Metal ion functionalized NT-proBNP photoelectrochemical sensor and preparation method thereof

Abstract

The invention discloses a metal ion functionalized NT-proBNP photoelectrochemical sensor and a preparation method, and belongs to the technical field of photoelectrochemical biosensors, the photoelectrochemical sensor comprises a Fe < 3 + >-coated BiVO4 electrode, and the preparation method of the electrode comprises the following steps: dispersing BiVO4 in deionized water, adding excessive Fe < 3 + > for adsorption, removing the excessive Fe < 3 + > after the adsorption is finished, and obtaining the metal ion functionalized NT-proBNP photoelectrochemical sensor. And then dispensing the Fe < 3 + >-coated BiVO4 on a conductive material, so as to obtain the Fe3 +-coated BiVO4 / Fe3 +-coated BiVO4 / Fe3 +-coated BiVO4 composite material. When the photoelectrochemical sensor provided by the invention is used for NT-proBNP detection, no trapping agent or bias voltage needs to be applied, measurable signal output is realized through enzyme-linked immunosorbent assay, and the detection scheme has good selectivity and stability, can realize accurate detection of NT-proBNP, and has good application prospects in clinical diagnosis.

Description

Technical Field

[0001] This invention relates to the field of photoelectrochemical biosensor technology, specifically to a metal ion-functionalized N-terminal probrain natriuretic peptide (NT-proBNP) photoelectrochemical sensor and its preparation method. Background Technology

[0002] NT-proBNP is an inactive fragment produced by the cleavage of the hormone precursor Pro-BNP in response to increased cardiac workload. As a key biomarker reflecting cardiac stress and ventricular tone, abnormal NT-proBNP levels are closely associated with various cardiac diseases such as heart failure and myocardial ischemia, and can also be used to assess disease severity and prognosis, playing a significant role in the clinical diagnosis and monitoring of heart failure. However, due to its low in vivo concentration and rapid dynamic changes, there is an urgent need to develop stable, sensitive, and rapid detection methods.

[0003] Currently, the main detection technologies for NT-proBNP include electrochemiluminescence immunoassay, enzyme-linked immunosorbent assay (ELISA), and fluorescence immunoassay. While these methods are widely used, they still suffer from drawbacks such as high instrument costs, cumbersome operation, long processing times, and insufficient sensitivity at ultra-low concentrations, making it difficult to meet the needs for accurate and rapid detection in emergency care. Therefore, developing new methods with high sensitivity, low background, and suitability for real-time analysis of trace samples has become a current research hotspot.

[0004] Photoelectrochemical biosensing technology, with its advantages of low power consumption, low detection limit, easy integration and signal readout, provides a promising new approach for the rapid and accurate detection of NT-proBNP. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention provides a metal ion functionalized NT-proBNP photoelectrochemical sensor and its preparation method, aiming to realize low-cost and high-sensitivity detection of NT-proBNP using photoelectrochemical biosensing technology.

[0006] The specific technical solution of the present invention is as follows: This invention provides an NT-proBNP photoelectrochemical sensor comprising Fe 3+ The BiVO4 electrode is prepared by the following method: BiVO4 was dispersed in deionized water, and an excess of Fe was added. 3+ Adsorption is performed, and after adsorption is complete, the mixture is washed multiple times to remove weakly adsorbed excess Fe. 3+ Thus, a surface with strong Fe adsorption is obtained. 3+ Functionalized semiconductor material Fe 3+ @BiVO4; Fe3+ @BiVO4 was drop-coated onto a conductive material to obtain Fe 3+ @BiVO4 electrode.

[0007] Preferably, BiVO4 is dendritic and can be hydrothermally synthesized from bismuth nitrate pentahydrate (Bi(NO3)3·5H2O) and ammonium metavanadate (NH4VO3) in a high-pressure reactor at 120-200°C, with the molar ratio of Bi(NO3)3·5H2O to NH4VO3 being 1:2-2:1.

[0008] Preferably, the conductive material is indium tin oxide (ITO) glass.

