Preparation method and application of self-powered photoelectrochemical aptamer sensor

Abstract

The application discloses a preparation method of a self-powered photoelectrochemical aptamer sensor and application of the sensor to PSA detection. ZIF-90@MB / PApt is used as a signal probe, Ag-CdIn2S4 is used as a photosensitive material, and the MB signal molecules released from the ZIF-90 channel based on the response of the target can produce a sensitization effect on the surface of the Ag-CdIn2S4 electrode, so that the photocurrent signal is enhanced. When the PSA target exists, the specific recognition of PApt and PSA makes the biological gate open, a large number of MB molecules are released from the ZIF-90, and the sensitization effect is produced on the surface of the Ag-CdIn2S4 photosensitive material modified electrode, so that the photocurrent signal is enhanced, and then the quantitative detection of PSA is realized. The sensor can realize the accurate determination of the PSA content in human serum samples, and provides a novel sensing strategy for the early diagnosis of clinical biomarkers.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of photoelectrochemical biosensors, and particularly relates to a self-powered photoelectrochemical aptamer sensor based on target response and Ag-CdIn2S4 photosensitive material for detection of prostate specific antigen. BACKGROUND

[0002] Prostate specific antigen (PSA, a tumor marker) exists in the prostate and plays an important role in the screening and early diagnosis of prostate cancer. Therefore, in clinical practice, it is of great significance to develop an accurate and sensitive method for detecting PSA in serum for the diagnosis of prostate cancer.

[0003] Photoelectrochemical (PEC) sensors are a new type of sensing technology that combines the advantages of electricity and optics. They have the advantages of easy operation, low background signal, strong anti-interference ability and fast response speed, and have therefore attracted widespread attention. Self-powered PEC sensors can work normally without an external voltage (E=0), making them a promising sensing strategy. They use photosensitive materials on the electrode surface as signal transducers to generate accurate photocurrent signals affected by the target under light. And at zero bias voltage, self-powered PEC sensors can effectively exclude interference from other oxidizing / reducing substances in the detection system, thereby reducing damage to biological molecules at the electrode interface and improving the accuracy of detection.

[0004] Target response controlled release is an effective strategy for constructing biosensors, which loads a large number of signal molecules inside a porous carrier and realizes signal transmission and amplification through biological gating and target response controlled release.

[0005] Among the currently disclosed patents for detecting prostate specific antigen, CN112526135A is a preparation method and application of a photoelectrochemical biosensor for detecting prostate specific antigen. Ag2S quantum dots are used to sensitize Ag / AgBr / BiOBr to enhance its visible light absorption, and its photoelectric activity is significantly improved. Prostate specific antibodies, bovine serum albumin and prostate specific antigen are assembled on the Ag / AgBr / BiOBr / Ag2S composite material, and the excellent photoelectric activity of Ag / AgBr / BiOBr and the specific binding between prostate specific antigen-antibody realize the ultra-sensitive analysis of prostate specific antigen, providing a new method for effectively detecting prostate specific antigen. However, the material needs to be self-assembled layer by layer, which is a cumbersome and time-consuming process.

[0006] CN110596364A A method for constructing a ratio electrochemical sensor for prostate-specific antigen detection and its application, Ag2S sensitized microspherical Ag / AgBr / BiOBr heterojunction is fixed on the surface of ITO electrode as an excellent light active substrate, which effectively improves the photocurrent response and its sensitivity. Mercapto acetic acid (TGA) is used as a connecting agent to fix prostate-specific antibody, through the specific binding between antigen and antibody, the photocurrent signal of different concentrations of antigen is detected, and the quantitative test of PSA is completed. But TGA is not easy to be fixed, and the first antibody and the second antibody may produce cross reaction, which can cause great interference to the result.

