Photoelectrochemical biosensor for detecting histone deacetylase and preparation method thereof

Abstract

The present invention discloses a photoelectrochemical biosensor for detecting histone deacetylase and a preparation method thereof, belonging to the field of photoelectrochemical analysis technology. The present invention comprises a base electrode, and CuS-BiVO4 and MnO2 sequentially modified on the surface of the base electrode. The present invention utilizes the energy band matching effect between CuS and BiVO4, and Sirt1 reacts with P Ac The specific deacetylation of MnO2 by NADH and the reduction of MnO2 by NADH enable the detection of the histone deacetylase Sirt1. This method is simple to operate and only requires simple modification of the ITO electrode surface to achieve Sirt1 detection.

Description

Technical Field

[0001] The present invention relates to the technical field of photoelectrochemical analysis, and in particular to a photoelectrochemical biosensor for detecting histone deacetylase and a preparation method thereof. Background Art

[0002] Histone deacetylation, a crucial step in maintaining the balance of histone acetylation levels, plays an irreplaceable role in regulating genome stability, maintaining chromatin structure, and modulating cellular metabolism. Histone acetylation levels are maintained in balance through the combined action of histone deacetylases (HDACs) and histone acetyltransferases (HATs). Abnormal histone deacetylation is also associated with a variety of human diseases, such as cancer, liver fibrosis, and cardiovascular disease.

[0003] Sirtuin 1 (Sirt1) is an important histone deacetylase that uses nicotinamide adenine dinucleotide (NAD+) as a cofactor instead of zinc to remove acetyl groups from lysine residues. Sirt1 is involved in regulating various physiological processes, including mitochondrial function, metabolism, aging, and inflammatory response. Abnormal expression of Sirt1 is also associated with cancer, neurological diseases, and type 2 diabetes. The level of Sirt1 can significantly affect the expression level of histone deacetylation. Therefore, it is of great significance to detect the level of Sirt1 and study its biological function.

[0004] Currently, most Sirt1 detection methods rely on high-performance liquid chromatography, mass spectrometry, and fluorescence sensors. These early approaches have advanced Sirt1 research. However, these methods are limited by expensive equipment and complex procedures. Therefore, developing a simple, sensitive detection method is crucial.

[0005] Photoelectrochemical biosensors are effective tools for recording cellular biological events. They convert biological phenomena into photocurrent signals through biorecognition elements and signal converters, and the changes in the photocurrent signals are used to enable qualitative and quantitative analysis of targets. Compared to fluorescence, electrochemistry, and electrochemiluminescence techniques, photoelectrochemical detection technology has become an analytical technique with great application value due to its independent excitation light source and signal acquisition system, resulting in low background signals and high detection sensitivity. However, there are currently no reports on the detection of Sirt1 using photoelectrochemical analysis methods. Summary of the Invention

[0006] In view of the above-mentioned prior art, the object of the present invention is to provide a photoelectrochemical biosensor for detecting histone deacetylase and a preparation method thereof.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a photoelectrochemical biosensor comprising a base electrode, and CuS-BiVO4 and MnO2 sequentially modified on the surface of the base electrode.

[0009] Preferably, the base electrode is an ITO electrode.

[0010] A second aspect of the present invention provides a method for preparing the above-mentioned photoelectrochemical biosensor, comprising the following steps:

[0011] (1) adding the CuS-BiVO4 dispersion dropwise onto the pretreated base electrode surface and drying to obtain a CuS-BiVO4 / electrode;

[0012] (2) Add MnO2 dispersion dropwise onto the CuS-BiVO4 / electrode surface and dry to obtain MnO2 / CuS-BiVO4 / electrode; use it as a photoelectrochemical biosensor.

[0013] Preferably, in step (1), the CuS-BiVO4 dispersion is obtained by dispersing CuS-BiVO4 nanomaterials in deionized water, and the concentration of the CuS-BiVO4 dispersion is 0.2-5 mg / ml.

