A photoelectric biosensor based on integrated electrodes and its preparation method

Abstract

The present invention provides an integrated electrode-based photoelectric biosensor and its preparation method, addressing the technical issues of prior art photoelectric biosensors, such as cumbersome assembly and operation, and low sensitivity in detecting protein kinase activity. The preparation method comprises the following steps: S1. preparing a kemptide-modified integrated printed electrode; S2. dripping a buffer solution containing protein kinase and ATP onto the electrode for reaction, then dripping an Au@MXenes / BiOCl probe solution onto the electrode. After the reaction is complete, the photoelectric biosensor is prepared. Under visible light excitation, the Au in the Au@MXenes / BiOCl probe generates free electrons due to localized plasma. Simultaneously, the MXenes and BiOCl semiconductors synergistically generate photoexcited electrons. These two electrons jointly transition to the electrode surface, generating a photocurrent. This further increases the current generated by the probe under visible light, effectively improving the sensitivity and linear range of detection. The sensor can be widely used in photoelectric sensing and biochemical analysis.

Description

Technical Field

[0001] The present invention relates to the field of photoelectric sensing and biochemical analysis, and in particular to a photoelectric biosensor based on an integrated electrode and a preparation method thereof. Background Art

[0002] Protein phosphorylation regulated by protein kinases (PKA) plays an important role in metabolism and cell signaling pathways. Protein kinases can catalyze protein phosphorylation reactions, and this protein phosphorylation process plays an important regulatory role in a series of physiological processes such as cell signal transmission, cell proliferation, differentiation and aging, and gene delivery. When errors occur in protein activation or the sites where protein phosphorylation occurs, it can lead to the occurrence of various diseases, such as tumors, diabetes, and Alzheimer's disease. In the field of biochemistry, the detection of protein kinase activity and its inhibitors can elucidate the molecular mechanisms of signal transduction. In clinical medicine and drug delivery, early detection of abnormal kinase expression activity is also beneficial for disease prevention and treatment. Therefore, sensitive detection of protein kinase activity is extremely important.

[0003] In the prior art, photoelectric sensing methods are generally used to detect the activity of protein kinases. In these methods, most of the electrodes used are ITO conductive glass, glassy carbon electrodes, or gold electrodes as working electrodes. Although relatively good detection results are achieved, these working electrodes still need to be washed and dried with strong alkali, ethanol, acetone, etc. before use, or they need to be polished and rinsed for a long time before continuing the sensor assembly steps. At the same time, the counter electrode and reference electrode are also required as auxiliary electrodes during testing, and the volume of electrolyte solution used is relatively large. Such methods have many steps, are relatively lengthy, and are expensive to operate. In addition, the probes used in most photoelectric sensors are connected to the substrate peptide phosphorylated by the protein kinase through metal ions / oxides, aptamers, or through the combination of antigens and antibodies. The connection efficiency is not high. At the same time, biological molecules such as aptamers, antigens, and antibodies are greatly affected by changes in external environmental conditions, which will have a certain impact on the detection results.

[0004] Therefore, there is an urgent need for a photoelectric biosensor that is simple to operate and assemble and can improve the sensitivity of protein kinase activity detection and a preparation method thereof. Summary of the Invention

[0005] In view of the shortcomings and deficiencies in the prior art, the present invention provides a photoelectric biosensor based on integrated electrodes and a preparation method thereof, which has high sensitivity in detecting protein kinase activity, a simple preparation method, low cost, a wide range of applications and strong practicality.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: The method for preparing a photoelectric biosensor based on an integrated electrode provided by the present invention comprises the following steps:

[0007] S1. Preparation of kemptide-modified integrated printed electrodes;

[0008] S2. A buffer solution containing protein kinase and ATP is added to the electrode for reaction, and then the Au@MXenes / BiOCl probe solution is added to the electrode. After the reaction is complete, a photoelectric biosensor based on an integrated electrode is prepared.

[0009] Preferably, the reaction temperature when the buffer solution and the Au@MXenes / BiOCl probe solution are added dropwise to the electrode is 36° C.-37.5° C., and the reaction time is 60 min-120 min.

