 and thrombin, respectively. It can be found that the photocurrent decreased sharply before 40 min. The reason was that more successful hybridization was formed with the
3.5. Optimization of experimental condition In order to achieve the best sensitization effect of CdS:Mn to TiO2, the concentration of TiO2 suspension and the coating number of CdS:Mn were optimizated. As seen from Fig. 6A, with the increased concentration of TiO2, the maximum photocurrent of CdS:Mn/TiO2/ ITO with five cycles of CdS deposition was obtained at 1.0 mg/mLTiO2. 307
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sensitivity, low detection limit, good selectivity and excellent stability. The quenched sandwich-type PEC aptamer sensing platform could be easily expanded to detect other proteins with relevant aptamers, showing great potential in bioanalysis.
Table 2 Results of thrombin detection in 100-Fold-Diluted Serum Samples (n = 3). Samples
Added TB (pM)
Found TBa (pM))
Recovery
RSD (%)
1 2 3
0.5 5 50
0.476 4.906 50.9
0.96 0.981 1.018
4.89 2.31 3.26
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant numbers: U1504216, 21575130); Startup Research Fund of Zhengzhou University (Grant no. 1511316006).
TB: thrombin. TBa: The average value of three measurements.
increasing incubation time. Therefore, the incubation time of S1 and thrombin were all chosen as 60 min.
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3.6. Analytical performance The quenched photocurrent was directly related to the concentration of target protein thrombin and thus providing a sensitive method for PEC detection of thrombin. Thus, to get the relationship between the photocurrent response with the concentration of thrombin, a series of different thrombin concentrations were investigated. Fig. 7A shows that the photocurrent response of the biosensor decreased gradually with the increasement of the concentration of thrombin. As shown in Fig. 7B, the decrement of photocurrent is proportional to the logarithm of thrombin concentration ranged from 0.1 pM to 8 nM. The equation of the linear regression is ΔI = 5.8102 log c + 78.3021 with the correlation coefficient of 0.997. ΔI = I - I0, where I stands for the PEC response without thrombin, and I0 stands for the PEC response with different concentration of thrombin. Furthermore, the limit of detection (LOD) was 30 fM (S/N = 3). The quenched sandwich-type PEC aptasensor showed a wide response and low limit of detection, which was similar or even better than the many previously reported methods of thrombin detection (Table 1). The stability was a vital factor for the application of a PEC sensor. In our work, the final PEC response of was recorded under 10 on/off irradiation cycles. As shown in Fig. 7C, the photocurrent were observed without a obvious variation, indicating the stability of the sensor. The selectivity of this method was tested by other co-existing proteins in the serum, Fig. 7D including creactive protein (CRP), alkaline phosphatase (ALP) and myoglobin (Mb). It had no influence on the PEC sensing although the 100-fold of the interference compared with that of the sensor incubated with thrombin, which disclosed that the fabricated PEC biosensor showed good selectivity for thrombin because of the specific recognition between thrombin with S1 and AuNPs-S2. 3.7. Real sample analysis To estimate the applicability of this proposed approach, the survey was determined by the standard addition method in three human serum samples from The first affiliated hospital of Zhengzhou university. The serum samples were measured for determination of thrombin with 100 fold dilution. As listed in Table 2, the recovery of thrombin in serum samples ranged from 96% to 101.8%, the RSD are no more than 5%, indicating that the proposed approach has good potential for the analysis of thrombin in real samples. 