[0014] More preferably, the CuS-BiVO4 nanomaterial is prepared by the following method:

[0015] Bismuth nitrate pentahydrate is dissolved in dilute nitric acid to form liquid A, ammonium metavanadate is dissolved in sodium hydroxide solution to form liquid B, liquid A is added to liquid B, and a hydrothermal reaction is carried out after stirring. After the reaction is completed, the mixture is washed and centrifuged to collect the solid BiVO4 nanomaterial; the obtained BiVO4 nanomaterial is dispersed in ethanol, copper nitrate and sodium thiosulfate are added in sequence, and the obtained mixed solution is stirred at 70°C for 4 hours. After the reaction is completed, the mixture is washed and centrifuged to collect the CuS-BiVO4 nanomaterial.

[0016] Preferably, in step (2), the MnO2 dispersion is prepared by the following method:

[0017] Tetramethylammonium hydroxide, ultrapure water, and hydrogen peroxide are mixed, and then manganese chloride is added. The mixture is stirred at room temperature, and the precipitate is collected by centrifugation. The precipitate is washed twice with water and methanol, and then the solid is redispersed in ultrapure water to prepare a MnO2 dispersion.

[0018] Preferably, in step (2), the concentration of the MnO2 dispersion is 0.1-0.3 mg / ml.

[0019] The third aspect of the present invention provides the use of the above-mentioned photoelectrochemical biosensor in detecting histone deacetylase.

[0020] In the above application, the histone deacetylase is Sirt1.

[0021] A fourth aspect of the present invention provides a method for detecting histone deacetylase Sirt1, comprising the following steps:

[0022] The acetylated peptide chain (P Ac ), NAD + The reaction mixture consisting of , Sirt1 is modified onto the surface of the above-mentioned photoelectrochemical biosensor as a working electrode, a saturated calomel electrode is used as a reference electrode, and a Pt wire is used as an auxiliary electrode to form a three-electrode system. Photoelectrochemical signal detection is carried out in the electrode detection liquid, and a standard curve between current and Sirt1 enzyme concentration is established to detect the Sirt1 enzyme content in the test liquid.

[0023] Preferably, the reaction mixture consists of acetylated peptide chain solution, NAD + The solution and Sirt1 solution were prepared in equal volume ratio. Ac ) is structured as follows: CGK Ac GGK Ac GGK Ac GGK Ac G; Ac represents acetyl modification.

[0024] Preferably, the electrode detection solution is a Tris-HCl buffer solution with a pH of 5.5-8.5 and a concentration of 0.1-100 mmol·L -1 .

[0025] Beneficial effects of the present invention:

[0026] (1) The present invention utilizes Sirt1 enzyme to Ac The specific deacetylation effect of NADH and the reduction effect of NADH on MnO2 were used to construct a photoelectrochemical biosensor, which achieved specific quantitative detection of Sirt1 enzyme.

[0027] (2) The present invention utilizes the energy band matching effect between CuS and BiVO4 to achieve photoelectric signal amplification and improve the detection sensitivity of Sirt1 enzyme.

[0028] (3) The detection method of the present invention is simple, low-cost, and miniaturized. The Sirt1 enzyme can be detected by simply processing the surface of the ITO electrode. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1: Schematic diagram of the construction of the photoelectrochemical biosensor of the present invention and the principle of Sirt1 enzyme detection.

[0030] Figure 2 : Linear fitting curve of photocurrent intensity and logarithmic value of Sirt1 enzyme concentration.

[0031] Figure 3 : Bar graph showing the changes in photoelectrochemical responses under different enzyme treatment conditions. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0033] Terminology Notes:

[0034] The range of "room temperature" in the present invention is 20-30°C.

[0035] The "humid condition" in the present invention means that the humidity is greater than 90%; the preferred humidity is 95-99%.

[0036] As mentioned above, existing Sirt1 detection methods have problems such as expensive detection equipment and complex operations, which limit the research and application of Sirt1.

[0037] Based on this, the present invention has constructed a photoelectrochemical biosensor for detecting Sirt1 enzyme. The schematic diagram of the construction and detection principle of the photoelectrochemical biosensor of the present invention is shown in FIG. Figure 1 . The photoelectrochemical biosensor of the present invention uses an ITO electrode as a base electrode, and CuS-BiVO4 and MnO2 are modified on the electrode surface in sequence. Among them, CuS-BiVO4, as a two-dimensional layered material, can be used as an excellent photoactive material. The energy bands of CuS and BiVO4 match to form a heterojunction, which increases the photocurrent intensity. MnO2, as a water-insoluble nanomaterial, covers the surface of the photoactive material CuS-BiVO4, blocks the substrate signal, and due to the steric hindrance effect, hinders the migration of photogenerated electrons of the photoactive material on the electrode surface, and reduces the photocurrent intensity.