[0010] Preferably, in step S1, the step of preparing the kemptide-modified integrated printed electrode comprises the following:

[0011] S101. The chitosan-modified TiO2 solution is added dropwise to the working electrode surface of the integrated printed electrode and dried;

[0012] S102. Adding a glutaraldehyde solution dropwise to the electrode prepared in step S101 to carry out a cross-linking reaction, and then continuing to add a kemptide solution dropwise to the electrode to carry out a reaction so that the kemptide is connected to the electrode;

[0013] S103. After the electrode after the reaction in step S102 is cleaned and dried, an integrated printed electrode modified with kemptide is obtained;

[0014] The mass concentration of the chitosan-modified TiO2 solution is 0.2 mg / mL-4 mg / mL, the molar concentration of the glutaraldehyde solution is 4 mM-6 mM, and the molar concentration of the kemptide solution is 400 μM-600 μM.

[0015] Preferably, the preparation method of the Au@MXenes / BiOCl probe solution in step S2 includes the following:

[0016] S201. Preparation of Ti3C2 MXenes nanosheets:

[0017] Ti3AlC2 was added to a 48% HF solution, the reaction was stirred thoroughly, the solution was centrifuged and washed, and the resulting precipitate was dried to obtain layered Ti3C2 MXenes nanosheets. The mass / volume ratio of Ti3AlC2 to HF was 0.1 g-1 g:5 mL-15 mL.

[0018] S202. Preparation of BiOCl:

[0019] Bismuth nitrate and polydiallyldimethylammonium chloride were added to ethylene glycol, heated for reaction, cooled, centrifuged, washed, and the resulting precipitate was dried to obtain BiOCl, wherein the addition ratio of bismuth nitrate, polydiallyldimethylammonium chloride, and ethylene glycol was 0.196 g:9.6 mL:160 mL;

[0020] S203. Ultrasonic mixing of a Ti3C2 MXenes solution having a mass concentration of 0.15 mg / mL-0.25 mg / mL and a BiOCl solution having a mass concentration of 10 μg / mL-12 μg / mL is performed to prepare Ti3C2 MXenes / BiOCl, wherein the volume ratio of the Ti3C2 MXenes solution to the BiOCl solution is 1:5-7;

[0021] S204. The Ti3C2 MXenes / BiOCl solution synthesized above is added to a 0.01%-0.02% w / v chloroauric acid solution, and the mixture is stirred for reaction to obtain Au@MXenes / BiOCl. The volume ratio of the Ti3C2 MXenes / BiOCl solution to the chloroauric acid solution is 1:10-12.

[0022] Preferably, the volume ratio of the kemptide solution and the Au@MXenes / BiOCl probe solution added dropwise onto the electrode is 1:1.

[0023] Preferably, the time for the cross-linking reaction of adding glutaraldehyde solution to the electrode in S102 is 1 h-1.2 h; after adding the kemptide solution to the electrode, the electrode is placed in the dark to react for 8 h-14 h to connect the kemptide to the electrode.

[0024] Preferably, the reaction conditions in S202 are: heating under reflux at 190°C-210°C for 1.5h-2h.

[0025] Preferably, before adding the buffer solution to the electrode, mercaptoethanol with a molar concentration of 0.8 mM-1.2 mM is added to the electrode for 25 min-35 min to block the blank sites, and the Au@MXenes / BiOCl probe solution is added after cleaning the electrode.

[0026] Preferably, the buffer solution has a molar concentration of 0.005 U mL -1 -0.5U mL -1 The invention relates to a novel lysine-based protein kinase, 145 μM-155 μM ATP, 48 mM-53 mM Tris-HCl and 19 mM-21 mM MgCl2, wherein the pH value of the buffer solution is 7.3-7.5.

[0027] The photoelectric biosensor based on integrated electrodes is prepared by any of the above methods for preparing the photoelectric biosensor based on integrated electrodes.

[0028] Beneficial effects of the present invention:

[0029] (1) The photoelectric biosensor based on integrated electrodes and its preparation method of the present invention have simple preparation and assembly process, strong operability, wide application range, and ensure the sensitivity of detection while simplifying the experimental process and time, thereby improving work efficiency.

[0030] The present invention uses Ti3C2 MXenes to construct the probe. Ti3C2 MXenes has the metallic conductivity of transition metal carbides, and also has a large specific surface area and biocompatibility. Its surface contains a complete metal atomic layer - Ti 2+ The present invention utilizes the reducing property of Ti3C2 MXenes to reduce chloroauric acid into gold nanoparticles to obtain Au@MXenes.

