A method for detecting PSA based on black phosphorus / bismuth vanadate composite material

Abstract

This invention discloses a PSA detection method based on a black phosphorus / bismuth vanadate composite material, comprising: S1, coating a microplate with a PSA monoclonal antibody, blocking it, adding the PSA sample to be tested and incubating it, then incubating it with a PSA polyclonal antibody, a thiolized oligonucleotide sequence 1, and a gold nanoparticle conjugate to form a sandwich immune complex; S2, adding a circular DNA template, DNA ligase, and other reagents, and incubating to complete RCA amplification; S3, adding a thiolized oligonucleotide sequence 2 and a conjugate with alkaline phosphatase, and adding a substrate for hydrolysis to generate ascorbic acid; S4, using a biosensor to perform photoelectrochemical testing under simulated sunlight and voltage, recording the oxidation current signal to quantify the PSA concentration. The PSA detection method disclosed in this invention, using a biosensor modified with this composite material and employing a signal amplification strategy, achieves highly sensitive and stable detection of PSA, which has significant clinical implications for the early diagnosis of prostate cancer.

Description

Technical Field

[0001] This invention relates to the technical field of photoelectrochemical biosensors, and in particular to a PSA detection method based on a black phosphorus / bismuth vanadate composite material. Background Technology

[0002] Prostate cancer is a highly prevalent malignant tumor among men worldwide, with over 1.4 million new cases globally in 2023. Early diagnosis is crucial for improving cure rates. PSA (prostate-specific antigen) serves as a core clinical screening biomarker, with a normal range of <4 ng / mL and a diagnostic "gray zone" of 4-10 ng / mL. However, current testing technologies suffer from high false-positive rates, insufficient sensitivity, and high costs, making it difficult to meet the needs of early and accurate screening.

[0003] Photoelectrochemical biosensors have become a research hotspot for tumor marker detection due to their advantages such as ease of operation, rapid response, and portability. Black phosphorus, as a two-dimensional semiconductor material, possesses a unique wrinkled structure and tunable band gap, but its performance is easily degraded by environmental oxidation, limiting its applications. Bismuth vanadate (BiVO4), as a typical n-type semiconductor material, has become a research hotspot for PEC sensors due to its wide visible light absorption range (band gap of approximately 2.4 eV), excellent photoelectric conversion efficiency, and good chemical stability. However, single BiVO4 materials still face challenges such as severe photocorrosion and high recombination rates of photogenerated electron-hole pairs, requiring further improvement in its photocatalytic performance. The formation of PM covalent bonds between bismuth vanadate and black phosphorus not only inhibits black phosphorus oxidation but also enhances the photoelectrocatalytic activity of bismuth vanadate. Black phosphorus nanomaterials possess high conductivity and a large specific surface area, which can improve the active area of ​​the photoelectrode and electron transport efficiency.

[0004] Currently, black phosphorus-based composite materials are mostly used for small molecule detection, and a mature PSA detection solution has not yet been developed. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention discloses a PSA detection method based on a black phosphorus / bismuth vanadate composite material. A biosensor is prepared using a working electrode modified with the black phosphorus / bismuth vanadate composite material, and a signal amplification strategy is further employed to achieve highly sensitive and stable detection of PSA, which has significant clinical implications for the early diagnosis of prostate cancer.

[0006] The specific technical solution is as follows:

[0007] A black phosphorus / bismuth vanadate composite material includes the following steps:

[0008] S1. After coating microplates with PSA monoclonal antibody and blocking non-specific sites, add the PSA sample to be tested, incubate at 37°C, wash, and incubate with conjugates of PSA polyclonal antibody, thiolized oligonucleotide sequence 1 and gold nanoparticles to form a sandwich immune complex.

[0009] S2. Add circular DNA template, T4 DNA ligase, 10×T4 DNA ligase buffer, Phi29 DNA polymerase, 10×Phi29 DNA polymerase buffer and deoxyribonucleoside triphosphate to the complex obtained in step S1, and incubate at 37°C for RCA amplification.

[0010] S3. Add the conjugate of thiolized oligonucleotide sequence 2 and alkaline phosphatase to the complex obtained in step S2, hybridize to form an enzyme-labeled complex, wash and add substrate, and hydrolyze to generate ascorbic acid to obtain the reaction solution.

[0011] S4. Using a biosensor, the reaction solution obtained in step S3 is subjected to photoelectrochemical testing under simulated sunlight irradiation and applied voltage. The oxidation current signal is recorded, and the PSA concentration is quantitatively analyzed.

[0012] The biosensor is prepared as follows:

[0013] (1) Bismuth salt, vanadium salt and black phosphorus nanomaterials are dispersed in a solvent and then subjected to a solvothermal reaction to obtain bismuth vanadate modified black phosphorus nanomaterials;

[0014] (2) The black phosphorus nanomaterial modified with bismuth vanadate prepared in step (1) is mixed evenly with the binder solution to obtain a black phosphorus / bismuth vanadate composite modified solution;

[0015] (3) The black phosphorus / bismuth vanadate composite modification solution prepared in step (2) is coated on the surface of the substrate electrode and dried to obtain the working electrode;

[0016] (4) Assemble the working electrode prepared in step (3) with the reference electrode, counter electrode and electrolyte solution to obtain a biosensor.