[0038] During the detection, acetylated peptide chains (P Ac ), NAD + , Sirt1 enzyme, Sirt1 enzyme to P Ac The specific deacetylation of MnO2 produces a byproduct NADH during the deacetylation process, which can be used as a signal amplification unit because NADH can reduce MnO2 to Mn 2+, exposing the CuS-BiVO4 substrate, thereby enhancing the photocurrent intensity. The amount of NADH is determined by the Sirt1 enzyme concentration, so the linear relationship between Sirt1 enzyme and photocurrent intensity can be used to achieve quantitative detection of Sirt1 enzyme.

[0039] In one embodiment of the present invention, the construction process of the photoelectrochemical biosensor is as follows:

[0040] (1) Preparation of BiVO4: Dissolve bismuth nitrate pentahydrate in dilute nitric acid to form solution A, and dissolve ammonium metavanadate in sodium hydroxide solution to form solution B. Add solution A to solution B, stir, and perform a hydrothermal reaction. After the reaction is complete, wash and centrifuge to collect the solid BiVO4 nanomaterial.

[0041] (2) Preparation of CuS-BiVO4: The obtained BiVO4 was dispersed in ethanol, and copper nitrate and sodium thiosulfate were added in sequence. The resulting mixture was stirred at 70°C for 4 hours. After the reaction was completed, the CuS-BiVO4 composite material was washed and collected by centrifugation.

[0042] (3) Preparation of MnO2: Tetramethylammonium hydroxide, ultrapure water, and hydrogen peroxide were mixed, and then manganese chloride was added. The mixture was stirred at room temperature for 12 hours, and the precipitate was collected by centrifugation and washed twice with water and methanol to obtain MnO2 nanomaterials.

[0043] (4) Preparation of CuS-BiVO4 dispersion: Weigh 2-10 mg of the CuS-BiVO4 nanomaterial prepared in step (3), add it to 2-10 mL of deionized water, and ultrasonically disperse it for 0.5-3 hours.

[0044] (5) Preparation of MnO2 dispersion: The MnO2 solid prepared in step (3) was redispersed in ultrapure water to prepare a MnO2 nanomaterial dispersion with a concentration of 0.1-0.3 mg / ml;

[0045] (6) ITO electrode pretreatment: Split the ITO conductive glass into 5×1cm 2 , respectively, with acetone, 1-4M NaOH alcohol aqueous solution (V 无水乙醇 :V 二次水 =1:(1-6)), ultrasonically treat the ITO electrode with secondary water for 15-60 minutes, then rinse with secondary water, dry naturally, and set aside.

[0046] (7) Immobilization of CuS-BiVO4: 20-80 μL of CuS-BiVO4 dispersion was added dropwise to the pretreated ITO electrode surface and dried under infrared light. The electrode was then washed 1-5 times with electrode cleaning buffer. The electrode was dried under nitrogen. The prepared electrode was labeled CuS-BiVO4 / ITO.

[0047] (8) MnO2 immobilization: 20-80 μL of MnO2 dispersion was added dropwise to the surface of the CuS-BiVO4 / ITO electrode and dried under infrared light. The electrode was then washed 1-5 times with electrode cleaning buffer. The electrode was dried under nitrogen. The prepared electrode was labeled as MnO2 / CuS-BiVO4 / electrode, which is the antibody-free enzyme-assisted photoelectrochemical biosensor of the present invention.

[0048] In another embodiment of the present invention, a method for detecting Sirt1 enzyme using the photoelectrochemical biosensor is provided, comprising the following steps:

[0049] (1) Deacetylation reaction involving Sirt1 enzyme: 20-80 μL of P Ac 、Sirt1、NAD + Mix equal volumes and react at 37°C in a humidified environment for 0.5-3 h to obtain a mixed solution containing NADH.

[0050] (2) Modification of Sirt1 enzyme: 20-80 μL of the NADH-containing mixture obtained in step (1) was added dropwise to the surface of the photoelectrochemical biosensor (MnO2 / CuS-BiVO4 / electrode) prepared in the present invention and reacted at 37°C in a humidified environment for 0.5-3 h. The electrode was then washed 1-5 times with electrode cleaning buffer. The electrode was dried with nitrogen gas. The prepared electrode was labeled Sirt1 / MnO2 / CuS-BiVO4 / electrode and used as the working electrode.