[0031] The probe also includes BiOCl. By constructing a BiOCl heterostructure and using PDDA to impart a positive charge to the BiOCl, the probe's charge transport properties are modulated, increasing the current signal and achieving a high signal-to-noise ratio. The probe prepared in this invention exhibits high photoelectric conversion efficiency, combining the charge separation properties of BiOCl with the charge extraction and transport capabilities of Au@MXenes. The Au@MXenes / BiOCl structure is used as a photoresponsive probe for the detection of protein kinase activity, effectively improving the sensitivity of protein kinase activity detection.

[0032] (2) The Au@MXenes / BiOCl probe of the present invention is modified to the phosphorylated kemptide on the electrode through the complexation of a large amount of Ti on its surface with the phosphate on the kemptide. This design simplifies the detection process and time while effectively ensuring the sensitivity of the detection.

[0033] (3) Under the excitation of visible light, the Au in the Au@MXenes / BiOCl probe of the present invention generates free electrons due to localized plasma, and at the same time, the MXenes and BiOCl semiconductors generate photoexcited electrons. These two parts of electrons jump to the electrode surface to generate photocurrent, which further increases the current generated by the probe under visible light and effectively improves the detection sensitivity and linear range.

[0034] When protein kinase activity is high, the degree of kemptide phosphorylation increases, which in turn increases the number of Au@MXenes / BiOCl probes and the photocurrent. Conversely, when protein kinase activity is low, the number of linked probes decreases, resulting in a lower photocurrent under visible light. This change in photocurrent can be used to determine the activity of the protein kinase.

[0035] (4) The integrated electrode-based photoelectric biosensor of the present invention uses an integrated printed electrode. The use of the integrated printed electrode does not require complicated processing, simplifies the detection steps, reduces the detection cost, and has strong operability while ensuring excellent linear range and sensitivity of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a reaction principle diagram of the photoelectric biosensor of the present invention;

[0037] Figure 2 Scanning and element distribution electron microscopy images of the Au@MXenes / BiOCl probe of the present invention;

[0038] Figure 3 The photoelectric biosensor of the present invention is a graph showing the photocurrent change results and a linear curve graph corresponding to different concentrations of protein kinase PKA activity. In the figure, the inset is a linear curve graph;

[0039] Figure 4 This is a graph showing the current results of the photoelectric biosensor of the present invention detecting the activity of protein kinases in different tumor cells;

[0040] Figure 5 These are graphs showing the photocurrent changes generated by a photoelectric biosensor formed by the action of different probes on the protein kinase PKA at the same concentration. In the figure, a is a graph showing the current changes generated by the action of the Au@MXenes / BiOCl probe prepared in Example 1 of the present invention, and b is a graph showing the current changes generated by the action of the Au@MXenes probe prepared in Comparative Example 1. DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0042] Protein kinase PKA, cysteine-terminated kemptide (CLRRASLG), and ATP used in the examples of the present invention were purchased from Sangon Biotech (Shanghai) Co., Ltd.; HAuCl4·3H2O (48% w / w) and Ti3AlC2 (98%) were purchased from Forsman Technology (Beijing) Co., Ltd.; and other reagents such as ZrCl4 were purchased from MacLean Reagent Company.

[0043] The light source used in the optoelectronic system of the present invention is a xenon lamp, and a visible light filter with a wavelength of 460 nm or more is used. The optoelectronic experiments were performed on a CHI660E electrochemical workstation (Shanghai Chenhua). Disposable integrated printed electrodes were used: an Ag / AgCl reference electrode, a platinum wire electrode, and a carbon working electrode. The reaction solution was a PBS phosphate solution containing 0.1 M ascorbic acid as an electron donor. Scanning electron microscopy images were obtained using a Hitachi SU8010 (Japan). Example 1

[0044] Preparation of photoelectric biosensors based on integrated electrodes, the specific steps include the following:

[0045] 1. Preparation of Kemptide-modified Integrated Printed Electrodes

[0046] (1) A TiO2 solution modified with chitosan (TiO2 / CS-NH2) with a mass concentration of 2 mg / mL was added dropwise to the working electrode surface of the integrated printed electrode and dried.

[0047] (2) A 5 mM glutaraldehyde solution was added to the electrode for a cross-linking reaction for 1 hour. Then, 20 μL of a 500 μM kemptide solution was added to the electrode. After the addition, the electrode was placed in a dark place at room temperature for 12 hours to allow the kemptide to connect to the electrode.