[0007] CN116337850A An electrochemiluminescence sensor based on FeMoOv nanometer enzyme bipolar electrode and application for detecting H2O2 and PSA, a sandwich immunosensor with FeMoOv / AuNPs as recognition probe is designed, which mediates the ECL reaction on the anode, realizes the high sensitive detection of PSA, and a unique mobile phone ECL imaging system is developed for determining different concentrations of PSA. But the electrode modification step can reduce the biological recognition process on the electrode surface. But this method has poor specificity and is easy to cause false positive results. SUMMARY

[0008] The present application provides a self-powered photoelectrochemical (PEC) aptamer sensor based on target response and Ag-CdIn2S4 photosensitive material for prostate-specific antigen detection, which can realize accurate and sensitive detection of PSA and provide a new sensing strategy for early diagnosis of clinical biomarkers.

[0009] The present application adopts the following technical solutions:

[0010] A preparation method of a self-powered photoelectrochemical aptamer sensor, comprising the following steps:

[0011] (1) preparing photoelectric active material Ag-CdIn2S4;

[0012] (2) preparing ZIF-90@MB;

[0013] (3) preparing signal probe ZIF-90@MB / PApt;

[0014] (4) constructing GCE / Ag-CdIn2S4 sensing interface;

[0015] (5) Constructing a self-powered photoelectrochemical aptamer sensor to detect prostate-specific antigen, using ZIF-90@MB / PApt as a signal probe and Ag-CdIn2S4 as a photosensitive material to provide a stable background photocurrent signal. The MB signal molecules released from the ZIF-90 channel based on the target response can produce a sensitization effect on the surface of the Ag-CdIn2S4 electrode, thereby enhancing the photocurrent signal and achieving signal amplification.

[0016] Further, the preparation of the photoelectric active material Ag-CdIn2S4 in step (1) specifically comprises the following steps:

[0017] First, 50-500 mg of CdCl2, 200-800 mg of InCl3·4H2O, and 5-50 mg of AgNO3 are ultrasonically dissolved in 5-50 mL of water and stirred at room temperature;

[0018] Next, 10-100 mg of thioacetamide (TAA) is ultrasonically dissolved in 2-200 mL of water and quickly added to the above mixture and continues to be stirred at room temperature to obtain a brown-yellow precursor solution;

[0019] Finally, the obtained brown-yellow precursor solution is transferred into a high-pressure reaction kettle polytetrafluoroethylene liner, heated at 60-300°C for 12 h, and after the reaction is completed, it is naturally cooled to room temperature, washed with ultrapure water and ethanol for 3 times respectively, and then vacuum dried at 60°C to obtain a brown product Ag-CdIn2S4.

[0020] Further, the preparation of ZIF-90@MB in step (2) uses zeolite imidazole framework-90 (ZIF-90) porous material as a carrier and loads methylene blue (MB) photosensitizer as a signal molecule in the ZIF-90 channel to prepare a controlled release system (ZIF-90@MB), which specifically comprises the following steps:

[0021] 5-500 mg of Zn(CH3COOH)2·2H2O, 2-20 mg of MB, and 20-200 mg of imidazole-2-formaldehyde (2-ICA) are ultrasonically dissolved in 2-200 mL of DMF solution and stirred at room temperature;

[0022] The obtained product is washed with ultrapure water and ethanol by centrifugation until the supernatant is colorless, and vacuum dried at 60°C to obtain a light blue product ZIF-90@MB.

[0023] Further, the preparation of the signal probe ZIF-90@MB / PApt in step (3) specifically comprises the following steps:

[0024] 2~200 mg ZIF-90@MB was ultrasonically dispersed into 2~200 mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer (HEPES, 10 mmol / L), and 10~1000 μL of PApt was added, and incubated on a 37℃ shaker;

[0025] The obtained complex was then centrifuged, washed twice to remove excess unbound PApt, and redispersed in 2~200 mL of HEPES buffer to obtain a signal probe ZIF-90@MB / PApt, which was stored at 4℃ for subsequent use.