[0009] In the aforementioned NT-proBNP photoelectrochemical sensor, Fe... 3+ The BiVO4 electrode is the working electrode, and it also includes a reference electrode and a counter electrode. Preferably, the reference electrode is Ag / AgCl and the counter electrode is a platinum wire electrode.

[0010] Based on the above-mentioned NT-proBNP photoelectrochemical sensor, the present invention provides an NT-proBNP detection product, which includes at least: 1) Fe 3+ @BiVO4 electrode; 2) Solid-phase support, immobilized with NT-proBNP capture antibody (Ab1); 3) Enzyme-labeled beacon antibody (Ab2); 4) The substrate, under enzyme catalysis, generates a small molecule product with specific functional groups (such as thiol groups), and this small molecule product reacts with Fe adsorbed on the electrode surface. 3+ Coordination poisoning occurs, which causes changes in photocurrent.

[0011] Preferably, in the above-mentioned NT-proBNP detection product, the solid support is a polystyrene microplate (such as a 96-well plate) with Ab1 immobilized. In some embodiments of the present invention, the solid support is prepared by: dropping an Ab1 solution into a 96-well plate, incubating at low temperature, washing, and then blocking with BSA.

[0012] Preferably, in the above-mentioned NT-proBNP detection product, the enzyme is acetylcholinesterase (AChE), and its corresponding substrate is thioacetylcholine (ATch); more preferably, the AChE-labeled Ab2 is a conjugate formed by AChE, gold nanoparticles (AuNPs), and Ab2 antibody, denoted as AchE-AuNPs-Ab2 conjugate, wherein the size of AuNPs is 20-50 nm.

[0013] The method for performing NT-proBNP detection using the above-mentioned NT-proBNP detection product includes the following steps: S1. Add the sample to be tested to the solid support, then add enzyme-labeled Ab2. After the immune recognition reaction, add the substrate and use enzyme to catalyze the hydrolysis of the substrate. S2. Add the reaction solution obtained in step S1 dropwise to Fe. 3+ After the reaction is complete on the @BiVO4 electrode, the sample is washed, air-dried, and subjected to PEC detection. The NT-proBNP concentration is calculated based on the photoelectric signal.

[0014] The principle of NT-proBNP detection in this invention is as follows: In the Fe photoelectrochemical sensor... 3+ In the BiVO4 electrode, metallic Fe is strongly adsorbed on the BiVO4 surface. 3+ On the one hand, through Fe 3+ Effective coupling of solid-liquid interface reactions accelerates the in-situ extraction of photogenerated charges to enhance photocurrent, while also providing signal sites for thioglycolic coordination poisoning, thus enabling the detection of NT-proBNP. Simultaneously, this detection scheme does not apply any trapping agent or external bias voltage; the measurable signal is output via enzyme-linked immunosorbent assay (ELISA): Ab1 is immobilized on a carrier, and after Ab1 binds to NT-proBNP, AChE-labeled Ab2 is introduced to form an immune complex; the addition of ATch generates thiocholine (TCh) containing thiol groups, and the thiol molecules bind to Fe adsorbed on the electrode surface. 3+ Coordination occurs, thereby regulating electron transfer at the electrode interface and causing changes in photocurrent. The amount of photocurrent change is related to the content of AChE in the immune complex, and the content of AChE is positively correlated with the concentration of NT-proBNP, thus achieving linear quantification between the concentration of NT-proBNP and the photoelectric signal.

[0015] Compared with the prior art, the beneficial effects of this invention are as follows: (1) This invention introduces Fe based on BiVO4 3+ Based on Fe 3+ The coupling interface reaction amplifies and stabilizes the photocurrent signal, thereby significantly improving the performance of the Fe semiconductor material. 3+ The photoelectric properties of @BiVO4 improve the sensitivity and accuracy of NT-proBNP detection. Meanwhile, Fe... 3+ The preparation method of @BiVO4 is simple, convenient, mild, low-cost, and easy to industrialize.