[0051] Using an electrochemical workstation as the signal acquisition instrument, LED light as the light source, Sirt1 / MnO2 / CuS-BiVO4 / electrode as the working electrode, saturated calomel electrode as the reference electrode, Pt electrode as the counter electrode, Tris-HCl buffer solution as the electrode detection solution, and 0.1V as the working voltage, IT technology was used to conduct detection research on the analyte, and a standard curve between the photocurrent intensity and the Sirt1 enzyme concentration was established, thereby realizing the quantitative detection of Sirt1 enzyme.

[0052] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0053] The test materials used in the examples of the present invention are all conventional test materials in the field and can be purchased through commercial channels. Experimental methods without detailed conditions were carried out in accordance with conventional test methods or the operating instructions recommended by the supplier.

[0054] The electrode cleaning buffer used in the present invention comprises 3-15 mM Tris-HCl, pH 7.4, and the solvent is sterile water.

[0055] The Sirt1 enzyme used in the present invention was purchased from Enzo, production batch number BML-SE239-0100, GENE / PROTEIN IDENTIFIER: NM_012238 (RefSeq);

[0056] The Sirt1 enzyme is dissolved in a dissolving solution when used. The components of the dissolving solution are: 5-100 mM Tris, pH=7.5, and the solvent is sterile water.

[0057] Example 1:

[0058] (1) Preparation of BiVO4: Dissolve 10 mmol of bismuth nitrate pentahydrate in 25 ml of 1 M dilute nitric acid to form Solution A. Dissolve 10 mmol of ammonium metavanadate in 20 ml of 1 M sodium hydroxide solution to form Solution B. Add Solution A to Solution B, stir for 2 h, pour into the lining of a reactor, and react in an oven at 180°C for 6 h. After the reaction is complete, wash with water and ethanol, collect by centrifugation, and dry in an oven at 60°C to obtain BiVO4 nanomaterials.

[0059] (2) Preparation of CuS-BiVO4: 0.3 g of the prepared BiVO4 nanomaterial was dispersed in 60 ml of ethanol. 2 mmol of copper nitrate and 3 mmol of sodium thiosulfate were added sequentially. The resulting mixture was stirred at 70°C for 4 hours. After the reaction was complete, the mixture was washed, collected by centrifugation, dried in an oven at 60°C, and ground to obtain a CuS-BiVO4 composite material.

[0060] (3) Preparation of MnO2: 4.3 ml of tetramethylammonium hydroxide, 13.7 ml of ultrapure water, and 2 ml of hydrogen peroxide were mixed, and then 10 ml of 0.3 mol / l manganese chloride solution was quickly added. The mixture was stirred at room temperature for 12 hours, and the precipitate was collected by centrifugation at 8000 rpm for 30 minutes. The precipitate was then washed with water and methanol three times each to obtain MnO2 solid nanomaterials.

[0061] (4) Preparation of CuS-BiVO4 dispersion: 10 mg of the CuS-BiVO4 nanomaterial prepared in step (3) was weighed and added to 5 mL of deionized water, and ultrasonically dispersed for 0.5 h to prepare a CuS-BiVO4 dispersion.

[0062] (5) Preparation of MnO2 dispersion: 10 mg of the MnO2 solid nanomaterial prepared in step (3) was redispersed in 50 ml of ultrapure water and ultrasonically dispersed for 0.5 h to prepare a MnO2 dispersion.

[0063] (6) ITO electrode pretreatment

[0064] Cut the ITO conductive glass into 5×1cm 2, respectively, with acetone, 1M NaOH alcohol aqueous solution (V 无水乙醇 :V 二次水 =1:1), ultrasonically treat the ITO electrode with secondary water for 20 minutes, then rinse with secondary water, dry naturally and set aside.

[0065] (7) Immobilization of CuS-BiVO4: 40 μL of CuS-BiVO4 dispersion was added dropwise to the pretreated ITO electrode surface and dried under infrared light. The electrode was then washed three times with electrode cleaning buffer and dried under nitrogen. The prepared electrode was labeled CuS-BiVO4 / electrode.