[0026] Further, the step (4) for constructing the GCE / Ag-CdIn2S4 sensing interface specifically comprises the following steps:

[0027] The GCE electrode was pretreated with aluminum oxide powder on a suede, washed with ultrapure water after polishing, and then ultrasonically washed with ethanol and ultrapure water, and dried at room temperature for standby use;

[0028] 2~200 mg of Ag-CdIn2S4 nanomaterial was ultrasonically dispersed in 0.1~100 mL of ethanol solution, and 2~200 μL of Ag-CdIn2S4 dispersion suspension was uniformly dropped onto the surface of the above cleaned GCE electrode, and dried in a 37℃ air oven, and stored at room temperature in the dark for subsequent use.

[0029] Further, the step (5) for constructing the self-powered photoelectrochemical aptamer sensor to detect prostate specific antigen specifically comprises the following steps:

[0030] 10~1000 μL of the above prepared ZIF-90@MB / PApt signal probe, 10~100 μL of different concentrations of PSA target, and 2~200 μL of HEPES buffer (10 mmol / L) were added to a centrifuge tube, and incubated at 37℃, and then centrifuged, and the supernatant was reserved;

[0031] 2~20 μL of the supernatant was dropped onto the GCE / Ag-CdIn2S4 electrode, and incubated at room temperature in the dark, and then washed with ultrapure water twice to remove excess unreacted MB, and dried at room temperature and modified with Nafion to prevent the electrode material from falling off, and the obtained GCE / Ag-CdIn2S4@MB electrode was used for subsequent PEC detection;

[0032] A 500 W xenon lamp was used as an excitation light source, the switching interval was 20 s, the external potential was 0 V, and the electrolyte was a PBS (10 mmol / L) buffer solution containing 0.1 mol / L AA, and all experiments were carried out at room temperature.

[0033] Further, the self-powered photoelectrochemical aptamer sensor prepared by the preparation method is used for detecting prostate specific antigen PSA.

[0034] The sensor of the application uses ZIF-90@MB / PApt as a signal probe and Ag-CdIn2S4 as a photosensitive material to provide a stable background photocurrent signal, and constructs a self-powered PEC aptamer sensor based on target response for high-sensitivity detection of PSA. The MB signal molecules released from the ZIF-90 channel by the target response can produce a sensitization effect on the surface of the Ag-CdIn2S4 modified electrode, thereby enhancing the photocurrent signal and achieving signal amplification.

[0035] The sensor of the application selects a homogeneous system, and the target recognition process occurs in a homogeneous solution, avoiding the traditional cumbersome electrode modification steps and reducing the mutual interference between the electrode surface biological recognition process and the PEC signal detection. When used for prostate specific antigen detection, the PSA aptamer (PApt) is attached to the surface of ZIF-90@MB to form a simple biological gate through the action of π-π stacking and electrostatic adsorption. When the PSA target exists, the biological gate is opened due to the specific recognition of PApt and PSA, a large number of MB molecules are released from ZIF-90, and a sensitization effect is produced on the surface of the Ag-CdIn2S4 photosensitive material modified electrode, thereby enhancing the photocurrent signal and realizing accurate and sensitive quantitative detection of PSA. At the same time, the self-powered PEC sensor can be used for sensitive and accurate detection of the PSA content in serum samples, and provides a new sensing strategy for early diagnosis of clinical biomarkers. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 A photocurrent response curve of the self-powered photoelectrochemical aptamer sensor prepared for the example for detecting PSA of different concentrations;

[0037] Figure 2 A linear relationship graph of the photocurrent signal intensity of the sensor and the logarithm of the PSA concentration under different concentrations of PSA for the example. DETAILED DESCRIPTION

[0038] The content of the application will be further described below in combination with the examples and the drawings, but is not a limitation on the application. EXAMPLE

[0039] A preparation method of a self-powered photoelectrochemical aptamer sensor, comprising the following steps:

[0040] (1) preparing photoelectric active material Ag-CdIn2S4;

[0041] (2) preparing ZIF-90@MB;

[0042] (3) Preparation of signal probe ZIF-90@MB / PApt;

[0043] (4) Construction of GCE / Ag-CdIn2S4 sensing interface;

[0044] (5) Construction of self-powered photoelectrochemical aptamer sensor for detection of prostate specific antigen, ZIF-90@MB / PApt as signal probe, Ag-CdIn2S4 as photosensitive material to provide stable background photocurrent signal, the MB signal molecule released from the ZIF-90 channel based on the target response can produce a sensitization effect on the surface of Ag-CdIn2S4 electrode, so as to enhance the photocurrent signal and achieve signal amplification.

[0045] Step (1) Preparation of photoelectric active material Ag-CdIn2S4, specifically including the following steps:

[0046] 100 mg of CdCl2, 500 mg of InCl3·4H2O and 7 mg of AgNO3 were ultrasonically dissolved in 30 mL of water and stirred at room temperature;

[0047] Then, 60 mg of TAA was ultrasonically dissolved in 150 mL of water and quickly added to the above mixture and continued to be stirred at room temperature to obtain a brownish yellow precursor solution;

[0048] Finally, the obtained brownish yellow precursor solution was transferred into a high-pressure reaction kettle polytetrafluoroethylene liner, heated at 250℃ for 12 h, and after the reaction was completed and it was naturally cooled to room temperature, washed with ultrapure water and ethanol for 3 times respectively, and then vacuum dried at 60℃ to obtain a brown product Ag-CdIn2S4.

[0049] Step (2) Preparation of ZIF-90@MB, specifically including the following steps:

[0050] 200 mg of Zn(CH3COOH)2·2H2O, 5 mg of MB and 100 mg of 2-ICA were ultrasonically dissolved in 150 mL of DMF solution, and stirred at room temperature;

[0051] The obtained product was washed with ultrapure water and ethanol by centrifugation until the supernatant was colorless, and vacuum dried at 60℃ to obtain a light blue product ZIF-90@MB.

[0052] Step (3) Preparation of signal probe ZIF-90@MB / PApt, specifically including the following steps:

[0053] 50 mg ZIF-90@MB was ultrasonically dispersed into 180 mL HEPES buffer (10 mmol / L), and 50 μL PApt was added, and then incubated at 37℃ on a shaker;

[0054] Then the obtained complex was centrifuged, washed twice to remove excess unbound Apt, and then redispersed into 180 mL HEPES buffer to obtain a signal probe ZIF-90@MB / PApt, and stored at 4℃ for subsequent use.

[0055] Step (4) constructing a GCE / Ag-CdIn2S4 sensing interface, specifically comprising the following steps:

[0056] The GCE electrode was pretreated with alumina powder on a piece of chamois leather, and then polished and washed with ultrapure water, and then sequentially washed with ethanol and ultrapure water, and dried at room temperature for standby use;

[0057] 50 mg of Ag-CdIn2S4 nanomaterials was ultrasonically dispersed in 50 mL of ethanol solution, 150 μL of Ag-CdIn2S4 dispersion suspension was taken and uniformly drop-coated on the surface of the above cleaned GCE electrode, and then dried in a 37℃ air oven, and stored at room temperature in the dark for subsequent use.

[0058] Step (5) constructing a self-powered photoelectrochemical aptamer sensor to detect prostate specific antigen, specifically comprising the following steps: 10 μL of the above prepared ZIF-90@MB / PApt signal probe, 10 μL of different concentrations of PSA target and 180 μL of HEPES buffer (10 mmol / L) were added into a centrifuge tube, and then incubated at 37℃, and then centrifuged, and the supernatant was reserved;

[0059] 5 μL of the supernatant was drop-coated on the GCE / Ag-CdIn2S4 electrode, and then incubated at room temperature in the dark, and then washed with ultrapure water twice to remove excess unreacted MB, and then dried at room temperature and modified with Nafion to prevent the electrode material from falling off, and then the obtained GCE / Ag-CdIn2S4@MB electrode was used for subsequent PEC detection;

[0060] A 500 W xenon lamp was used as an excitation light source, the interval of switching on and off the lamp was 20 s, the applied potential was 0 V, and the electrolyte was a PBS (10 mmol / L) buffer solution containing 0.1 mol / L AA, and all experiments were carried out at room temperature.