4. Conclusion This work develops a novel sensitive sandwich-type photoelectrochemical aptamer sensing paltform for target protein. The specific recognition of target protein by aptamer enable the immobilization of target protein and signal aptamer on the electrode step-by-step, which brings AuNPs far from the CdS:Mn/TiO2/ITO electrode and thus produces a strong quenched photocurrent through the ET effect between the AuNPs and CdS:Mn QDs. Using thrombin as a target protein, resulting PEC aptasensor exhibited wide detectable range, high 308
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Y. Zhan et al. [23] X. Zeng, S. Ma, J. Bao, W. Tu, Z. Dai, Using graphene-based plasmonic nanocomposites to quench energy from quantum dots for signal-on photoelectrochemical aptasensing, Anal. Chem. 85 (2013) 11720–11724. [24] Y.C. Zhu, F. Xu, N. Zhang, W.W. Zhao, J.J. Xu, H.Y. Chen, DNA sequence functionalized with heterogeneous core–satellite nanoassembly for novel energy-transferbased photoelectrochemical bioanalysis, Biosens. Bioelectron. 91 (2017) 293–298. [25] J.J. Bock, L.C. Griffin, L.C. Latham, J.A. Vermaas, E.H. Toole, Selection of singlestranded DNA molecules that bind and inhibit human thrombin, Nature 355 (1992) 564–566. [26] D. Weihs, Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions, Nature 242 (1973) 117–118. [27] G.C. Fan, H. Zhu, D. Du, J.R. Zhang, J.J. Zhu, Y. Lin, Enhanced photoelectrochemical immunosensing platform based on CdSeTe@CdS:Mn Core-Shell quantum dots-sensitized TiO2 amplified by CuS nanocrystals conjugated signal antibodies, Anal. Chem. (2016) 3392–3399. [28] W. Yun, L. You, F. Li, H. Wu, L. Chen, L. Yang, Proximity ligation assay induced and DNAzyme powered DNA motor for fluorescent detection of thrombin, Spectrochim. Acta - Part A Mol. Biomol. Spectrosc. 207 (2019) 39–45. [29] S. Umrao, V. Jain, Anusha, B. Chakraborty, R. Roy, Protein-induced fluorescence
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Quenched sandwich-type photoelectrochemical aptasensor for protein detection based on exciton energy transfer
T
Yi Zhana, Jing Tangb, Di Huanga, Lina Zoua, , Baoxian Yea, ⁎
a b
⁎
College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, PR China Henan Provincial Institute of Cultural Relics and Archaeology, Zhengzhou 450001, PR China
ARTICLE INFO
ABSTRACT
Keywords: Photoelectrochemical aptasensor Energy transfer CdS:Mn AuNPs Thrombin
This work proposes a quenched photoelectrochemical sensing method for highly selective and sensitive detection of protein via Energy Transfer (ET) effect between the AuNPs and CdS:Mn quantum dots. This detection was performed on a sandwich-type aptamer sensing interface. Chitosan modified CdS:Mn/TiO2/ITO electrode was used to immobilize capture DNA (S1) via -CONH- bond. In the presence of target protein, AuNPs labeled DNA (AuNPs-S2) was further bonded to the protein to fabricate sandwich sensing platform, which forced the AuNPs away from the electrode surface. In this state, the photocurrent was greatly depressed, mainly due to two factors: (a) the ET effect produced by interparticle distance between CdS:Mn and AuNPs; (b) the steric hindrance of AuNPs-S2 partly obstructs the diffusion of the electron donor. The photocurrent decreased with the increasing concentration of the target protein. Using thrombin as a target, this sensitized method showed a detectable range of 0.1 pM to 8 nM and a detection limit of 30 fM. It possessed high selectivity and good stability for detection of thrombin. This method is extremely flexible and can be extended to varieties of protein targets.