[0048] (3) The electrode was washed with water and then dried with nitrogen to obtain a kemptide-modified electrode.

[0049] Most existing electrodes use ITO conductive glass, glassy carbon, or gold electrodes as working electrodes. While these electrodes achieve relatively good detection results, they still require washing and drying with strong alkali, ethanol, acetone, or other cleaning methods before use, or require lengthy polishing and rinsing before continuing with the sensor assembly process. Furthermore, counter and reference electrodes are required for testing, and the volume of electrolyte solution used is relatively large. The present invention uses integrated printed electrodes as working electrodes, simplifying the electrode processing steps while maintaining detection results, effectively improving efficiency and reducing costs.

[0050] 2. Preparation of Au@MXenes / BiOCl Probes

[0051] (1) Preparation of Ti3C2 MXenes nanosheets:

[0052] 1g of Ti3AlC2 powder was added to 15mL of 48% HF and stirred at 45°C for 24h. After the reaction, the solution was centrifuged at 5000 rpm and washed. The resulting precipitate was dried at room temperature to obtain layered Ti3C2MXene nanosheets.

[0053] (2) Preparation of BiOCl:

[0054] 196 mg of bismuth nitrate and 10 mL of polydiallyldimethylammonium chloride (PDDA, MW: 300,000-400,000) were added to 160 mL of ethylene glycol, refluxed at 200°C for 2 h, cooled to room temperature, centrifuged, and washed with water several times. The resulting precipitate was dried to obtain solid BiOCl.

[0055] (3) Preparation of Ti3C2 MXenes / BiOCl probe:

[0056] 5 mL of Ti3C2 MXenes solution with a mass concentration of 0.2 mg / mL was mixed with 25 mL of BiOCl solution with a concentration of 11 μg / mL by ultrasonic vibration for 2 h.

[0057] 10 mL of the MXenes / BiOCl aqueous solution synthesized above was added to 100 mL of 0.01% w / v chloroauric acid solution and stirred for 1 min to stop the reaction to obtain the Au@MXenes / BiOCl probe solution.

[0058] MXenes are a type of metal carbon / nitride with a two-dimensional layered structure, with hydroxyl groups or terminal oxygen on the surface. They have the metallic conductivity of transition metal carbides, and at the same time have a large specific surface area and biocompatibility. Among MXenes, Ti3C2 MXenes is the most common and applied, and its surface contains a complete metal atomic layer - Ti 2+ , and a rich array of functional groups such as hydroxyl, oxygen, and fluorine, making them suitable for photoelectrochemical sensing applications. This invention utilizes the reducing properties of Ti3C2 MXenes to reduce chloroauric acid to gold nanoparticles, yielding the Au@MXenes material. Furthermore, the Ti on its surface is used to link phosphates produced by kinase-catalyzed protein phosphorylation. This design simplifies the detection process and time while ensuring detection sensitivity.

[0059] BiOCl is an important ternary bismuth-based photoelectric material with a wide band gap and a stable layered structure. It is a typical p-type semiconductor that can absorb ultraviolet light to a large extent and achieve charge separation. It is used as a photocatalyst in photocatalytic water decomposition, gas reduction, pollutant degradation and other photoelectric fields. Although BiOCl is widely used in the field of energy catalysis, its application in the field of sensing probes has rarely been effectively utilized, which is mainly limited by the high resistance induced by the wide band gap. Therefore, external substances are needed to improve the conductivity, increase the current signal, and achieve a high signal-to-noise ratio. The present invention regulates the charge transfer performance of BiOCl by constructing a BiOCl heterostructure. The Au@MXenes / BiOCl probe of the present invention integrates the charge separation properties of BiOCl and the charge extraction and transfer capabilities of Au@MXenes, and uses Au@MXenes / BiOCl as a light-responsive probe for the detection of protein kinases.

[0060] 3. Assembling the Photoelectric Biosensor

[0061] The blank sites of the electrode were blocked with 1 mM mercaptoethanol for 30 min to reduce nonspecific adsorption. The electrode was cleaned and a buffer solution containing PKA and ATP (0.5 U mL -1 Protein kinase, 150 μM ATP, 50 mM Tris-HCl, 20 mM MgCl2, pH 7.4) was added dropwise to the electrode. After reacting at 37°C, 20 μL of Au@MXenes / BiOCl probe solution was added dropwise and reacted for 100 minutes. After the reaction was complete, it was washed with buffer solution and dried with nitrogen to obtain the prepared photoelectric biosensor.