[0017] In step S1:

[0018] Preferably, the microplate is selected from 96-well microplates;

[0019] Preferably, the PSA monoclonal antibody is selected from mAb1;

[0020] Preferably, the PSA polyclonal antibody is selected from pAb2;

[0021] Preferably, the thiolized oligonucleotide sequence 1 is 5'-HS-(CH2)6-CCGGTCGAAATAGTGAGT-3';

[0022] Preferably, the amount of the coupling agent is 50-200 μL / pore, wherein the concentration of gold nanoparticles is 8 nM.

[0023] In step S2:

[0024] For a 96-well plate, in each microplate reaction system:

[0025] Preferably, the concentration of the circular DNA template is 10 μM and the volume is 2 μL;

[0026] Preferably, the concentration of T4 DNA ligase is 5 U / μL, and the amount used is 2 μL;

[0027] Preferably, the volume of 10×T4 DNA ligase buffer used is 2 μL;

[0028] Preferably, the concentration of Phi29 DNA polymerase is 10 U / μL, and the amount used is 1 μL;

[0029] Preferably, the volume of 10×Phi29 DNA polymerase buffer is 2 μL;

[0030] Preferably, the concentration of deoxyribonucleoside triphosphate is 10 mM and the dosage is 2 μL;

[0031] Preferably, the pure water is 9 μL;

[0032] Preferably, the specific sequence of the circular DNA template is: 5'-p-TTCGACCGGAACTGTCTTAGCAAAAACTGTCTTAGCAAACTCACTAT-3'.

[0033] In step S3:

[0034] Preferably, the amount of the conjugate of thiolized oligonucleotide sequence 2 and alkaline phosphatase added to each well is 100 μL, and the concentration is 83.5 μg / mL;

[0035] The thiolized oligonucleotide sequence 2 is 5'-HS-(CH2)6-CACTATTTCGAC-3';

[0036] Preferably, the substrate is selected from L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate and / or L-ascorbic acid-2-phosphate trisodium salt;

[0037] Preferably, the substrate concentration is 5-40 mM, and the dosage is 200 μL per microwell.

[0038] In step S4:

[0039] The biosensor includes a working electrode, a reference electrode, a counter electrode, and an electrolyte solution;

[0040] The working electrode includes a base electrode and a surface modification layer containing a black phosphorus / bismuth vanadate composite material uniformly loaded on the base electrode;

[0041] Preferably, in the black phosphorus / bismuth vanadate composite material, bismuth vanadate is grown in situ on the surface of black phosphorus nanomaterial, the particle size of bismuth vanadate is <50nm, and the mass ratio of black phosphorus nanomaterial is 50~90%.

[0042] Further optimization yields bismuth vanadate material with a particle size of 1~20nm.

[0043] Further preferably, in the black phosphorus / bismuth vanadate composite material, the mass percentage of black phosphorus nanomaterials is 55-85%; more preferably, it is 67.3-82.7%.

[0044] Through continuous optimization of the above parameters, the surface modification layer of the prepared black phosphorus / bismuth vanadate composite material has both high conductivity and fully exposed bismuth vanadate active sites.

[0045] Preferably, the modification amount of the modification layer on the upper surface of the substrate electrode is 0.05~2.0 mg / cm³. 2 Further preferably, it is 0.1~1.0 mg / cm³. 2 More preferably, it is 0.2~0.5 mg / cm³. 2 The optimal value is 0.22 mg / cm³. 2 With this continuously optimized modification amount, sufficient active sites can be ensured while maintaining good mass transfer efficiency, resulting in a stable PSA response signal.

[0046] In step (1):

[0047] The bismuth salt is selected from common types in the art, such as one or more of bismuth chloride, bismuth nitrate, bismuth sulfate, and bismuth acetylacetonate;

[0048] The vanadium salt is selected from common types in the art, such as one or more of vanadium chloride, vanadium sulfate, vanadium acetylacetone, sodium vanadate, and potassium vanadate.

[0049] The black phosphorus nanomaterials are selected from common types in the field, such as one or more of black phosphorus nanosheets, black phosphorus quantum dots, and black phosphorus olefins.

[0050] The solvent is selected from common types in the art, such as one or more of N,N-dimethylformamide, N-methylpyrrolidone, ethanol, and isopropanol.

[0051] Preferably, the molar ratio of bismuth salt to vanadium salt is 1:(0.25~2.0); more preferably, it is an equimolar ratio.

[0052] Preferably, the total molar ratio of bismuth salt to vanadium salt to black phosphorus nanomaterial is (1~40):1; more preferably (1~5):1.

[0053] Preferably, the concentration of bismuth salt in the solvent is 0.1~50.0 mmol / L; the concentration of vanadium salt is 0.1~50.0 mmol / L; more preferably, the concentrations of the two salts are 0.1~5.0 mmol / L respectively.

[0054] Preferably, the solvothermal reaction is carried out at a temperature of 120~250℃; more preferably, at 150~180℃.