[0016] (2) The photoelectrochemical biosensor for detecting NT-proBNP provided by this invention adsorbs Fe 3+Using BiVO4 as the electrode material, with the concentration of NT-proBNP within 1 - 5000 pg / mL, the photocurrent of the sensor shows a good linear relationship with the concentration of NT-proBNP. In addition, the sensor has good selectivity and stability, and the sensor system has a fast response, which is suitable for the detection of NT-proBNP in clinical emergencies such as heart failure and has good application prospects in clinical diagnosis. Brief Description of the Drawings

[0017] To more clearly illustrate the technical solutions of the present invention, the drawings used in the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0018] Figure 1 Semiconductor material Fe provided by the present invention 3+ @BiVO4, the mechanism diagram of Fe 3+ coupling solid-liquid interface reaction to enhance and stabilize the optoelectronic signal.

[0019] Figure 2 For the optoelectrochemical biosensor based on Fe 3+ @BiVO4 for detecting NT-pro BNP in the present invention.

[0020] Figure 3 For the scanning electron microscope images and transmission electron microscope images of BiVO4 and Fe 3+ @BiVO4 in Example 1, a is the scanning electron microscope image of BiVO4, b is the transmission electron microscope image of BiVO4, and c is the transmission electron microscope image of Fe 3+ @BiVO4. <00001​​​​​​​​​​​​​​​​​​​​​​The linear graph of the @BiVO4 photoelectrochemical biosensor for detecting NT-proBNP is shown, where a is the photocurrent response of the sensor when the NT-proBNP concentration is between 0-5000 pg / mL, and b is the linear relationship between photocurrent intensity and NT-proBNP concentration.

[0025] Figure 8 As in Example 3, based on Fe 3+ Selectivity test diagram of NT-proBNP for the @BiVO4 photoelectrochemical biosensor. Detailed Implementation

[0026] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification and claims of this invention are intended to cover non-exclusive inclusion.

[0028] To address the technical challenge of achieving high-sensitivity detection of NT-proBNP using photoelectrochemical biosensing technology, this invention develops a metal ion-functionalized NT-proBNP photoelectrochemical sensor, which utilizes surface Fe... 3+ Functionalized BiVO4 semiconductor materials (i.e., Fe) 3+ @BiVO4), combined with the double-antibody sandwich method, enables the specific detection of NT-proBNP. This detection method exhibits good detection accuracy and significant stability, and requires no capture agent or external bias voltage, thus showing good application prospects in clinical diagnosis.

[0029] In a first aspect, embodiments of the present invention provide a semiconductor material Fe 3+ @BiVO4, its preparation includes the following steps: 1) BiVO4 was synthesized from Bi(NO3)3·5H2O and NH4VO3 via a hydrothermal method; 2) The oxidation potentials of different metal ions were obtained by electrochemical cyclic voltammetry, and metal ions with oxidation potentials lower than the valence band value of BiVO4 were selected. Based on the determination, Fe was selected in this invention. 3+ The BiVO4 was treated by dispersing it in deionized water and adding excess Fe. 3+ Vigorous stirring was applied to induce adsorption, followed by multiple washings to remove weakly adsorbed Fe. 3 + .

[0030] Compared to BiVO4, the Fe provided by this invention 3+ The photocurrent of @BiVO4 increased by 5 times, and the attenuation was significantly reduced, indicating a significant improvement in its photocurrent output intensity and stability; this is because Fe... 3+ The coupling of solid-liquid interface reactions amplifies and stabilizes the photoelectric signal; the specific mechanism is as follows: Figure 1 As shown.

[0031] Secondly, embodiments of the present invention provide a photoelectrochemical biosensor comprising Fe 3+ @BiVO4 electrode, the preparation method of this electrode is as follows: Fe 3+ A dispersion of BiVO4 was drop-coated onto ITO glass, and after drying, Fe was obtained. 3+ @BiVO4 electrode.

[0032] Thirdly, embodiments of the present invention improve a Fe-based 3+ The @BiVO4 photoelectrochemical biosensor scheme for detecting NT-proBNP includes the following reagents: a solid support containing Ab1 immobilized with NT-proBNP, AchE, AchE-AuNPs-Ab2 conjugates, ATch, and Fe. 3+ @BiVO4 electrode.