[0062] In the presence of ATP and magnesium ions, protein kinases catalyze the replacement of the hydroxyl groups on kemptide with the phosphate groups in ATP, leading to phosphorylation and formation of phosphate radicals. The Au@MXenes / BiOCl probe of the present invention complexes the phosphate radicals on the kemptide peptide through the large amount of titanium present on its surface, allowing the Au@MXenes / BiOCl probe to be modified onto the phosphorylated kemptide peptide on the electrode. This design simplifies the detection process and time, while effectively ensuring detection sensitivity.

[0063] Figure 2 Scanning electron microscopy image of the Au@MXenes / BiOCl probe prepared in Example 1. Example 2

[0064] Preparation of photoelectric biosensors based on integrated electrodes, the specific steps include the following:

[0065] 1. Preparation of Kemptide-modified Integrated Printed Electrodes

[0066] (1) A TiO2 solution modified with chitosan (TiO2 / CS-NH2) with a mass concentration of 0.2 mg / mL was added dropwise to the working electrode surface of the integrated printed electrode and dried.

[0067] (2) A 4 mM glutaraldehyde solution was added dropwise to the electrode for a cross-linking reaction for 1 hour. Then, 20 μL of a 400 μM kemptide solution was added dropwise to the electrode. After the addition, the electrode was placed in a dark place at room temperature for a reaction of 8 hours to allow the kemptide to connect to the electrode.

[0068] (3) The electrode was washed with water and then dried with nitrogen to obtain a kemptide-modified electrode.

[0069] 2. Preparation of Au@MXenes / BiOCl Probes

[0070] (1) Preparation of Ti3C2 MXenes nanosheets:

[0071] 0.2 g of Ti3AlC2 powder was added to 5 mL of 48% HF and stirred at 45°C for 24 hours. After the reaction, the solution was centrifuged at 5000 rpm and washed. The resulting precipitate was dried at room temperature to obtain layered Ti3C2 MXene nanosheets.

[0072] (2) Preparation of BiOCl:

[0073] 196 mg of bismuth nitrate and 10 mL of polydiallyldimethylammonium chloride (PDDA, MW: 300,000-400,000) were added to 160 mL of ethylene glycol, refluxed at 190°C for 1.5 h, cooled to room temperature, centrifuged, and washed with water several times. The resulting precipitate was dried to obtain solid BiOCl.

[0074] (3) Preparation of Ti3C2 MXenes / BiOCl probe:

[0075] 5 mL of Ti3C2 MXenes solution with a mass concentration of 0.25 mg / mL was mixed with 25 mL of BiOCl solution with a concentration of 10 μg / mL by ultrasonic vibration for 2 h.

[0076] 10 mL of the MXenes / BiOCl aqueous solution synthesized above was added to 100 mL of 0.02% w / v chloroauric acid solution and stirred for 1 min to stop the reaction to obtain the Au@MXenes / BiOCl probe solution.

[0077] 3. Assembling the Photoelectric Biosensor

[0078] The blank sites of the electrode were blocked with 0.8 mM mercaptoethanol for 25 min to reduce nonspecific adsorption. The electrode was cleaned and a buffer solution containing PKA and ATP (0.01 U mL -1 Protein kinase, 145 μM ATP, 48 mM Tris-HCl, 19 mM MgCl2, pH 7.3) was added dropwise to the electrode. After reacting at 36°C, 20 μL of Au@MXenes / BiOCl probe solution was added dropwise and reacted for 70 minutes. After the reaction was complete, it was washed with buffer solution and dried with nitrogen to obtain the prepared photoelectric biosensor. Example 3

[0079] Preparation of photoelectric biosensors based on integrated electrodes, the specific steps include the following:

[0080] 1. Preparation of Kemptide-modified Integrated Printed Electrodes

[0081] (1) A TiO2 solution modified with chitosan (TiO2 / CS-NH2) with a mass concentration of 0.3 mg / mL was added dropwise to the working electrode surface of the integrated printed electrode and dried.

[0082] (2) A 6 mM glutaraldehyde solution was added dropwise to the electrode for a cross-linking reaction for 1 hour. Then, 20 μL of a 600 μM kemptide solution was added dropwise to the electrode. After the addition, the electrode was placed in a dark place at room temperature for a reaction of 14 hours to allow the kemptide to connect to the electrode.

