Multifunctional ceo2 / cds and interface etching-based ratiometric photoelectrochemical analysis method

Abstract

The application belongs to the technical field of nanomaterials and biological analysis, and particularly relates to a ratio type photoelectrochemical analysis method based on multifunctional CeO2 / CdS and interface etching. First, a multifunctional CeO2 / CdS nanocomposite is synthesized by an in-situ growth method, and the nanocomposite has photoelectric properties of CdS and CeO2 peroxidase activity; second, a beacon antibody labeled with nanogold and glucose oxidase is prepared by an electrostatic adsorption method, a target protein, the beacon antibody and an antibody coated on an enzyme-labeled microplate are mixed and incubated to form an immune complex, and after glucose is added, H2O2 is generated by catalysis of the glucose oxidase on the beacon antibody; the generated H2O2 is catalyzed by CeO2 in the CeO2 / CdS nanocomposite to generate hydroxyl radicals, and in-situ etching of CdS in the electrode interface reduces the photocurrent, and a photocurrent intensity ratio (Iw / Ic) of a double-channel electrode has a linear relationship with the concentration of the target protein, so that quantitative analysis can be realized. The application has a sensitive response to the detection of proteins and has a fast detection speed.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials and bioanalysis technology, specifically relating to a ratiometric photoelectrochemical analysis method based on multifunctional CeO2 / CdS and interface etching. Background Technology

[0002] Photoelectrochemical (PEC) sensing methods generate electrical signals through photo-to-electric conversion using photoactive materials. Involving both photoelectric conversion and electrochemical processes, it has become an independent detection method capable of highly sensitive analysis of biological enzymes, heavy metal ions, and nucleic acids. PEC sensing methods consist of three parts: a light source (external physical or chemiluminescent), a photoelectric detection platform (photosensitive electrode and electrolytic cell), and an electrical signal output device (electrochemical workstation, handheld digital multimeter, etc.). The primary function of the sensor's light source system is to excite the photosensitive electrode to generate a photocurrent. The photosensitive electrode loaded with photoelectric material is a crucial component of the photoelectric detection system. After absorbing photons, electrons in the valence band of the photoelectric material are excited to the conduction band, leaving holes in the valence band, generating electron-hole pairs. The resulting photogenerated electrons are transferred to the electrode and the electrolyte, and the corresponding electrical signal is output by the signal output device. Quantitative analysis of the analyte can be achieved by adding a analyte to the sensing system, causing changes in the electrolyte composition, light source intensity, and photoelectric sensing interface composition, leading to changes in output current and voltage.

[0003] The most widely used signal modulation strategy currently is to utilize the specific recognition of the analyte to trigger a series of reactions, causing changes in the concentration of electron donors (reducing substances) or acceptors (oxidizing substances) in the electrolyte composition. Under photoexcitation, these changes in the concentration of electron acceptors or donors directly affect the reaction process with electrons or holes generated by photoelectric materials, causing changes in the output electrical signal and enabling quantitative analysis of protein analytes. However, this detection method is susceptible to the influence of redox substances, light intensity, and electrolyte concentration fluctuations in complex detection environments. Summary of the Invention

[0004] The technical problem solved by this invention is to provide a ratiometric photoelectrochemical analysis method based on multifunctional CeO2 / CdS and interface etching. By utilizing the ratio of the output signals of the working electrode to the internal reference electrode (Iw / Ic), the influence of external environmental changes on the photocurrent signal can be effectively reduced, thereby achieving highly sensitive detection of target proteins.

[0005] The technical solution of this invention is as follows:

[0006] The ratiometric photoelectrochemical analysis method based on multifunctional CeO2 / CdS and interface etching described in this invention includes the following steps:

[0007] (1) Preparation of multifunctional CeO2 / CdS nanocomposites;

[0008] (2) Prepare beacon probes labeled with gold nanoparticles (Au NPs) and glucose oxidase (GOx), add the target protein and beacon probes to enzyme-labeled microplates coated with the first antibody of the target protein (Ab1) and incubate to construct a sandwich complex. After washing, add glucose to obtain the catalytic reaction solution.