[0055] In step (2):

[0056] Preferably, the adhesive solution uses one or more of Nafion, chitosan, and conductive polymer as solute, and water, ethanol, or an aqueous acetic acid solution as solvent, with a concentration of 0.01~4.0 wt%.

[0057] Preferably, the total mass of the bismuth vanadate-modified black phosphorus nanomaterial and the binder solution is 100%, and the amount of binder solution added is 1.0~10.0 wt%.

[0058] In step (3), the substrate electrode is selected from common types in the art, such as glassy carbon electrode, graphite electrode, ITO / FTO / ATO electrode, paper-based electrode, flexible conductive material electrode or noble metal electrode;

[0059] In step (4), the reference electrode is selected from common types in the art, such as silver / silver chloride electrode or saturated calomel electrode;

[0060] The counter electrode is selected from common types in the art, such as platinum wire electrode, carbon electrode or platinum sheet electrode;

[0061] The electrolyte solution is a PBS buffer solution containing electrolytes, which are selected from inorganic salts, such as common types like KCl and NaCl.

[0062] Preferably, the concentration of the electrolyte solution is 0.01~1M, more preferably 0.05~0.5M; and even more preferably 0.1M.

[0063] Preferably, the electrolyte solution is selected from a neutral to slightly alkaline buffer solution, and more preferably, the pH value is 7-8. Experiments have shown that within the continuously optimized pH range, the working electrode disclosed in this invention exhibits higher photoelectrocatalytic activity and application stability.

[0064] In step S4, preferably, photoelectrochemical testing is performed under 300W simulated sunlight irradiation with a voltage of 0.2V applied.

[0065] The detection principle is as follows: mAb1 captures PSA in the sample, and T1-AuNPs-pAb2 binds to it to form a sandwich complex; oligonucleotide T1 initiates the RCA reaction, generating a large amount of long-chain DNA containing the complementary sequence of oligonucleotide T2; after specific hybridization of T2-ALP conjugate, it catalyzes the formation of AA from AAP; AA undergoes photoelectrocatalytic oxidation on the surface of black phosphorus / bismuth tungstate electrode, generating an oxidation current positively correlated with the PSA concentration, which is then quantitatively detected by a standard curve.

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] (1) This invention discloses a PSA detection method based on black phosphorus / bismuth vanadate composite material. It introduces a three-stage system of immune sandwich reaction, rolling cycle amplification and enzyme catalytic signal amplification for tumor marker detection. Combined with the high catalytic activity of the black phosphorus / bismuth vanadate composite material itself, it can effectively improve the sensitivity and specificity of PSA detection of biological macromolecules and reduce the detection limit.

[0068] (2) The biosensor used in the detection method disclosed in this invention uses a black phosphorus / bismuth vanadate composite material as an electrode modification material. The bismuth vanadate and black phosphorus form a PM covalent bond, which can inhibit the oxidative degradation of black phosphorus. At the same time, black phosphorus and bismuth vanadate form a heterojunction, which synergistically enhances the absorption of light by the photoelectric material and inhibits the recombination of photogenerated electron-hole pairs, thereby enhancing the photoelectrocatalytic activity. Compared with the existing single-material modified electrodes, which have problems such as poor stability, poor conductivity and insufficient photoelectrocatalytic activity, this biosensor has better detection stability and electrocatalytic sensitivity, and has good reproducibility and long-term stability, making it suitable for large-scale production and actual clinical testing scenarios. Attached Figure Description

[0069] Figure 1 SEM image of the bismuth vanadate-modified black phosphorus nanocomposite material prepared in Example 1;

[0070] Figure 2 The elemental distribution (a~e) and energy spectrum (f) of the bismuth vanadate-modified black phosphorus nanocomposite material prepared in Example 1 are shown.

[0071] Figure 3 The image shows the XRD pattern of the bismuth vanadate-modified black phosphorus nanocomposite material prepared in Example 1.

[0072] Figure 4 Cyclic voltammetry curves of the working electrodes prepared in Example 1 and Comparative Examples 1-2 before and after the addition of 1.0 mM ascorbic acid (AA);

[0073] Figure 5 The photoelectrocatalytic response performance of the six independent electrodes modified with the bismuth vanadate-modified black phosphorus composite material prepared in Example 1 was tested.

[0074] Figure 6 Standard curves for detecting different concentrations of AA using the bismuth vanadate-modified black phosphorus composite electrode prepared in Example 1;

[0075] Figure 7 Standard curves for detecting different concentrations of PSA using the bismuth vanadate-modified black phosphorus composite electrode prepared in Example 1;

[0076] Figure 8 SEM image of the bismuth vanadate nanomaterial prepared in Comparative Example 1;

[0077] Figure 9 The photoelectrocatalytic response performance of the working electrodes prepared in Examples 1-4 after the addition of 1.0 mM AA;

[0078] Figure 10 The photoelectrocatalytic performance of the working electrodes prepared in Examples 1, 5-9 after the addition of 1.0 mM AA is shown.

[0079] Figure 11 The photoelectrocatalytic performance of the working electrodes prepared in Examples 1, 10-13 after the addition of 1.0 mM AA is shown. Detailed Implementation

[0080] The present invention will be further illustrated below with reference to specific embodiments. Those skilled in the art will understand that the embodiments described below are only some, not all, embodiments of the present invention, and are used merely to illustrate the invention, and should not be considered as limiting the scope of the invention.