1. Introduction Protein detection plays an important role in the disease prevention, health care, medical diagnosis and disease treatment [1,2]. Highly sensitive and selective analytical technology of special proteins is greatly desirable. Aptamer assay is one of the hot research areas for specific protein detection, which is developed on the basis of immunoassay. Compared with traditional recognition element antibodies, aptamers not only have high specificity comparable to antibodies, but also have features of easy synthesis, high stability, easy to label and nonimmunogenicity [3,4]. Therefore, various strategies of biosensors for protein detection based on aptamers have been developed, such as fluorescence [5], colorimetry [6], electrochemiluminescence [7], electrochemical method [8] and photoelectrochemical technique [9]. Among these biosensors, photoelectrochemical (PEC) analysis is a recently developed technology for rapid and sensitive bioassays, in which light excite photoactive materials to generate photocurrent. Due to the complete separation of the excitation signal (light) and the detection signal (current), PEC method perform a lower background signal and higher sensitivity compared to the other conventional methods. Thus PEC holds great promise and has attracted considerable attention in biological assays for its simplicity, low cost,simple sample preparation and potentially high sensitivity [10,11]. ⁎
For PEC bioanalysis, photoactive material with high photoelectric conversion efficiency plays a major role, meanwhile, the PEC sensing system with dramatically changed photocurrent is also necessary for highly sensitive detection. To achieve this aim, a sensitized structure, which is composed of large band gap photoactive material and one or more semiconductors with narrow band gap, is very effective [12]. TiO2 only absorb the ultraviolet light (< 387 nm) for the wide band gap of 3.2 eV, while the absorption range of narrow band gap quantum dots CdS (∼ 2.4 eV) extends to the wavelength of 550 nm [13,14]. Coupled TiO2 with Mn-doped CdS QDs to reduce the recombination of e--h+ pairs and increase the photocurrent intensity, for the doped Mn2+ ion could create a new band gap in the midst of CdS [15]. Therefore, coupling TiO2 with QDs would extend the absorption range and improve the photoelectric conversion efficiency. Moreover, Doping metal ions in quantum dots can further improve photoelectric conversion efficiency. After doping with optically active transition metal ions Mn2+, the electronic and photophysical properties of quantum dots vary greatly and can be used as photoelectrochemical enhancers [16]. Since Kamat et al. [17] first proposed Mn2+-doped CdS as a photoanode, it not only improves the electron transfer and photophysical properties of quantum dots, but also opens up new models for exploring new materials and methods. In view of the above report, by doping Mn2+, the intermediate region of the quantum dot produces an
Corresponding authors. E-mail addresses: [email protected] (L. Zou), [email protected] (B. Ye).
https://doi.org/10.1016/j.talanta.2019.02.019 Received 18 September 2018; Received in revised form 30 January 2019; Accepted 3 February 2019 Available online 06 February 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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electronic state that facilitates electron transfer, thereby Mn-doped CdS quantum dots can be used to sensitize TiO2 for further analysis. The changed photocurrent after biological recognition is the basis of PEC bioanalysis. Signal quenching strategy is one of the easy to use and sensitive methods, which can be realized via the consumption of electron donor or acceptor [18], steric hindrance effect [19], or energy transfer (ET) [20]. A classical and strong ET effect between CdS QDs and gold nanoparticles (AuNPs) for PEC biosensing application was firstly reported [21]. The quenched photocurrent caused by ET effect should meet two factors: (a) the emission spectrum of CdS QDs should overlap with the absorption spectrum of AuNPs, which would induce the surface plasmon resonance of the AuNPs; (b) there are should certain distance between the CdS QDs and AuNPs, making ET play a dominant