[0066] (8) MnO2 immobilization: 40 μL of MnO2 dispersion was added dropwise to the surface of the CuS-BiVO4 / ITO electrode and dried under infrared light. The electrode was then washed three times with electrode cleaning buffer. The electrode was dried under nitrogen gas. The prepared electrode was labeled MnO2 / CuS-BiVO4 / electrode. This is the photoelectrochemical biosensor of the present invention.

[0067] Example 2: Photoelectrochemical detection

[0068] 60 μM P Ac , a series of concentrations of Sirt1 enzyme, 600 μM NAD + 40 μL of each were mixed and reacted at 37°C in a humidified environment for 2 h to obtain a mixed solution containing NADH. Ac The peptide chain structure is: CGK Ac GGK Ac GGK Ac GGK Ac G; Ac represents acetyl modification.

[0069] 40 μL of the NADH-containing mixture was dropwise added to the MnO2 / CuS-BiVO4 / ITO electrode surfaces, and the reaction was allowed to proceed at 37°C in a humidified environment for 40 minutes. The electrodes were then washed three times with electrode cleaning buffer and dried under nitrogen. The resulting electrodes were labeled Sirt1 / MnO2 / CuS-BiVO4 / ITO and used as the working electrodes of the present invention.

[0070] Using an electrochemical workstation as the signal acquisition instrument, a 3W LED lamp as the light source, Sirt1 / MnO2 / CuS-BiVO4 / electrode as the working electrode, a saturated calomel electrode as the reference electrode, a Pt electrode as the counter electrode, and a Tris-HCl buffer solution as the electrode detection solution, the working voltage was 0.1V, and the IT technology was used to detect the analyte. A standard curve between the photocurrent intensity and the Sirt1 enzyme concentration was established ( Figure 2 ), with a detection limit of 3.38 pM.

[0071] Example 3: Detection selectivity experiment

[0072] Selectivity is an important indicator of the performance of photoelectrochemical sensors. In order to study the specificity of the constructed sensor, alkaline phosphatase (ALP), histone acetyltransferase (HAT), bovine serum albumin (BSA), demethylase (MBD2), and endoribonuclease (MazF) at a concentration of 3 nM were selected as interfering substances to study the selectivity of the sensor prepared in Example 1. The photocurrent change values ​​(ΔI = I2-I1, I1 is the photocurrent change after the addition of P Ac 、NAD + The photocurrent value of the electrode treated by the mixed solution of MnO2 / CuS-BiVO4 / electrode, I2 is the result of different interferences and P Ac 、NAD + The results showed that the change in the current value of the sensor constructed with the interference was significantly lower than that of the Sirt1 enzyme, indicating that the constructed sensor has good specificity ( Figure 3 ).

[0073] Example 4: Stability test

[0074] The Sirt1 / MnO2 / CuS-BiVO4 / ITO sensor was prepared using the method of Example 1 and continuously measured for 10 cycles. The photoelectrochemical signal was detected in the detection liquid, and the standard deviation of the photocurrent was 1.3%, indicating that the method has good stability.

[0075] Comparative Example 1:

[0076] (1) Preparation of BiVO4 dispersion:

[0077] 10 mmol of bismuth nitrate pentahydrate was dissolved in 25 ml of 1M dilute nitric acid to form Solution A. 10 mmol of ammonium metavanadate was dissolved in 20 ml of 1M sodium hydroxide solution to form Solution B. Solution A was added to Solution B, stirred for 2 hours, and then poured into the lining of a reactor. The mixture was then reacted in an oven at 180°C for 6 hours. After completion of the reaction, the mixture was washed with water and ethanol, collected by centrifugation, and dried in an oven at 60°C to obtain the BiVO4 nanomaterial.

[0078] 10 mg of the prepared BiVO4 nanomaterial was weighed, added to 5 mL of deionized water, and ultrasonically dispersed for 0.5 h to prepare a BiVO4 dispersion.

[0079] (2) ITO electrode pretreatment

[0080] Cut the ITO conductive glass into 5×1cm 2, respectively, with acetone, 1M NaOH alcohol aqueous solution (V 无水乙醇 :V 二次水 =1:1), ultrasonically treat the ITO electrode with secondary water for 20 minutes, then rinse with secondary water, dry naturally and set aside.