[0081] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Any processes in the embodiments not specifically described in detail are those that can be implemented or understood by those skilled in the art by referring to existing technology. Reagents or instruments used without specifying the manufacturer are considered to be conventional products that can be purchased commercially.

[0082] Example 1

[0083] (1) Preparation of black phosphorus nanomaterials: Black phosphorus crystals were ground and pulverized in a mortar. The resulting black phosphorus powder was transferred to N-methylpyrrolidone solvent and ultrasonically pulverized with a probe ultrasonic pulverizer at an ultrasonic power of 500W for more than 10 hours. The resulting suspension was centrifuged at 1000rpm to remove the precipitate. The resulting supernatant was then centrifuged at 12000rpm. The precipitate was washed with anhydrous ethanol and vacuum dried to obtain black phosphorus nanomaterials.

[0084] (2) Preparation of bismuth vanadate-modified black phosphorus nanomaterials: Weigh 0.0026 g (0.0066 mmol) of anhydrous bismuth nitrate, 0.0023 g (0.0066 mmol) of vanadium acetylacetonate and 4.0 mg (0.013 mmol) of black phosphorus nanomaterials, disperse them in 20 mL of DMF, and sonicate for 1 hour to dissolve the metal salts; transfer to a reaction vessel, and hydrothermally react at 160 °C for 3 hours; centrifuge at 8000 rpm for 5 minutes, wash 3 times with anhydrous ethanol, and freeze-dry to obtain bismuth vanadate-modified black phosphorus nanomaterials, denoted as BiVO4@BPNSs.

[0085] Figure 1 The SEM image of the BiVO4@BPNSs prepared in this embodiment shows that the BiVO4@BPNSs nanocomposite material has a two-dimensional sheet structure with lateral dimensions ranging from hundreds of nanometers to several micrometers and a thickness of several nanometers. The material surface is relatively rough and has obvious particle loading. Figure 2 The elemental distribution (a~e) and energy spectrum (f) of the BiVO4@BPNSs prepared in this embodiment show that Bi, V and P elements are uniformly distributed, and the black phosphorus content in the material is 82.7 wt%.

[0086] Figure 3 The XRD pattern of the BiVO4@BPNSs prepared in this embodiment shows that the diffraction peaks of the synthesized BiVO4@BPNSs nanocomposite material are attributed to black phosphorus crystals and bismuth vanadate crystals, respectively. The intensity of the diffraction peaks of the black phosphorus nanosheets is significantly reduced, indicating that the black phosphorus nanosheets are covered by bismuth vanadate particles.

[0087] (3) Preparation of black phosphorus / bismuth vanadate composite modification solution: 5.0 mg of black phosphorus nanomaterial modified with bismuth vanadate was dispersed in 0.97 mL of pure water and sonicated for 30 minutes; 30 μL of 1 wt% Nafion solution was added and sonicated for another 30 minutes to obtain the composite modification solution, which was denoted as BiVO4@BPNSs composite modification solution.

[0088] (4) The ITO electrode was washed with 75% ethanol, dried at room temperature, and then 20 μL of BiVO4@BPNSs composite modification solution was dropped onto it. After natural drying, it was stored for later use and designated as BiVO4@BPNSs / ITO-1. The modification amount obtained at this drop volume was calculated to be 0.22 mg / cm³. 2 .

[0089] (5) Using BiVO4@BPNsS / ITO-1 prepared in step (4) as the working electrode, a platinum wire electrode as the counter electrode, an Ag / AgCl electrode as the reference electrode, and a PBS buffer electrolyte containing 0.1M KCl pH=7.4, a three-electrode system was obtained after assembly.

[0090] Photoelectrochemical tests were conducted under simulated sunlight (300W). The cyclic voltammetry test potential range was -0.5V to 1.2V, and the chronoamperometry test potential was 0.3V. Figure 4 The figure shows the photoelectrocatalytic cyclic voltammetry curves of the working electrode prepared in Example 1. The figure also includes the photoelectrocatalytic cyclic voltammetry curves of the working electrodes prepared in Comparative Examples 1 and 2, and the bare ITO electrode, for comparison. It was observed that the bismuth vanadate-modified black phosphorus nanomaterials begin to oxidize AA at 0 V, with an oxidation peak appearing at 0.55 V. Within the range of 0 V to 1.2 V, the oxidation peak current is significantly higher than the photocurrent signals of pure bismuth vanadate and black phosphorus nanomaterials, indicating that the bismuth vanadate-modified black phosphorus nanomaterials have better photoelectrocatalytic response performance to AA. Considering that excessively high voltage may promote the oxidation of other molecules in the electrolytic cell and cause interference, 0.3 V was used as the potential for detection and analysis.

[0091] Figure 5 To repeat steps (1) to (5) five times, a total of six working electrodes were obtained. The photoelectrocatalytic oxidation response current under simulated sunlight (300W) irradiation and a voltage of 0.3V was measured after adding 1.0 mM AA. Observations showed that the working electrode modified with the BiVO4@BPNsS nanocomposite material disclosed in this invention exhibited good reproducibility and stability. The response current of the six independent BiVO4@BPNSs to 1.0 mM AA was 136.38 ± 4.98 μA.