role and further reducing the photocurrent significantly. The PEC bioanalysis based on ET effect have been reported in the detection of microRNA [22], carcinoembryonic antigen (CEA) [23], prostate specific antigen (PSA) [24]. High sensitivity and selectivity are the common characteristics of these systems. As a promising strategy, the research about ET-based PEC bioanalysis should be extended to various biological recognition systems. Herein, on the basis of the sensitization of CdS:Mn quantum dots on TiO2 and the ET effect between AuNPs and CdS:Mn QDs, we presented a sandwich-type PEC quenched assay for simple but high sensitive detection of protein, where thrombin (TB) was employed as the target. The PEC bioassay composed of the following steps (Scheme 1): coating TiO2 nanoparticles on the ITO surface and forming CdS:Mn/TiO2 hybrid structure; assembling capture DNA (S1) for the specific recognizing TB; on this biosensor surface, the TB could further induce the probe DNA functionalized AuNPs (AuNPs-S2) to from sandwich-type. In this state, a strong ET effect occurs under light illumination, combining with the
steric hindrance effect, the PEC current decreases dramatically then the concentration of target TB should be detected. The prepared biosensor was successfully applied for the determination of TB in real samples. To the best of our knowledge, this is the first report of ET-based PEC aptasensor for TB detection. Because common sandwich-type recognition mode was applied in this strategy, it can be extended to other protein detection easily and flexibly. 2. Experiment section 2.1. Materials and experiment measurements ITO slices (type JH52, ITO coating 30 ± 5 nm, sheet resistance ≤ 10 Ω/square) was purchased from Zhuhai Kaivo Optoelectronic Technology Co., Ltd. (China). Methanol, cadmium nitrate (Cd (NO3)2·4H2O), sodium sulfide (Na2S·9H2O) and manganese acetate (Mn (Ac)2·4H2O) were purchased from Tianjin Chemical Reagent Co., Ltd. (China). TiO2 powder (P25) was purchased from the Degussa Co. (Germany). Bovine serum albumin (BSA,98%; EMD Chemicals Inc.) and tris (2-carboxyethyl)-phosphine (TCEP) were obtained from SigmaAldrich. Ascorbic acid (AA) was purchased from Sinopharm Chemical Reagent Co., Ltd. (China). All other reagents were of analytical grade and used as received. All aqueous solutions were prepared with deionized water (DI water, 18 MΩ/cm), which was obtained from a Milli-Q water purification system. 0.01 M phosphate buffer solution (PBS, pH=7.5) was prepared with K2HPO4·3H2O, NaH2PO4·2H2O and KCl, being employed for washing buffer solution and detecting bottom liquid which contained 0.1 M AA. The oligonucleotides [25] with following sequences (5′ to 3′) were from Sangon Biotechnology Inc. (Shanghai, China):
Scheme 1. The fabrication of PEC biosensor. 303
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Thrombin aptamer(S1): 5′-GGT TGG TGT GGT TGG-(CH2)6-NH2-3′; Thrombin aptamer(S2): 5′-GGT TGG TGT GGT TGG-(CH2)6-SH-3′. A PEAC200A photochemical reaction apparatus (Tianjin Aida Hengsheng technology development Co.，LTD) was used as the irradiation source, The photocurrent was recorded on a RST5210F electrochemical workstation (Zhengzhou Shiruisi Instrument Technology Co., Ltd) through a three-electrode system with a 0.25 cm2 ITO as working electrode, a Pt wire as counter electrode, and a saturated Ag/ AgCl electrode as the reference electrode. Scanning electron microscopy was conducted using (SEM, JEOL JSM-6700, Japan) equipped with an energy-dispersive spectroscopy (EDS, Oxford Inca Energy 400, UK). UV–Vis spectra was obtained from a Lambda 35 UV–Vis spectrometer (Pgeneral General Instrument, Beijing, China). Electrochemical impedance spectroscopy (EIS) was performed on an RST5210F electrochemical workstation (Zhengzhou Shiruisi Instrument Technology Co., Ltd) via a three-electrode system in 0.1 M KCl solution containing 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture as a redox probe and recorded in the frequency range of 0.01 Hz to 100 kHz with an amplitude of 50 mV.
BSA solution was dropped on the electrode for 1 h to block the unmodified sites. After being washed with 10 mM PBS (pH = 7.4), the PEC biosensor was obtained and stored at 4 °C before use.