[0033] In some embodiments of the present invention, the solid support is a 96-well plate connected with Ab1.

[0034] The detection steps of the above scheme are as follows: The sample to be tested is added to a solid support for reaction, then the AchE-AuNPs-Ab2 conjugate is added, and after the immunorecognition reaction, ATch is added for further reaction; the resulting reaction solution is then dropwise added to Fe... 3+ After the reaction is complete on the @BiVO4 electrode, the sample is washed, air-dried, and then subjected to PEC detection. The NT-proBNP concentration is calculated based on the photoelectric signal.

[0035] like Figure 2 As shown, the principle of the above NT-proBNP detection scheme is as follows: Ab1 on the solid support binds to NT-proBNP in the sample to be tested. Subsequently, NT-proBNP binds to the AchE-AuNPs-Ab2 conjugate via Ab2. Then, AchE catalyzes the hydrolysis of ATch to generate TCh. Finally, TCh is used to detect Fe. 3+ Coordination poisoning effect, inhibiting Fe 3+The coupled interfacial reaction process leads to changes in photocurrent. Higher concentrations of NT-proBNP in the sample result in greater amounts of AuNPs loaded with AchE bound to the solid support, leading to increased TCh production and more significant inhibition of the interfacial reaction process. Based on this principle, a quantitative correlation between the photocurrent signal and the NT-proBNP concentration in the sample is ultimately achieved.

[0036] It is understandable that, referring to the NT-proBNP detection scheme, the Fe-based detection method provided in this invention... 3+ The @BiVO4 photoelectrochemical biosensor can also be used to detect other target substances by replacing Ab1 and Ab2 with antibody pairs targeting other target substances.

[0037] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0038] Example 1 This example provides a metal ion-functionalized photoelectrochemical sensor, the preparation method of which is as follows: (1) Fe 3+ Preparation of @BiVO4.

[0039] Take 1 mmol Bi(NO3)3·5H2O and 1 mmol NH4VO3, dissolve them in 50 mL of deionized water and 20 mL of deionized water respectively, and stir at 60℃ until completely dissolved to obtain solutions A and B respectively. Under stirring, solution B is rapidly added dropwise to solution A to obtain a mixed solution with pH 2. The solution is then transferred to a 100 mL stainless steel high-pressure reactor with a polytetrafluoroethylene liner and hydrothermally reacted at 180℃ for 12 h. After the reaction is completed and naturally cooled, the solution is centrifuged and washed three times with deionized water, and then dried at 60℃ for 12 h to obtain BiVO4.

[0040] BiVO4 was dispersed in deionized water, and an excess of Fe was added. 3+ The mixture was vigorously stirred for 24 hours, and then thoroughly washed five times with deionized water to remove weakly adsorbed metal ions, ultimately yielding a product with strongly adsorbed Fe on the surface. 3+ Functionalized semiconductor materials, denoted as Fe 3+ @BiVO4.

[0041] The BiVO4 and Fe prepared above 3+ Electron microscopy analysis of @BiVO4 yielded the following results: Figure 1 As shown, BiVO4 and Fe 3+ @BiVO4 exhibits a tree-like shape.

[0042] Simultaneously, BiVO4 and Fe 3+ X-ray powder diffraction analysis of BiVO4 showed that BiVO4 and Fe were successfully prepared in this example. 3+ @BiVO4, its characteristic peaks are consistent with BiVO4 (JCPDS No. 14-0688). Figure 2 ).

[0043] (2) Fe 3+ Preparation of @BiVO4 electrode.

[0044] Take 10 μL of 5 mg / mL Fe 3+ @BiVO4 dispersion was drop-coated onto an area of ​​0.071 cm² 2 Fe was obtained by drying on indium tin oxide (ITO) glass at 50°C. 3+ @BiVO4 electrode. The BiVO4 electrode was prepared using the same method.