[0092] Figure 6 The photoelectrocatalytic performance of the working electrode modified with the BiVO4@BPNSs nanocomposite material prepared in Example 1 for different concentrations of AA is shown in Table 1 below.

[0093] Table 1

[0094]

[0095] observe Figure 6 It can be seen that the detection linear range of the working electrode modified with BiVO4@BPNSs nanocomposite material disclosed in this invention is 0.002 mM to 0.5 mM, the linear equation is y=67.881x+0.2645 (R²=0.997), the detection sensitivity is 67.881 μA / mM, and the detection limit is 1.15 μM. The above characterization shows that the BiVO4@BPNSs nanocomposite material modified electrode lays the foundation for high sensitivity in subsequent PSA detection.

[0096] PSA testing

[0097] (a) Preparation of T1-AuNPs-pAb2 conjugates:

[0098] Boil 200 mL of a solution containing 1 mM HAuCl4, then quickly add 20 mL of a solution containing 38.8 mM sodium citrate, and continue boiling for 20 min to obtain a colloidal solution of gold nanoparticles.

[0099] Take 20 μL of 100 μM thiolated oligonucleotide sequence 1 (denoted as T1, sequence 5'-3': 5'-HS-(CH2)6-CCGGTCGAAATAGTGAGT-3') and 10 μL of 0.01 M TCEP (tris(2-carboxyethyl)phosphine) and add them to 280 μL of HEPES buffer. Mix and react at room temperature with shaking for 1 h under nitrogen protection. Purify 2-3 times with a 3 kDa filter tube. Finally, add HEPES buffer to make up to 300 μL to obtain TCEP-activated thiolated T1.

[0100] Add 5 μM TCEP-activated thiolized T1 to 0.5 mL of AuNPs at a concentration of 1.5 μg / L, incubate at 4˚C for 4 h, then adjust the NaCl concentration stepwise to a final concentration of 0.1 M, centrifuge and resuspend to obtain T1-AuNPs; add 25 μg PSA polyclonal antibody (pAb2, Shanghai Lingchao Biotechnology Co., Ltd., L1C00402) and incubate for 2 h, centrifuge, and resuspend in PBS to obtain the final T1-AuNPs-pAb2 conjugate.

[0101] (b) Preparation of T2-ALP conjugates:

[0102] 0.5 mg of alkaline phosphatase (ALP, Maclean's, A796559) was dissolved in 250 μL of 10 mM, pH 7.4 HEPES buffer, and 8.2 μL of 5 mg / mL sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester (Sulfo-SMCC) solution was added. The mixture was vortexed and shaken at 22˚C for 2 h. The solution was purified by ultrafiltration and then redispersed in 250 μL of HEPES solution to obtain Sulfo-SMCC activated ALP.

[0103] Take 20 μL of 100 μM thiolated oligonucleotide sequence 2 (denoted as T2, sequence 5'-HS-(CH2)6-CACTATTTCGAC-3') and 10 μL of 0.01 M TCEP and add them to 280 μL of HEPES buffer. Mix and react at room temperature with shaking for 1 h under nitrogen protection. Purify 2-3 times with a 3 kDa filter tube. Finally, add HEPES buffer to make up to 300 μL to obtain TCEP-reduced thiolated T2.

[0104] Sulfo-SMCC-activated ALP and TCEP-reduced thiolated T2 were mixed at a molar ratio of 1:2 and reacted at 16˚C for 8 h. The mixture was then purified using a 30 kDa filter and finally dispersed in HEPES buffer to a concentration of 83.5 μg / mL.

[0105] S1. Coat each well of a 96-well microplate (Jeter, FEP101896) with 10 μg / mL mAb1 (Shanghai Lingchao Biotechnology Co., Ltd., L1C00401), 100 μL per well, and incubate overnight at 4°C. After washing, add PBS buffer containing 1% bovine serum albumin (BSA, Beyotime, ST025) as blocking solution, incubate at 37°C for 1 hour, wash with PBS washing buffer containing 0.05% Tween 20, and pat dry. Add 100 μL of PSA (Shanghai Lingchao Biotechnology Co., Ltd., L2C001, 0.1 ng / mL~100 ng / mL) per well, incubate at 37°C for 2 hours, wash with washing buffer, and pat dry. Add 100 μL of T1-AuNPs-pAb2 conjugate per well, incubate at 37°C for 2 hours to form a sandwich complex, wash with washing buffer, and pat dry.

[0106] S2. Add circular DNA template (sequence: 5'-p-TTCGACCGGAACTGTCTTAGCAAAAACTGTCTTAGCAAACTCACTAT-3'), T4 DNA ligase, 10×T4 DNA ligase buffer, Phi29 DNA polymerase, 10×Phi29 DNA polymerase buffer, and deoxyribonucleoside triphosphate. Incubate at 37°C for RCA amplification. For a 96-well plate, the reaction system for each well contains the following amounts: circular DNA template (10 μM, 2 μL), T4 DNA ligase (5 U / μL, 2 μL), 10×T4 DNA ligase buffer (2 μL), Phi29 DNA polymerase (10 U / μL, 1 μL), 10×Phi29 DNA polymerase buffer (2 μL), deoxyribonucleoside triphosphate (10 mM, 2 μL), and 9 μL of pure water.