2.2. Preparation of AuNPs-S2
SEM was utilized to characterize the surface topographies of TiO2 and CdS:Mn/TiO2, Fig. 1A and B exhibit the typical SEM images of TiO2 and CdS:Mn/TiO2, respectively. As shown in Fig. 1A, scattered TiO2 nanoparticles with a average diameter of 21 nm have been successfully fabricated on the ITO. From Fig. 1B, after CdS:Mn deposited, a great deal of small particles were distributed on the TiO2 nanoparticles. For further investigating the situation of CdS:Mn coating on the TiO2 nanoparticles, the corresponding EDX spectra were obtained. As demonstrated by Fig. 1C, Ti and O existed, indicating that TiO2 has been successfully loaded on ITO electrode. From Fig. 1D, the existence of Cd, Mn and S showed that CdS:Mn has been assembled on TiO2/ITO. In Fig. 2, curve b shows that the synthetic AuNPs has the maximum absorption peak at 520 nm, which have aquasi-spherical structure with a diameter of about 12 nm (insert in Fig. 2). The PL spectrum of CdS:Mn (curve a) has a peak at 524 nm. Obviously the PL spectrum of CdS:Mn overlap with the UV–vis absorption of Au NPs, which is essential for the strong ET effect.
2.5. Measurement procedure A volume of 10 μL different concentration of target thrombin solution was dropped on the PEC biosensor surface for 1 h incubation, followed by washing with PBS buffer (pH = 7.4). At last, 10 μL AuNPsS2 was fabricated on the electrode through an immune binding. Finally, the PEC detection was performed at room temperature in 4 mL of PBS buffer (10 mM, pH = 7.4), which contained 0.1 M AA as a sacrificial electron donor. White light, with a spectral range of 400–700 nm, was utilized as excitation light and was switched on and off at the interval of 10 s. The applied potential was selected as 0.0 V. 3. Results and discussion 3.1. Characterization of TiO2, CdS:Mn/TiO2, AuNPs and CdS:Mn QDs
Brifely, A typical method for synthesizing AuNPs [26] was to add 2.5 mL 1% weight ratio sodium citrate solution into 100 mL of HAuCl4 (0.01% weight ratio), stirring the mixture until it turned to orange-red. After cooling down to room temperature, the AuNPs solution was obtained and stored at 4 °C for later use. Then, 10 μL of S2 (100 μM) was slowly added into the mixture which contained 500 μL of above prepared AuNPs solution, 10 μL of tris (2-carboxyethyl)- phosphine hydrochloride (TCEP; 10 mM), and 100 μL of NaCl (0.5 M). After stirred in the dark for 16 h, the above obtained solution was centrifuged under 14000 rpm for 15 min, after the suspension suspended, the red-oil precipitation was dispersed in 1 mL 10 mM PBS contained 0.1 M NaCl, the AuNPs-S2 was obtained and stored at 4 °C for later use. 2.3. Preparation of modified electrode Prior to use, The ITO electrode was sequentially ultrasonic with acetone, de-ionized water, ethyl alcohol and de-ionized water for 15 min respectively, then dried with N2 for later use. 10 mg of TiO2 powder was ultrasonically dispersed in 10 mL de-ionized water, and then 20 μL of this homogeneous suspension (1.0 mg/mL) was dropped onto a piece of ITO slice with a fixed area of 0.25 cm2. After dried in air, the film was sintered at 450 °C for 30 min to obtain a stable TiO2/ITO electrode. The deposition of CdS on the TiO2/ITO electrode was performed with a successive ionic layer adsorption and reaction (SILAR) method [27]. Briefly, the TiO2/ITO electrode was first dipped into the mixture methanol solution which contained 0.1 M Cd(NO3)2 and 0.08 M Mn(Ac)2 for 1 min and rinsed with methanol, Subsequently dipping into 0.1 M Na2S methanol/water mixture (1:1, v/v) for 1 min, after each soaked in solution, the electrode was carefully washed with methanol. This SILAR cycle was repeated five times to acquire the CdS:Mn/TiO2/ ITO electrode.