[0009] (3) The multifunctional CeO2 / CdS nanocomposite was drop-coated onto the surface of the working electrode and the internal reference electrode, and incubated with the supernatant of the catalytic reaction solution in step (2);

[0010] (4) Photoelectric quantitative detection was performed using an electrochemical workstation and a xenon lamp.

[0011] Preferably, step (1) specifically includes the following steps:

[0012] a. Mix Ce(NO3)3 solution and NaOH solution at a molar ratio of 1: (12~15) until homogeneous, carry out hydrothermal reaction, and obtain solid powder after washing with water and drying.

[0013] b. Take 100-200 mg of the product obtained in step a, disperse it in 100-200 mL of deionized water, add 150-300 μL of 1 M CdCl2 solution, and react to obtain the CeO2 / CdS nanocomposite.

[0014] The hydrothermal reaction temperature is 170~190℃, and the time is 20~28h;

[0015] The size of the CeO2 / CdS nanocomposite is 20~50 nm.

[0016] Preferably, step (2) specifically includes the following steps:

[0017] a. Add the target protein secondary antibody (Ab2) and GOx to Au NPs and incubate. Add BSA to block and centrifuge to collect the beacon probe (AuNPs / Ab2 / GOx).

[0018] b. Add 0.1 ng / mL target protein solution, target protein solutions of varying concentrations and test sample solutions to enzyme-labeled microplates coated with Ab1 and incubate at room temperature. After washing, add AuNPs / Ab2 / GOx and incubate at room temperature to construct sandwich complexes. After washing, add PBS solution containing glucose, adjust the pH, and incubate at 37°C to obtain the catalytic reaction solution.

[0019] The dosage of Au NPs is 1-5 mL, and the pH is 6.5-8.5; the concentration of Ab2 is 180-220 μg / mL, and the dosage is 40-50 μL; the concentration of GOx is 5-7 mg / mL, and the dosage is 100-150 μL.

[0020] The mass concentration of BSA is 1%, and the dosage is 90-110 μL.

[0021] The washing buffer was 10 mM, pH 7.0 PBS buffer.

[0022] The amount of beacon probe used is 100~200μL.

[0023] The glucose concentration in the PBS solution is 10-20 mM, the PBS concentration is 100 mM, and the pH is 7.0.

[0024] When adjusting the pH, use an acetate buffer solution with a pH of 4.5 to adjust the solution pH to 4.5-6.

[0025] Preferably, step (3) specifically includes the following steps:

[0026] a. Take 8-12 μL of CeO2 / CdS nanocomposite with a concentration of 20-30 mg / mL and drop it onto the surface of the working electrode and the internal reference electrode, and let it dry.

[0027] b. Take the supernatant of the catalytic reaction solution corresponding to the gradient concentration target protein solution and the sample solution to be tested in step (2), drop it onto the surface of the working electrode for incubation, and take the supernatant of the catalytic reaction solution corresponding to the 0.1 ng / mL target protein solution in step (2), drop it onto the surface of the internal reference electrode for incubation.

[0028] Preferably, step (4) specifically includes the following steps:

[0029] Na2SO4 solution was used as the electrolyte, and photoelectric detection was performed using a xenon lamp and an electrochemical workstation. The xenon lamps of both the internal reference electrode and the working electrode were turned on for 9-11 seconds, and the output signals were Ic and Iw, respectively. A standard working curve was obtained using the Ic / Iw signal ratio, and the target protein was quantified.

[0030] Further preferably, the Na2SO4 solution concentration is 0.2~0.4 M, and the xenon lamp power is 300~500 W.