[0107] S3. Add T2-ALP conjugate and incubate at 37°C for 60 minutes; after washing, add 200 μL of 40 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AAP) as substrate and react at 37°C for 60 minutes to obtain the reaction solution;

[0108] S4. Take 0.2 mL of reaction solution and add it to 7.8 mL of PBS buffer with pH=7.38. Use the three-electrode system prepared in Example 1 to perform chronoamperometry under simulated sunlight (300W) and 0.30 V, record the oxidation current signal, and calculate the PSA concentration according to the standard curve.

[0109] Figure 7 The chronoamperometry response curves of the bismuth vanadate-modified black phosphorus composite electrode prepared in Example 1 for different concentrations of PSA are shown in the figure. The detection range of the BiVO4@BPNsS nanocomposite modified electrode for PSA is 0.1 ng / mL-10 ng / mL, and the linear equation is y=0.1094x+0.1231, R 2 =0.9754; detection sensitivity is 0.1094 μA / (ng‧mL) -1 The detection limit is 0.07 ng / mL.

[0110] Comparative Example 1

[0111] The preparation process is basically the same as in Example 1, except that the bismuth vanadate-modified black phosphorus composite material is replaced with an equal mass of bismuth vanadate nanomaterials. The working electrode prepared is denoted as BiVO4 / ITO.

[0112] The preparation of bismuth vanadate nanomaterials is as follows:

[0113] Weigh 0.0026 g (0.0066 mmol) of anhydrous bismuth nitrate and 0.0023 g (0.0066 mmol) of vanadium acetylacetonate and disperse them in 20 mL of DMF. Sonicate for 1 hour to dissolve the metal salts. Transfer to a reaction vessel and hydrothermally react at 160 °C for 3 hours. Centrifuge at 8000 rpm for 5 minutes, wash three times with anhydrous ethanol, and freeze-dry to obtain bismuth vanadate nanomaterials.

[0114] Figure 8 The image shows a SEM image of the bismuth vanadate nanomaterial prepared in this comparative example. The material has a nanosphere structure with nanoparticles having a size of approximately 50 nm.

[0115] Comparative Example 2

[0116] The preparation process is basically the same as in Example 1, except that the bismuth vanadate-modified black phosphorus composite material is replaced with the black phosphorus nanomaterial prepared in step (1) of equal mass. The working electrode prepared is denoted as BPNSs / ITO.

[0117] Example 2

[0118] The preparation process is basically the same as in Example 1, except that:

[0119] In step (2), the mass of black phosphorus nanosheets is replaced with 16.0 mg (0.052 mmol).

[0120] Characterization showed that the BiVO4@BPNSs nanocomposite material prepared in this embodiment has a two-dimensional sheet structure with lateral dimensions ranging from hundreds of nanometers to several micrometers and a thickness of several nanometers. The material surface is relatively rough and has particle loading. The black phosphorus content in the composite material is 73.4 wt%.

[0121] In steps (3) to (5), the bismuth vanadate-modified black phosphorus nanomaterials are replaced with the BiVO4@BPNSs nanocomposite material prepared in this embodiment, and the resulting working electrode is denoted as BiVO4@BPNSs / ITO-2.

[0122] According to the test, the photoelectric response current of the working electrode prepared in this embodiment, after adding 1.0 mM AA, is 85.83 ± 4.85 μA under simulated sunlight (300W) irradiation and 0.3V voltage.

[0123] Example 3

[0124] The preparation process is basically the same as in Example 1, except that:

[0125] In step (2), the mass of anhydrous bismuth nitrate and vanadium acetylacetonate is replaced with 0.0040 g (0.010 mmol) and 0.0035 g (0.010 mmol), respectively.

[0126] Characterization showed that the BiVO4@BPNSs nanocomposite material prepared in this embodiment has a two-dimensional sheet structure with lateral dimensions ranging from hundreds of nanometers to several micrometers and a thickness of several nanometers. The material surface is relatively rough and has particle loading. The black phosphorus content in the composite material is 67.3 wt%.

[0127] In steps (3) to (5), the bismuth vanadate-modified black phosphorus nanomaterials are replaced with the BiVO4@BPNSs nanocomposite material prepared in this embodiment, and the resulting working electrode is denoted as BiVO4@BPNSs / ITO-3.

[0128] Example 4

[0129] The preparation process is basically the same as in Example 1, except that:

[0130] In step (2), the mass of anhydrous bismuth nitrate and vanadium acetylacetonate is replaced with 0.0080 g (0.020 mmol) and 0.007 g (0.020 mmol), respectively.

[0131] Characterization showed that the BiVO4@BPNSs nanocomposite material prepared in this embodiment has a two-dimensional sheet structure with lateral dimensions ranging from hundreds of nanometers to several micrometers and a thickness of several nanometers. The material surface is relatively rough and has particle loading. The black phosphorus content in the composite material is 54.8 wt%.