3.2. PEC behavior of CdS:Mn/TiO2/ITO electrode TiO2 have a wide energy band gap (∼ 3.2 eV), which absorbs the ultraviolet light (< 387 nm), while CdS has a narrow energy band gap (∼ 2.4 eV) with the absorption range extending to the wavelength of 550 nm. Therefore, combining CdS with TiO2 to form a hybrid structure can evidently extend the absorption range, improving the utilization of light energy. Meanwhile, the doped Mn2+ in CdS can effectively extend the life of carriers, delay the recombination of electrons with holes, which will improve the photoelectric conversion efficiency. The photocurrent response of ITO, TiO2/ITO, CdS/TiO2/ITO and CdS:Mn/TiO2/ ITO were showed in Fig. 3, the photocurrent of CdS:Mn/TiO2/ITO was the highest. Eventually, CdS:Mn/TiO2/ITO was used as the working electrode for its excellent PEC performance in this work. 3.3. Characterization of the PEC biosensor
2.4. Fabrication of the PEC aptasensor
As is shown in Fig. 4, the preparation of PEC biosensor was characterized with electrochemical impedance spectroscopy (EIS) in 0.1 M KCl containing 5 mM K3Fe(CN)6 and K4Fe(CN)6. Compared with the CdS:Mn/TiO2/ITO electrode (curve a), the chitosan assembled CdS:Mn/ TiO2/ITO electrode (curve b) showed a much larger electron transfer impedance Ret (curve b) due to the insulativity of chitosan. After incubation of S1, Ret increased (c) a lot for negatively charged oligonucleotide repelling the [Fe(CN)6]3−/4−. After the electrode surface was incubated with BSA (curve d), target thrombin (curve e), and AuNPs-S2 (curve f) gradually, the Ret increased step and step, implying the successful hybridization of target thrombin with S1 and AuNPs-S2.
First of all, the CdS:Mn/TiO2/ITO electrode electrode was treated with 20 μL of 2% acetic acid solution containing 0.1 mg/m L CS to acquire a -COOH modified interface and dried at 60 °C. Then 10 μL of 2.5% GLD was covered onto the electrode surface and remained for 1 h at room temperature. After rinsed with de-ionized water, a volume of 10 μL 1 μM capture DNA S1 solution was dropped directly onto the CdS:Mn/TiO2/ITO electrode and incubated for 1 h in the dark to form a self-assembled monolayer (SAM) via -CONH- chemical bond. The excess capture-DNA was removed via a room temperature de-ionized water rinse (∼ 20 s). For avoiding the nonspecific adsorption, 10 μL of 1% 304
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Fig. 1. SEM image of (A) TiO2/ITO and (B) CdS:Mn/TiO2/ITO; EDS image of (C) TiO2/ITO and (D) CdS:Mn/TiO2/ITO.
Fig. 4. EIS of (a) CdS:Mn/TiO2/ITO, (b) CS assembly, (c) Glutarldehyde and S1 immobilization, (d) BSA immobilization, (e) incubated with 10 μL of 8 nM thrombin and (f) incubated with AuNPs-S2.
Fig. 2. Fluorescence spectrum of CdS:Mn (curve a) and UV–vis spectrum of AuNPs-S2 (curve b), insert: TEM image of AuNPs.
Fig. 5. Photocurret responses of (a) ITO/TiO2, (b) CdS:Mn/TiO2/ITO, (c) CS assembly, (d) glutarldehyde and S1 immobilization, (e) BSA immobilization, (f) incubation of 10 μL of 8 nM thrombin, (g) incubation of S2 and (h) the immobilization of AuNPs-S2.
Fig. 3. Photocurret responses of (a) ITO, (b) TiO2/ITO, (c) CdS/TiO2/ITO and (d) CdS:Mn/TiO2/ITO.
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Scheme 2. Energy transfer mechanism of the operating PEC system in 0.1 M AA electrolyte.
Fig. 6. Photocurrent response of (A) the CdS:Mn/TiO2/ITO electrode with different TiO2 concentration, the SILAR of CdS:Mn is 5, (B) the different SILAR of CdS:Mn, C(TiO2) = 1.0 mg/mL, (C) the different incubation time of 10 μL 1 μM S1 and (D) the different incubation time of 10 μL 8 nM thrombin.