[0061] In the method, different concentrations of PSA were analyzed and detected by using a self-powered photoelectrochemical aptamer sensor. Figure 1The self-powered photoelectrochemical aptamer sensor prepared in the embodiment detects the photocurrent response curves of different concentrations of PSA, wherein the concentrations of PSA are 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1 and 10 ng / mL in turn.

[0062] As shown in Figure 1 , with the increase of the concentration of PSA, the photocurrent signal gradually increases, because of the specific recognition of PSA and PApt, the increase of the amount of released MB, and the certain energy level matching relationship between MB and Ag-CdIn2S4, which effectively realizes the separation of photoinduced electron-hole pairs, so as to achieve the effect of signal enhancement. Within the concentration range of 0.005-10 ng / mL, the photocurrent signal has a linear relationship with the logarithm of the concentration of PSA, as shown in Figure 2 , the linear equation is: I =143.04×logC [PSA] +686.53 (R 2 =0.9984), and the lowest detection limit (LOD) is 2.35 pg / mL (3SD / K, SD is the standard deviation of the PEC current signal of 11 blank samples, and K is the slope of the linear equation).

[0063] Experimental example:

[0064] Detection of serum samples: 6 groups of clinical serum samples were taken from the Guilin Hospital of Integrated Traditional Chinese and Western Medicine, 6 groups of human serum samples containing different concentrations of PSA were determined by using the self-built PEC aptamer sensor, and the measured results were compared with the results determined by the hospital (Antu A2000-2 chemiluminescence immunoassay analyzer). The concentrations of PSA in the collected serum samples were 0.007, 0.101, 0.381, 0.739, 1.482 and 5.765 ng / mL, and the results are shown in Table 1. The experimental results of the PEC aptamer sensor constructed in Table 1 in the analysis of PSA in serum samples

[0065]

[0066] As can be seen from Table 1, the measured results are close to the ECL determination results, and the relative standard deviation (RSD) is between 1.6% and 7.1%. The above results show that the self-powered PEC aptamer sensing platform based on Ag-CdIn2S4 has satisfactory accuracy and practical applicability for detecting PSA.

Claims

1. A method for preparing a self-powered photoelectrochemical aptamer sensor, characterized in that, It comprises the following steps: (1) preparing photoelectric active material Ag-CdIn2S4; (2) preparing ZIF-90@MB; (3) preparing signal probe ZIF-90@MB / PApt; (4) constructing GCE / Ag-CdIn2S4 sensing interface; (5) constructing self-powered photoelectrochemical aptamer sensor for detecting prostate specific antigen, taking ZIF-90@MB / PApt as signal probe and Ag-CdIn2S4 as photosensitive material to provide stable background photocurrent signal, and the signal molecule MB released from ZIF-90 channel based on target response can produce sensitization effect on the surface of Ag-CdIn2S4 electrode, so as to enhance the photocurrent signal and achieve signal amplification.

2. The preparation method of the self-powered photoelectrochemical aptamer sensor according to claim 1, characterized in that: step (1) of preparing photoelectric active material Ag-CdIn2S4 specifically comprises the following steps: first, 50-500 mg of CdCl2, 200-800 mg of InCl3·4H2O and 5-50 mg of AgNO3 are ultrasonically dissolved in 5-50 mL of water and stirred at room temperature; then, 10-100 mg of thioacetamide is ultrasonically dissolved in 2-200 mL of water and quickly added to the above mixture and continuously stirred at room temperature to obtain a brownish yellow precursor solution; finally, the obtained brownish yellow precursor solution is transferred into a high-pressure reaction kettle polytetrafluoroethylene liner, heated at 60-300℃ for 12h, after the reaction is completed and it is naturally cooled to room temperature, washed with ultrapure water and ethanol for 3 times respectively, and then vacuum dried at 60℃ to obtain a brown product Ag-CdIn2S4.