[0045] Using Fe 3+ A three-electrode system was constructed using a BiVO4 electrode or a BiVO4 electrode as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum wire electrode as the counter electrode. A PBS solution with pH 7.2-7.4 was selected as the buffer solution, and PEC tests were performed under conditions without trapping agents or bias voltage, and the it curves were recorded.

[0046] Test results as follows Figure 3 As shown, Fe 3+ @BiVO4 achieved a 5-fold increase in photocurrent of BiVO4 without trapping agent or external bias voltage. More importantly, the attenuation decreased from 36% and 50% to 1.4% in 5 cycles of testing (two parallel tests), which greatly overcame photocorrosion and solved the important problem of photocurrent output intensity and stability.

[0047] Example 2 This example investigates the effect of in-situ infrared spectroscopy on Fe. 3+ The mechanism by which @BiVO4 exhibits superior photoelectric performance.

[0048] Take 100 μL of 5 mg / mL Fe 3+ @BiVO4 was dropped onto the substrate for in-situ infrared testing, and in-situ infrared spectra were measured under illumination for 0, 1, 2, 3, 4, 7, and 10 minutes, respectively.

[0049] Test results are as follows Figure 4 As shown: During illumination for 0-4 minutes, the Fe substrate... 3+The BiVO4 solution was not yet dry when adsorbed oxygen was produced, and the peak intensity increased with increasing light exposure time, proving that the oxygen production under light corresponds to water oxidation; however, after 7 and 10 minutes of light exposure, the water dried up, and the peak intensity at 845 cm⁻¹... -1 High-priced iron-oxygen species (Fe) have emerged. Ⅳ The peak of Fe(=O) increases with time. These results indicate that Fe... 3+ During the photoelectrochemical process of @BiVO4, Fe adsorbed on the surface 3+ In-situ extraction of photogenerated holes to produce Fe Ⅳ =O, Fe Ⅳ =O undergoes water oxidation to produce oxygen ( Figure 1 ); this process Fe 3+ A redox cycle occurred, which accelerated the in-situ extraction of holes through the coupling of the solid-liquid interface, avoided photocorrosion, and improved and stabilized the photocurrent.

[0050] Example 3 Use Fe 3+ @BiVO4 electrode, this example provides a method for detecting NT-proBNP, including the following steps: (1) Preparation of AchE-AuNPs-Ab2 conjugate.

[0051] First, 40 mL of 10 ng / mL AuNPs, 1.6 mL of 10 U / mL AchE, and 400 μL of 50 μg / mL Ab2 solution were placed in a 50 mL centrifuge tube and gently shaken (130 r / min) for 1 h at 4°C. Then, 800 μL of 1.0 wt / % PEG solution was added to the suspension and incubated at 4°C for 2 h. The AchE-AuNPs-Ab2 conjugate was obtained by centrifugation (8000 r / min, 10 min). Finally, the conjugate was redispersed in 8 mL of a solution containing 1.0 wt% bovine serum albumin (BSA) and stored at 4°C for later use.

[0052] (2) Preparation of NT-pro BNP detection solution.

[0053] Add 50 μL of the prepared 20 μg / mL Ab1 solution to a 96-well plate and incubate at 4 °C for 24 h, then wash three times with Tween 20 solution. Next, add 100 μL of 1 wt% BSA to the plate and incubate at 37 °C for 1 h. Then add 50 μL of the test sample and gently shake at 37 °C for 1 h. Following this, add 50 μL of the AchE-AuNPs-Ab2 conjugate to the plate and incubate at 37 °C for 1 h, then wash three times with PBS. Finally, add 150 μL of 10 mM ATch solution and incubate at 37 °C for 1 h.

[0054] (3) Detection of NT-proBNP.

[0055] Fe prepared using Example 1 3+ @BiVO4 electrode, take 10 μL of the NT-pro BNP detection solution obtained in step (2) and add it to the electrode, incubate at 37℃ for 30 min, so that the thiol-rich TCh reacts with the Fe on the electrode surface. 3+ Fully coordinate and bind; wash with deionized water, air dry, and perform PEC testing.