3.4. Feasibility of the PEC biosensor
recombination. As compared to the CdS:Mn/TiO2/ITO electrode, the immobilization of CS (curve c) and capture DNA S1 (curve d) caused gradually decrease of photocurrent intensity which shown the successful fabrication of DNA S1 using CS as cross linker, due to the poor conductivity and steric hindrance. After the incubation with BSA (curve e) and target thrombin (curve f), the photocurrent intensity decreased due to the insulativity of protein. In order to research with the PEC mechanism of this biosensor, S2 (curve g) and AuNPs-S2 (curve h) were further incubated respectively, it was found that the photocurrent
The validation of the designed strategy was further performed via photocurrent characterization of the PEC biosensing process. As shown in Fig. 5, the TiO2/ITO electrode displays a small photocurrent intensity (curve a), because TiO2 could only absorb UV light so that the photoelectric conversion efficiency is low. After deposition of CdS:Mn, the photocurrent intensity (curve b) had a sharp rise, revealing that CdS:Mn could promote charge separation and depress the electron-hole 306
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Fig. 7. (A) Photocurrent responses of thrombin at 10–13,10–12,10–11,10−10,10−9,10−8.3 M respectively. (B) Calibration curve for thrombin. (C) The stability of the developed biosensor. (D) Selectivity of the sensor incubated with CRP, ALP and Mb.
Table 1 Comparison of different methods for the determination of thrombin. Method
Platform
Fluorescence
Aptamer, Aptamer, Aptamer, Aptamer, Aptamer, Aptamer, Aptamer, Aptamer,
colorimetric sensor Electrochemical Aptasensor Photoelectrochemical Sensing
DNAzyme powered DNA motor Protein-induced gold nanoparticle aggregation cationic polythiophene derivative Co-based metal-organic frameworks rolling circle amplification Au nanoparticles modified nanoporous BiVO4 CdS:Mn/TiO2/ITO
intensity of assembling with AuNPs-S2 (curve h) showed a larger decrease than S2 (curve g). Therefore, the PEC mechanism was shown in Scheme 2: AuNPs-S2 bonded on target thrombin will force the AuNPs away from the electrode surface. In this state, strong quenching of photocurrent intensity will be realized for the energy transfer effect between AuNPs and CdS:Mn. All of these results suggest the successful fabrication of the PEC aptasensor as designed.
Linear range
Detection limit
Ref.
10 pM~10 nM 0.25 pM~25 nM 10 nM~5 μM 0.01–1 nM 1 pM to 30 nM 0.1 pM~10 nM 1 pM~10 nM 0.1 pM~ 8 nM
4 pM 8.9 pM 7.5 nM 4 pM 0.32 pM 35 fM 0.5 pM 30 fM
[28] [29] [30] [31] [32] [33] [34] This work
In the low concentration range of TiO2 (0.3–1.0 mg/mL), more amount of CdS:Mn could be absorbed on the surface of TiO2, which benefit for photocurrent intensity increase. However, In the high concentration range of TiO2 (1.0–2.0 mg/mL), excessive TiO2 hinder the electron transfer, which leads to the gradual decrease of the photocurrent intensity. Therefore, a 1.0 mg/mL TiO2 suspension was used to make the electrodes. The deposition of CdS:Mn can be adjusted by the period of SILAR. Similarly, the photocurrent of the CdS:Mn/TiO2/ITO electrode was investigated with different CdS:Mn deposition period at 1.0 mg/mL TiO2. which reached the maximum at 5 cycles (Fig. 6B). For best signal amplification, 5 SILAR cycles of CdS:Mn deposition and 1.0 mg/mL TiO2 were chosen to make the electrodes. Fig. 6C and Fig. 6D showed the optimization incubation time for S1 and thrombin, respectively. It can be found that the photocurrent decreased sharply before 40 min. The reason was that more successful hybridization was formed with the
3.5. Optimization of experimental condition In order to achieve the best sensitization effect of CdS:Mn to TiO2, the concentration of TiO2 suspension and the coating number of CdS:Mn were optimizated. As seen from Fig. 6A, with the increased concentration of TiO2, the maximum photocurrent of CdS:Mn/TiO2/ ITO with five cycles of CdS deposition was obtained at 1.0 mg/mLTiO2. 307
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sensitivity, low detection limit, good selectivity and excellent stability. The quenched sandwich-type PEC aptamer sensing platform could be easily expanded to detect other proteins with relevant aptamers, showing great potential in bioanalysis.