[0132] In steps (3) to (5), the bismuth vanadate-modified black phosphorus nanomaterials are replaced with the BiVO4@BPNSs nanocomposite material prepared in this embodiment, and the resulting working electrode is denoted as BiVO4@BPNSs / ITO-4.

[0133] Figure 9 The working electrodes prepared in Examples 1-4, after adding 1.0 mM AA, exhibit photoelectrocatalytic performance under simulated sunlight (300W) irradiation and a voltage of 0.3V, as shown in the figure. The influence of black phosphorus content in the composite material on photoelectrochemical performance is shown. Higher catalytic activity is observed at a mass content of 67.3-82.7%, with the optimal content being 82.7%. At this content, the working electrode also possesses high photoelectrocatalytic performance. Considering production costs and dispersion issues, the black phosphorus content is further optimized to 82.7%.

[0134] Example 5

[0135] The preparation process is basically the same as in Example 1, except that:

[0136] In step (5), the electrolyte is replaced with PBS buffer containing 0.1 M KCl at pH=5.

[0137] Example 6

[0138] The preparation process is basically the same as in Example 1, except that:

[0139] In step (5), the electrolyte is replaced with PBS buffer containing 0.1 M KCl at pH 6.

[0140] Example 7

[0141] The preparation process is basically the same as in Example 1, except that:

[0142] In step (5), the electrolyte is replaced with PBS buffer containing 0.1 M KCl at pH=8.

[0143] Example 8

[0144] The preparation process is basically the same as in Example 1, except that:

[0145] In step (5), the electrolyte is replaced with PBS buffer containing 0.1 M KCl at pH=9.

[0146] Example 9

[0147] The preparation process is basically the same as in Example 1, except that:

[0148] In step (5), the electrolyte is replaced with PBS buffer containing 0.1 M KCl at pH=10.

[0149] Figure 10 The photoelectrocatalytic performance of the working electrodes prepared in Examples 1, 5-9 after the addition of 1.0 mM AA was compared. It was found that the working electrode modified with BiVO4@BPNSs nanocomposite material had better electrocatalytic activity in a neutral to slightly alkaline buffer solution. Considering the stability in practical applications, a neutral buffer solution with a pH of 7-8 was used for testing.

[0150] Examples 10-13

[0151] The preparation process is basically the same as in Example 1, except that:

[0152] In step (4), the volume of the BiVO4@BPNSs composite modification solution is replaced with 10 μL, 30 μL, 40 μL and 50 μL in sequence.

[0153] The modification amounts on the prepared working electrodes were 0.11 mg / cm², 0.33 mg / cm², 0.44 mg / cm², and 0.55 mg / cm², respectively.

[0154] Figure 11 The photoelectrocatalytic current response of the working electrodes prepared in Examples 1, 10-13 after the addition of 1.0 mM AA was investigated. The effect of the modification amount of BiVO4@BPNSs nanocomposite on the performance of the working electrodes was studied. When the material modification amount increased, the oxidation current also increased. The performance was optimal when the modification amount was 0.22 mg / cm² to 0.44 mg / cm². Considering both signal and background, the modification amount of 0.22 mg / cm² prepared in Example 1 can ensure sufficient active sites and maintain good mass transfer efficiency, thus obtaining a stable oxidation current response signal.

Claims

1. A PSA detection method based on black phosphorus / bismuth vanadate composite material, characterized in that, Includes the following steps: S1. After coating microplates with PSA monoclonal antibody and blocking non-specific sites, add the PSA sample to be tested, incubate at 37°C, wash, and incubate with conjugates of PSA polyclonal antibody, thiolized oligonucleotide sequence 1 and gold nanoparticles to form a sandwich immune complex. S2. Add circular DNA template, T4 DNA ligase, 10×T4 DNA ligase buffer, Phi29 DNA polymerase, 10×Phi29 DNA polymerase buffer and deoxyribonucleoside triphosphate to the complex obtained in step S1, and incubate at 37°C for RCA amplification. S3. Add the conjugate of thiolized oligonucleotide sequence 2 and alkaline phosphatase to the complex obtained in step S2, hybridize to form an enzyme-labeled complex, wash and add substrate, and hydrolyze to generate ascorbic acid to obtain the reaction solution. S4. Using a biosensor, the reaction solution obtained in step S3 is subjected to photoelectrochemical testing under simulated sunlight irradiation and applied voltage. The oxidation current signal is recorded, and the PSA concentration is quantitatively analyzed. The biosensor is prepared as follows: (1) Bismuth salt, vanadium salt and black phosphorus nanomaterials are dispersed in a solvent and then subjected to a solvothermal reaction to obtain bismuth vanadate modified black phosphorus nanomaterials; (2) The black phosphorus nanomaterial modified with bismuth vanadate prepared in step (1) is mixed evenly with the binder solution to obtain a black phosphorus / bismuth vanadate composite modified solution; (3) The black phosphorus / bismuth vanadate composite modification solution prepared in step (2) is coated on the surface of the substrate electrode and dried to obtain the working electrode; (4) Assemble the working electrode prepared in step (3) with the reference electrode, counter electrode and electrolyte solution to obtain a biosensor.