[0031] The principle of this invention is to modify the surface of a dual-channel electrode with CeO2 / CdS, and after adding the analyte, form an immune sandwich structure. An antibody labeled with glucose oxidase is introduced into the detection system. The peroxidase-like properties of CeO2 are used to construct a GOx / CeO2 catalytic system, in which H2O2 generated from glucose by GOx can be catalyzed by CeO2 to generate hydroxyl radicals, which etch the CdS on the CeO2 surface, resulting in a decrease in the photocurrent of the working electrode. Quantitative analysis of the analyte is achieved based on Iw / Ic.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The analytical method described in this invention overcomes the influence of factors such as light intensity and electrolyte concentration on the single-channel photoelectrode during the measurement process;

[0034] 2. The method described in this invention exhibits the characteristics of fast response, high sensitivity, and good selectivity;

[0035] 3. This invention utilizes a multifunctional CeO2 / CdS heterojunction to improve the photoelectric response of CeO2 and reduce the photocorrosion of CdS.

[0036] 4. This invention utilizes an immunoassay mode for quantitative analysis of proteins and also has good universality for the detection of tumor markers, providing a new approach for the detection of tumor markers. Attached Figure Description

[0037] Figure 1 A schematic diagram of a ratiometric photoelectrochemical analysis method based on multifunctional CeO2 / CdS and interface etching;

[0038] Figure 2 Scanning electron microscope image of CeO2 / CdS nanocomposite;

[0039] Figure 3 The UV-Vis absorption spectra of TMB catalyzed by different concentrations of c-CeO2 are shown in the figure. From bottom to top, the concentrations are 0, 2, 4, 5, 6, 8, and 10 mg / mL.

[0040] Figure 4 A is the photoelectric response diagram, and B is the standard working curve diagram;

[0041] Figure 5 UV-Vis absorption spectra of AuNPs (a), beacon probe AuNPs / Ab2 / GOx (b), and mixture of GOx and Ab2 (c). Detailed Implementation

[0042] The present invention will be further described below with reference to embodiments.

[0043] It should be noted that all raw materials used in the examples, unless otherwise specified, were commercially available.

[0044] Example 1

[0045] The ratiometric photoelectrochemical analysis method based on multifunctional CeO2 / CdS and interface etching described in this invention includes the following steps:

[0046] (1) Preparation of multifunctional CeO2 / CdS nanocomposites

[0047] Ce(NO3)3 solution and NaOH solution were mixed at a molar ratio of 1:12, stirred thoroughly, and then placed in a high-pressure reactor and reacted at 180℃ for 24 h. 100 mg of the generated product was dispersed in 100 mL of water, and 300 μL of 1M CdCl2 solution was added. The reaction yielded the CeO2 / CdS nanocomposite. Figure 2 As shown, the size of cubic CeO2 is 20-50 nm. After CdS nanoparticles are deposited on the surface of cubic CeO2, irregular CdS particles appear on the CeO2 surface. Figure 3 As shown, using TMB as a chromogenic substrate, the catalytic oxidation of the substrate TMB in the presence of both CeO2 and H2O2 was monitored. With the increase of cubic CeO2 concentration, the absorption of oxidized TMB at 652 nm gradually increased, indicating that the amount of hydroxyl radicals generated is CeO2 concentration-dependent.

[0048] (2) Preparation of beacon probes

[0049] Take 1 mL of the prepared Au NPs, adjust the pH to 7, add 40 μL of CEA Ab2 (200 μg / mL) and 150 μL of GOx (6 mg / mL). After incubation for 2 h, add 100 μL of BSA (1%, w / v) to block for 1 h, and collect the beacon probes (AuNPs / Ab2 / GOx) by centrifugation. Add CEA (0.1 ng / mL) and gradient concentrations of CEA to 96-well microplates coated with Ab1, and incubate at room temperature for 1.5 h. Wash three times with PBS (10 mM, pH 7.0) buffer, add 150 μL of beacon probes, incubate at room temperature for 1 h, and wash again. Add PBS solution (100 mM, pH 7.0) containing glucose (15 mM) to 96-well microplates, adjust the pH of the solution to 4.5 with acetate buffer (pH 4.5), and incubate at 37 °C for 25 min.