Table 2 Results of thrombin detection in 100-Fold-Diluted Serum Samples (n = 3). Samples
Added TB (pM)
Found TBa (pM))
Recovery
RSD (%)
1 2 3
0.5 5 50
0.476 4.906 50.9
0.96 0.981 1.018
4.89 2.31 3.26
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant numbers: U1504216, 21575130); Startup Research Fund of Zhengzhou University (Grant no. 1511316006).
TB: thrombin. TBa: The average value of three measurements.
increasing incubation time. Therefore, the incubation time of S1 and thrombin were all chosen as 60 min.
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3.6. Analytical performance The quenched photocurrent was directly related to the concentration of target protein thrombin and thus providing a sensitive method for PEC detection of thrombin. Thus, to get the relationship between the photocurrent response with the concentration of thrombin, a series of different thrombin concentrations were investigated. Fig. 7A shows that the photocurrent response of the biosensor decreased gradually with the increasement of the concentration of thrombin. As shown in Fig. 7B, the decrement of photocurrent is proportional to the logarithm of thrombin concentration ranged from 0.1 pM to 8 nM. The equation of the linear regression is ΔI = 5.8102 log c + 78.3021 with the correlation coefficient of 0.997. ΔI = I - I0, where I stands for the PEC response without thrombin, and I0 stands for the PEC response with different concentration of thrombin. Furthermore, the limit of detection (LOD) was 30 fM (S/N = 3). The quenched sandwich-type PEC aptasensor showed a wide response and low limit of detection, which was similar or even better than the many previously reported methods of thrombin detection (Table 1). The stability was a vital factor for the application of a PEC sensor. In our work, the final PEC response of was recorded under 10 on/off irradiation cycles. As shown in Fig. 7C, the photocurrent were observed without a obvious variation, indicating the stability of the sensor. The selectivity of this method was tested by other co-existing proteins in the serum, Fig. 7D including creactive protein (CRP), alkaline phosphatase (ALP) and myoglobin (Mb). It had no influence on the PEC sensing although the 100-fold of the interference compared with that of the sensor incubated with thrombin, which disclosed that the fabricated PEC biosensor showed good selectivity for thrombin because of the specific recognition between thrombin with S1 and AuNPs-S2. 3.7. Real sample analysis To estimate the applicability of this proposed approach, the survey was determined by the standard addition method in three human serum samples from The first affiliated hospital of Zhengzhou university. The serum samples were measured for determination of thrombin with 100 fold dilution. As listed in Table 2, the recovery of thrombin in serum samples ranged from 96% to 101.8%, the RSD are no more than 5%, indicating that the proposed approach has good potential for the analysis of thrombin in real samples. 4. Conclusion This work develops a novel sensitive sandwich-type photoelectrochemical aptamer sensing paltform for target protein. The specific recognition of target protein by aptamer enable the immobilization of target protein and signal aptamer on the electrode step-by-step, which brings AuNPs far from the CdS:Mn/TiO2/ITO electrode and thus produces a strong quenched photocurrent through the ET effect between the AuNPs and CdS:Mn QDs. Using thrombin as a target protein, resulting PEC aptasensor exhibited wide detectable range, high 308
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