[0056] The linearity of the above method was analyzed, specifically, the test samples in step (2) were a series of NT-pro BNP solutions with different concentrations (0.1, 1, 5, 10, 50, 100, 500, 1000, 2000, 5000, 10000 pg / mL). The detection results are as follows: Figure 7 As shown: the photocurrent response gradually decreases with NT-pro BNP concentration between 0-5000 pg / mL. Figure 7 a); the photocurrent and the logarithm of NT-pro BNP concentration showed a linear correlation in the range of 1-5000 pg / mL ( Figure 7 b), and the linear fitting equation is I(μA) = 928.1 + 105.4 C NT-pro BNP (R²=0.991), the limit of detection (S / N=3) was 0.33 pg / mL.

[0057] To simulate the actual testing environment, the inventors screened several substances that might interfere with NT-proBNP, namely carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), immunoglobulin G (IgG), insulin, and glucose (Glu), to verify the specificity of the above method. Referring to step (2), the sample to be tested was replaced with 2500 ng / mL of interfering substance, and then the PEC test was performed. The test results are as follows: Figure 8 As shown, the prepared photoelectrochemical biosensor exhibits excellent selectivity for potential interfering substances in the human body, indicating that this method can achieve specific detection of NT-pro BNP in clinical samples.

[0058] In summary, this invention uses Fe adsorption 3+ A photoelectrochemical sensor was successfully established using BiVO4 as the electrode material, and the sensor exhibited good detection accuracy and significant stability for NT-proBNP.

[0059] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A method for preparing a photoelectrochemical sensor, characterized in that, Includes the following steps: BiVO4 was dispersed in deionized water, and an excess of Fe was added. 3+ Adsorption is carried out, and excess Fe is removed after adsorption is complete. 3+ Thus, Fe adsorbed on the surface is obtained. 3+ Functionalized semiconductor material Fe 3+ @BiVO4; Fe 3+ @BiVO4 was drop-coated onto a conductive material to obtain Fe 3+ @BiVO4 electrode.

2. The preparation method according to claim 1, characterized in that, The BiVO4 is dendritic and is hydrothermally synthesized from bismuth nitrate pentahydrate and ammonium metavanadate in a high-pressure reactor at 120-200°C.

3. The photoelectrochemical sensor obtained by the preparation method according to claim 1 or 2.

4. The photoelectrochemical sensor according to claim 3, characterized in that, With Fe 3+ The @BiVO4 electrode is the working electrode, and it also includes a reference electrode and a counter electrode.

5. The application of the photoelectrochemical sensor as described in claim 3 in the detection of N-terminal probrain natriuretic peptide.

6. A detection product for N-terminal probrain natriuretic peptide, characterized in that, Including the Fe as described in claim 1 3+ @BiVO4 electrode.

7. The testing product according to claim 6, characterized in that, It also includes a solid-phase carrier, an acetylcholinesterase-labeled beacon antibody, and thioacetylcholine, wherein the solid-phase carrier is immobilized with a capture antibody containing an N-terminal probrain natriuretic peptide.

8. The testing product according to claim 7, characterized in that, The acetylcholinesterase-labeled beacon antibody is a conjugate formed by acetylcholinesterase, gold nanoparticles, and the beacon antibody.

9. The testing product according to claim 7, characterized in that, The solid support is a polystyrene microplate immobilized with capture antibodies.

10. A method for detecting N-terminal probrain natriuretic peptide, characterized in that, Using the testing product of claim 7, and comprising the following steps: S1. Add the sample to be tested to the solid-phase carrier, then add the beacon antibody labeled with acetylcholinesterase. After the immune recognition reaction, add thioacetylcholine and use acetylcholinesterase to catalyze the hydrolysis of thioacetylcholine to generate thiocholine. S2. Add the reaction solution obtained in step S1 dropwise to Fe. 3+ After the reaction is complete on the @BiVO4 electrode, the sample is washed, air-dried, and subjected to PEC detection. The NT-proBNP concentration is calculated based on the photoelectric signal.

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