3. The preparation method of the self-powered photoelectrochemical aptamer sensor according to claim 1, characterized in that: step (2) of preparing ZIF-90@MB specifically comprises the following steps: 5-500 mg of Zn(CH3COOH)2·2H2O, 2-20 mg of MB and 20-200 mg of imidazole-2-formaldehyde are ultrasonically dissolved into 2-200 mL of DMF solution and stirred at room temperature; the obtained product is centrifugally washed with ultrapure water and ethanol until the supernatant is colorless, and vacuum dried at 60℃ to obtain a light blue product ZIF-90@MB.

4. The preparation method of the self-powered photoelectrochemical aptamer sensor according to claim 1, characterized in that: step (3) of preparing signal probe ZIF-90@MB / PApt specifically comprises the following steps: 2-200 mg of ZIF-90@MB is ultrasonically dispersed into 2-200 mL of HEPES buffer solution with a concentration of 10 mmol / L, and then 10-1000 μL of PApt is added and incubated on a shaking bed at 37℃; The obtained complex was then centrifuged again, washed twice to remove excess unbound PApt, and redispersed in 2-200 mL of HEPES buffer to obtain the signal probe ZIF-90@MB / PApt, which was stored at 4°C for subsequent use.

5. The preparation method of the self-powered photoelectrochemical aptamer sensor according to claim 1, characterized in that: Step (4) of constructing the GCE / Ag-CdIn2S4 sensing interface specifically comprises the following steps: The GCE electrode is pretreated with aluminum oxide powder on a suede, washed clean with ultrapure water after polishing, and then ultrasonically washed with ethanol and ultrapure water, respectively, and dried at room temperature for standby use; 2-200 mg of Ag-CdIn2S4 nanomaterials are ultrasonically dispersed in 0.1-100 mL of an ethanol solution, 2-200 μL of Ag-CdIn2S4 dispersion suspension is uniformly dropped onto the surface of the above-mentioned clean GCE electrode, and it is completely dried in a 37°C air oven, and stored in the dark at room temperature for subsequent use.

6. The preparation method of the self-powered photoelectrochemical aptamer sensor according to claim 1, characterized in that: Step (5) of constructing the self-powered photoelectrochemical aptamer sensor for detecting prostate specific antigen specifically comprises the following steps: 10-1000 μL of the above-mentioned prepared ZIF-90@MB / PApt signal probe, 10-100 μL of different concentrations of PSA target, and 2-200 μL of 10 mmol / L HEPES buffer are added to a centrifuge tube, incubated at 37°C, then centrifuged, and the supernatant is reserved; 2-20 μL of the supernatant is dropped onto the GCE / Ag-CdIn2S4 electrode, incubated in the dark at room temperature, then washed twice with ultrapure water to remove excess unreacted MB, dried at room temperature and modified with Nafion to prevent the electrode material from falling off, and the obtained GCE / Ag-CdIn2S4@MB electrode is used for subsequent PEC detection; A 500 W xenon lamp is used as the excitation light source, the switching interval is 20 s, the applied potential is 0 V, the electrolyte is a 10 mmol / L PBS buffer solution containing 0.1 mol / L AA, and all detections are carried out at room temperature.

7. A self-powered photoelectrochemical aptamer sensor prepared by the method of any one of claims 1-6, wherein the photoelectrochemical aptamer sensor is capable of detecting an analyte. The self-powered photoelectrochemical aptamer sensor is used for detecting prostate specific antigen PSA.

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