[0050] (3) Establish a ratiometric photoelectrochemical sensing system

[0051] CeO2 / CdS nanocomposite was coated onto both the working electrode and the internal reference electrode. Supernatant from reaction solutions corresponding to gradient concentrations of CEA was dropped onto the working electrode surface and incubated. The internal reference electrode was incubated at room temperature with supernatant from a 0.1 ng / mL CEA reaction solution. Na2SO4 solution (0.4 M) was used as the electrolyte, and photoelectric detection was performed using a xenon lamp (400 W) and an electrochemical workstation. The xenon lamp on-time for both the internal reference electrode and the working electrode was 10 s, and the output signals were Ic and Iw, respectively. CEA was quantified using the Ic / Iw signal ratio, and the resulting standard working curve is shown below. Figure 4 As shown in B.

[0052] Example 2

[0053] (1) Preparation of multifunctional CeO2 / CdS nanocomposites

[0054] Ce(NO3)3 solution and NaOH solution were mixed at a molar ratio of 1:12, stirred thoroughly, and then placed in a high-pressure reactor and reacted at 180℃ for 24 h. 100 mg of the generated product was dispersed in 100 mL of water, and 300 μL of 1M CdCl2 solution was added. The reaction yielded the CeO2 / CdS nanocomposite. Figure 2 As shown, the size of cubic CeO2 is 20~50 nm. After CdS nanoparticles are deposited on the surface of cubic CeO2, irregular CdS particles appear on the surface of CeO2.

[0055] (2) Preparation of beacon probes

[0056] Take 1 mL of the prepared Au NPs, adjust the pH to 8, add 50 μL of ovalbumin (food allergen) Ab2 (200 μg / mL) and 100 μL of GOx (6 mg / mL). After incubation for 2 h, add 100 μL of BSA (1%, w / v) to block for 1 h, and collect the beacon probes (AuNPs / Ab2 / GOx) by centrifugation. Add ovalbumin (0.1 ng / mL) and gradient concentrations of ovalbumin to 96-well plates coated with Ab1, and incubate at room temperature for 1.5 h. Wash three times with PBS (10 mM, pH 7.0) buffer, add beacon probes (200 μL), incubate at room temperature for 1 h, and then wash again. Add PBS solution (100 mM, pH 7.0) containing glucose (15 mM) to 96-well plates, adjust the pH of the solution to 4.5 with acetate buffer (pH 4.5), and incubate at 37 ℃ for 25 min. like Figure 5 As shown, in the UV-Vis absorption spectrum, the characteristic peak of the mixture of GOx and Ab2 appears at 280 nm. Compared with pure GOx and Ab2, the AuNPs surface is closer to the tryptophan residues, causing a change in the microenvironment around the chromophore, thus resulting in a blue shift of the absorption peak. Furthermore, with further broadening of the absorption band, the surface plasmon resonance peak of Au NPs red-shifts from 520 nm to 526 nm.

[0057] (3) Establish a ratiometric photoelectrochemical sensing system

[0058] CeO2 / CdS nanocomposite was coated onto both the working electrode and the internal reference electrode. Supernatant from reaction solutions corresponding to gradient concentrations of ovalbumin was dropped onto the working electrode surface and incubated. The internal reference electrode was incubated with 0.1 ng / mL ovalbumin reaction solution supernatant at room temperature. Na2SO4 solution (0.4 M) was used as the electrolyte, and photoelectric detection was performed using a xenon lamp (400 W) and an electrochemical workstation. The xenon lamp on-time for both the internal reference electrode and the working electrode was 10 s, and the output signals were Ic and Iw, respectively. Ovalbumin was quantified using the Ic / Iw signal ratio. The results obtained by the recovery method are shown in Table 1, demonstrating a high recovery rate.