[0081] (3) Fixation of BiVO4:

[0082] 40 μL of BiVO4 dispersion was added dropwise to the pretreated ITO electrode surface and dried under infrared light. The electrode was then rinsed three times with electrode cleaning buffer and dried under nitrogen. The resulting electrode was labeled BiVO4 / electrode.

[0083] (4) Fixation of MnO2:

[0084] Mix 4.3 ml of tetramethylammonium hydroxide, 13.7 ml of ultrapure water, and 2 ml of hydrogen peroxide. Then quickly add 10 ml of a 0.3 mol / l manganese chloride solution. Stir at room temperature for 12 hours. Centrifuge at 8,000 rpm for 30 minutes to collect the precipitate. Wash it three times each with water and methanol to obtain a MnO2 solid nanomaterial. Redisperse 10 mg of the MnO2 solid nanomaterial in 50 ml of ultrapure water to prepare a MnO2 dispersion.

[0085] 40 μL of the MnO₂ dispersion was added dropwise to the BiVO₄ / ITO electrode surface and dried under infrared light. The electrode was then rinsed three times with electrode cleaning buffer and dried under nitrogen. The resulting electrode was labeled MnO₂ / BiVO₄ / electrode.

[0086] Comparative Example 2:

[0087] (1) Preparation of CuS dispersion:

[0088] Preparation of CuS: Disperse 2 mmol of copper nitrate in 60 ml of ethanol and stir for 30 minutes. Then, add 3 mmol of sodium thiosulfate. The resulting mixture is stirred at 70°C for 4 hours. After the reaction is complete, wash with water and ethanol, collect by centrifugation, and dry in an oven at 60°C to obtain CuS nanomaterials.

[0089] 10 mg of the prepared CuS nanomaterial was weighed, added to 5 mL of deionized water, and ultrasonically dispersed for 0.5 h to prepare a CuS dispersion.

[0090] (2) ITO electrode pretreatment

[0091] Cut the ITO conductive glass into 5×1cm 2 , respectively, with acetone, 1M NaOH alcohol aqueous solution (V 无水乙醇 :V 二次水=1:1), ultrasonically treat the ITO electrode with secondary water for 20 minutes, then rinse with secondary water, dry naturally and set aside.

[0092] (3) Fixation of CuS:

[0093] 40 μL of CuS dispersion was added dropwise to the pretreated ITO electrode surface and dried under infrared light. The electrode was then rinsed three times with electrode cleaning buffer and dried under nitrogen. The resulting electrode was labeled CuS / electrode.

[0094] (4) Fixation of MnO2:

[0095] Mix 4.3 ml of tetramethylammonium hydroxide, 13.7 ml of ultrapure water, and 2 ml of hydrogen peroxide. Then quickly add 10 ml of a 0.3 mol / l manganese chloride solution. Stir at room temperature for 12 hours. Centrifuge at 8,000 rpm for 30 minutes to collect the precipitate. Wash it three times each with water and methanol to obtain a MnO2 solid nanomaterial. Redisperse 10 mg of the MnO2 solid nanomaterial in 50 ml of ultrapure water to prepare a MnO2 dispersion.

[0096] 40 μL of the MnO2 dispersion was added dropwise to the surface of the CuS / ITO electrode and dried under infrared light. The electrode was then rinsed three times with electrode cleaning buffer and dried under nitrogen. The resulting electrode was labeled MnO2 / CuS / electrode.

[0097] Test example:

[0098] The photocurrent changes of the MnO2 / CuS-BiVO4 / electrode prepared in Example 1, the MnO2 / BiVO4 / electrode prepared in Comparative Example 1, and the MnO2 / CuS / electrode prepared in Comparative Example 2 were investigated when treated with a Sirt1 mixed solution and when not treated with a Sirt1 mixed solution. The specific process is as follows:

[0099] 40 μL of P Ac 、Sirt1、NAD + A 1:1:1 volume ratio of the mixed solution was added dropwise to the surface of each electrode and allowed to react at 37°C in a humidified environment for 40 minutes. The electrode was then washed three times with electrode cleaning buffer. The electrode was dried under nitrogen and used as the working electrode, a saturated calomel electrode as the reference electrode, a Pt electrode as the counter electrode, and a Tris-HCl buffer solution as the electrode detector. The photocurrent (I²) was measured using an electrochemical workstation as the signal acquisition instrument and a 3W LED lamp as the light source at a working voltage of 0.1V.