2. The method for PSA detection based on black phosphorus / bismuth vanadate composite material according to claim 1, characterized in that, In step S1: The microplate is selected from 96 microplates; The PSA monoclonal antibody was selected from mAb1; The PSA polyclonal antibody was selected from pAb2; The thiolized oligonucleotide sequence 1 is 5'-HS-(CH2)6-CCGGTCGAAATAGTGAGT-3'; The amount of the coupling agent used is 50~200 μL / well, wherein the concentration of gold nanoparticles is 8 nM.

3. The method for PSA detection based on black phosphorus / bismuth vanadate composite material according to claim 1, characterized in that, In step S2: For a 96-well plate, in each microplate reaction system: The concentration of the circular DNA template was 10 μM, and the volume was 2 μL. The concentration of T4 DNA ligase is 5 U / μL, and the volume is 2 μL; The volume of 10×T4 DNA ligase buffer used is 2 μL; The concentration of Phi29 DNA polymerase is 10 U / μL, and the amount used is 1 μL; The volume of 10×Phi29 DNA polymerase buffer used is 2 μL; The concentration of deoxynucleoside triphosphate is 10 mM, and the dosage is 2 μL; The pure water volume is 9 μL; The specific sequence of the circular DNA template is: 5'-p-TTCGACCGGAACTGTCTTAGCAAAAACTGTCTTAGCAAACTCACTAT-3'.

4. The method for PSA detection based on black phosphorus / bismuth vanadate composite material according to claim 1, characterized in that, In step S3: The amount of the conjugate between thiolized oligonucleotide sequence 2 and alkaline phosphatase was 100 μL per well, with a concentration of 83.5 μg / mL. The thiolized oligonucleotide sequence 2 is 5'-HS-(CH2)6-CACTATTTCGAC-3'; The substrate is selected from L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate and / or L-ascorbic acid-2-phosphate trisodium salt; The substrate concentration is 5-40 mM, and the dosage is 200 μL per microwell.

5. The method for PSA detection based on black phosphorus / bismuth vanadate composite material according to claim 1, characterized in that, In step S4: The biosensor includes a working electrode, a reference electrode, a counter electrode, and an electrolyte solution; The working electrode includes a base electrode and a surface modification layer containing a black phosphorus / bismuth vanadate composite material uniformly loaded on the base electrode; In the black phosphorus / bismuth vanadate composite material, bismuth vanadate is grown in situ on the surface of black phosphorus nanomaterials, the particle size of bismuth vanadate is <50nm, and the mass ratio of black phosphorus nanomaterials is 50~90%.

6. The method for PSA detection based on black phosphorus / bismuth vanadate composite material according to claim 5, characterized in that, The modification amount of the upper surface modification layer of the base electrode is 0.05-2.0 mg / cm2 2 .

7. The method for PSA detection based on black phosphorus / bismuth vanadate composite material according to claim 1, characterized in that, In step (1): The bismuth salt is selected from one or more of bismuth chloride, bismuth nitrate, bismuth sulfate, and bismuth acetylacetonate; The vanadium salt is selected from one or more of vanadium chloride, vanadium sulfate, vanadium acetylacetone, sodium vanadate, and potassium vanadate. The black phosphorus nanomaterial is selected from one or more of black phosphorus nanosheets, black phosphorus quantum dots, and black phosphorus olefins. The solvent is selected from one or more of N,N-dimethylformamide, N-methylpyrrolidone, ethanol, and isopropanol.

8. The method for PSA detection based on black phosphorus / bismuth vanadate composite material according to claim 1, characterized in that, In step (1): The molar ratio of bismuth salt to vanadium salt is 1:(0.25~2.0). The total molar ratio of bismuth salt and vanadium salt to black phosphorus nanomaterial is (1~40):1; In the solvent, the concentration of bismuth salt is 0.1~50.0 mmol / L; the concentration of vanadium salt is 0.1~50.0 mmol / L. The solvothermal reaction is carried out at a temperature of 120~250℃.

9. The method for PSA detection based on black phosphorus / bismuth vanadate composite material according to claim 1, characterized in that, In step (2): The adhesive solution uses one or more of Nafion, chitosan, and conductive polymers as solutes, and water, ethanol, or an aqueous acetic acid solution as solvents, with a concentration of 0.01~4.0 wt%. The total mass of the bismuth vanadate-modified black phosphorus nanomaterials and the binder solution is 100%, and the amount of binder solution added is 1.0~10.0 wt%.

10. The PSA detection method based on black phosphorus / bismuth vanadate composite material according to claim 1, characterized in that: In step (3), the substrate electrode is selected from glassy carbon electrode, graphite electrode, ITO / FTO / ATO electrode, paper-based electrode, flexible conductive material electrode or noble metal electrode; In step (4), the reference electrode is selected from a silver / silver chloride electrode or a saturated calomel electrode; The counter electrode is selected from platinum wire electrode, carbon electrode, or platinum sheet electrode; The electrolyte solution is a PBS buffer solution containing an electrolyte, which is selected from inorganic salts.

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