[0059] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

[0060] Table 1

[0061]

Claims

1. A ratiometric photoelectrochemical analysis method based on multifunctional CeO2 / CdS and interface etching, characterized in that: Includes the following steps: (1) Preparation of multifunctional CeO2 / CdS nanocomposites; (2) Prepare beacon probes labeled with gold nanoparticles and glucose oxidase. Add the target protein and beacon probes to enzyme-labeled microplates coated with the first antibody of the target protein and incubate to construct a sandwich complex. After washing, add glucose to obtain the catalytic reaction solution. (3) The multifunctional CeO2 / CdS nanocomposite was drop-coated onto the surface of the working electrode and the internal reference electrode, and incubated with the supernatant of the catalytic reaction solution in step (2); (4) Photoelectric quantitative detection was performed using an electrochemical workstation and a xenon lamp. Na2SO4 solution was used as the electrolyte. Photoelectric detection was performed using a xenon lamp and an electrochemical workstation. The xenon lamps of the internal reference electrode and the working electrode were turned on for 9-11 seconds. The output signals were Ic and Iw, respectively. A standard curve was obtained using the Ic / Iw signal ratio to quantify the target protein.

2. The ratiometric photoelectrochemical analysis method based on multifunctional CeO2 / CdS and interface etching according to claim 1, characterized in that: Step (1) specifically includes the following steps: a. Mix Ce(NO3)3 solution and NaOH solution at a molar ratio of 1: (12~15) until homogeneous, carry out hydrothermal reaction, and obtain solid powder after washing with water and drying. b. Take 100-200 mg of the product obtained in step a, disperse it in 100-200 mL of deionized water, add 150-300 μL of 1 M CdCl2 solution, and react to obtain the CeO2 / CdS nanocomposite.

3. The ratiometric photoelectrochemical analysis method based on multifunctional CeO2 / CdS and interface etching according to claim 1, characterized in that: Step (2) specifically includes the following steps: a. Add the target protein secondary antibody and glucose oxidase to the gold nanoparticles and incubate. Add BSA to block the incubation and collect the beacon probe by centrifugation. b. Add the target protein solution (0.1 ng / mL), target protein solutions of varying concentrations, and test sample solutions to enzyme-labeled microplates coated with the first antibody against the target protein, and incubate at room temperature. After washing, add the beacon probe and incubate at room temperature to construct a sandwich complex. After washing, add PBS solution containing glucose, adjust the pH, and incubate at 37°C to obtain the catalytic reaction solution.

4. The ratiometric photoelectrochemical analysis method based on multifunctional CeO2 / CdS and interface etching according to claim 3, characterized in that: The amount of gold nanoparticles used is 1-5 mL, and the pH is 6.5-8.5; the concentration of the secondary antibody against the target protein is 180-220 μg / mL, and the amount used is 40-50 μL; the concentration of glucose oxidase is 5-7 mg / mL, and the amount used is 100-150 μL.

5. The ratiometric photoelectrochemical analysis method based on multifunctional CeO2 / CdS and interface etching according to claim 3, characterized in that: The amount of beacon probe used is 100~200μL.

6. The ratiometric photoelectrochemical analysis method based on multifunctional CeO2 / CdS and interface etching according to claim 3, characterized in that: The glucose concentration in the PBS solution is 10-20 mM, the PBS concentration is 100 mM, and the pH is 7.

0.

7. The ratiometric photoelectrochemical analysis method based on multifunctional CeO2 / CdS and interface etching according to claim 3, characterized in that: Step (3) specifically includes the following steps: a. Take 8-12 μL of CeO2 / CdS nanocomposite with a concentration of 20-30 mg / mL and drop it onto the surface of the working electrode and the internal reference electrode, and let it dry. b. Take the supernatant of the catalytic reaction solution corresponding to the gradient concentration target protein solution and the sample solution to be tested in step (2), drop it onto the surface of the working electrode for incubation, and take the supernatant of the catalytic reaction solution corresponding to the 0.1 ng / mL target protein solution in step (2), drop it onto the surface of the internal reference electrode for incubation.

8. The ratiometric photoelectrochemical analysis method based on multifunctional CeO2 / CdS and interface etching according to claim 1, characterized in that: The Na2SO4 solution concentration is 0.2~0.4M, and the xenon lamp power is 300~500W.

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