[0100] 40 μL of P Ac 、NAD +The mixed solution with a volume ratio of 1:1 was added dropwise to the surface of the above electrodes and reacted in a humidified environment at 37°C for 40 minutes. The electrodes were then washed three times with the electrode cleaning buffer. The electrodes were dried with nitrogen and used as the working electrode, a saturated calomel electrode as the reference electrode, a Pt electrode as the counter electrode, and a Tris-HCl buffer solution (pH 7.5, 50 mmol L -1 ) was used as the electrode detection liquid, 0.1 V was used as the working voltage, an electrochemical workstation was used as the signal acquisition instrument, and a 3 W LED lamp was used as the light source to measure the photocurrent value I1.

[0101] The above-mentioned P Ac The peptide chain structure is: CGK Ac GGK Ac GGK Ac GGK Ac G; Ac represents acetyl modification.

[0102] Calculate the photocurrent change ΔI for different electrodes, ΔI = I2 - I1. The results are as follows:

[0103] electrode ΔI <![CDATA[MnO2 / CuS-BiVO4 / electrode]]> 13.6μA <![CDATA[MnO2 / BiVO4 / Electrode]]> 0.1μA <![CDATA[MnO2 / CuS / Electrode]]> 0.04μA

[0104] The results showed that when BiVO4 or CuS were modified on the surface of a base electrode, both then blocked with MnO2, and used as a photoelectrochemical biosensor, the photocurrent change caused by Sirt1 enzyme treatment or absence was relatively small. However, when BiVO4 and CuS were combined, the photocurrent change caused by Sirt1 enzyme treatment or absence was greatly improved.

[0105] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A photoelectrochemical biosensor, characterized in that: It includes a base electrode and a base electrode modified on the surface of the base electrode and ; The photoelectrochemical biosensor is used to detect histone deacetylase Sirt1 and is prepared by the following method: (1) The dispersion was added dropwise to the pre-treated surface of the base electrode and dried to obtain / electrode; (2) Add the dispersion dropwise to / Electrode surface, dried, obtained / / electrode; using it as a photoelectrochemical biosensor; In step (1), the The dispersion is The nanomaterial is dispersed in deionized water to obtain The concentration of the dispersion is 0.2-5 mg / ml; In step (2), the The dispersion was prepared by the following method: Tetramethylammonium hydroxide, ultrapure water, and hydrogen peroxide were mixed, and then manganese chloride was added, stirred at room temperature, and the precipitate was collected by centrifugation, washed with water and methanol twice, and then the solid was redispersed in ultrapure water to prepare dispersion; The concentration of the dispersion was 0.2 mg / ml.

2. The photoelectrochemical biosensor according to claim 1, wherein The base electrode is an ITO electrode.

3. The photoelectrochemical biosensor according to claim 1, characterized in that described Nanomaterials are prepared by the following methods: Dissolve bismuth nitrate pentahydrate in dilute nitric acid to form liquid A, dissolve ammonium metavanadate in sodium hydroxide solution to form liquid B, add liquid A to liquid B, stir and perform hydrothermal reaction, wash and centrifuge after the reaction is completed, and collect the solid Nanomaterials; The nanomaterials were dispersed in ethanol, copper nitrate and sodium thiosulfate were added in sequence, and the resulting mixture was stirred at 70 ° C for 4 hours. After the reaction was completed, the mixture was washed and centrifuged, and the mixture was collected. Nanomaterials.

4. Use of the photoelectrochemical biosensor according to any one of claims 1 to 3 in detecting histone deacetylase Sirt1.

5. A method for detecting histone deacetylase Sirt1, characterized in that: The following steps are involved: The acetylated peptide chain, , and Sirt1 are modified onto the surface of the photoelectrochemical biosensor according to any one of claims 1 to 3, using a saturated calomel electrode as a reference electrode and a Pt wire as an auxiliary electrode to form a three-electrode system, performing photoelectrochemical signal detection in an electrode detection solution, establishing a standard curve between current and Sirt1 enzyme concentration, and detecting the Sirt1 enzyme content in the test solution; The reaction mixture consists of an acetylated peptide chain solution, The solution and Sirt1 solution were composed in an equal volume ratio.

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