[0083] (3) The electrode was washed with water and then dried with nitrogen to obtain a kemptide-modified electrode.

[0084] 2. Preparation of Au@MXenes / BiOCl Probes

[0085] (1) Preparation of Ti3C2 MXenes nanosheets:

[0086] 0.5 g of Ti3AlC2 powder was added to 10 mL of 48% HF and stirred at 45°C for 24 hours. After the reaction, the solution was centrifuged at 5000 rpm and washed. The resulting precipitate was dried at room temperature to obtain layered Ti3C2 MXene nanosheets.

[0087] (2) Preparation of BiOCl:

[0088] 196 mg of bismuth nitrate and 10 mL of polydiallyldimethylammonium chloride (PDDA, MW: 300,000-400,000) were added to 160 mL of ethylene glycol, refluxed at 210°C for 2 h, cooled to room temperature, centrifuged, and washed with water several times. The resulting precipitate was dried to obtain solid BiOCl.

[0089] (3) Preparation of Ti3C2 MXenes / BiOCl probe:

[0090] 5 mL of Ti3C2 MXenes solution with a mass concentration of 0.25 mg / mL was mixed with 25 mL of BiOCl solution with a concentration of 11 μg / mL by ultrasonic vibration for 2 h.

[0091] 10 mL of the synthesized MXenes / BiOCl aqueous solution was added to 100 mL of 0.01% w / v chloroauric acid solution and stirred for 1 min to stop the reaction to obtain the Au@MXenes / BiOCl probe solution.

[0092] 3. Assembling the Photoelectric Biosensor

[0093] The blank sites of the electrode were blocked with 1.2 mM mercaptoethanol for 35 min to reduce nonspecific adsorption. The electrode was cleaned and a buffer solution containing PKA and ATP (0.03 U mL -1 Protein kinase, 155 μM ATP, 52 mM Tris-HCl, 20 mM MgCl2, pH 7.5) was added dropwise to the electrode, and after reacting at 37.5°C, 20 μL of Au@MXenes / BiOCl probe solution was added dropwise and reacted for 80 minutes. After the reaction was complete, it was washed with buffer solution and dried with nitrogen to obtain the prepared photoelectric biosensor. Example 4

[0094] Preparation of photoelectric biosensors based on integrated electrodes, the specific steps include the following:

[0095] 1. Preparation of Kemptide-modified Integrated Printed Electrodes

[0096] (1) A TiO2 solution modified with chitosan (TiO2 / CS-NH2) with a mass concentration of 3 mg / mL was added dropwise to the working electrode surface of the integrated printed electrode and dried.

[0097] (2) A 5 mM glutaraldehyde solution was added to the electrode for a cross-linking reaction for 1 hour. Then, 20 μL of a 500 μM kemptide solution was added to the electrode. After the addition, the electrode was placed in a dark place at room temperature for 12 hours to allow the kemptide to connect to the electrode.

[0098] (3) The electrode was washed with water and then dried with nitrogen to obtain a kemptide-modified electrode.

[0099] 2. Preparation of Au@MXenes / BiOCl Probes

[0100] (1) Preparation of Ti3C2 MXenes nanosheets:

[0101] 0.8 g of Ti3AlC2 powder was added to 12 mL of 48% HF and stirred at 45°C for 24 hours. After the reaction, the solution was centrifuged at 5000 rpm and washed. The resulting precipitate was dried at room temperature to obtain layered Ti3C2 MXene nanosheets.

[0102] (2) Preparation of BiOCl:

[0103] 196 mg of bismuth nitrate and 10 mL of polydiallyldimethylammonium chloride (PDDA, MW: 300,000-400,000) were added to 160 mL of ethylene glycol, refluxed at 200°C for 1.5 h, cooled to room temperature, centrifuged, and washed with water several times. The resulting precipitate was dried to obtain solid BiOCl.

[0104] (3) Preparation of Ti3C2 MXenes / BiOCl probe:

[0105] 5 mL of Ti3C2 MXenes solution with a mass concentration of 0.2 mg / mL was mixed with 35 mL of BiOCl solution with a concentration of 12 μg / mL by ultrasonic vibration for 2 h.

[0106] 10 mL of the MXenes / BiOCl aqueous solution synthesized above was added to 120 mL of 0.01% w / v chloroauric acid solution and stirred for 1 min to stop the reaction to obtain the Au@MXenes / BiOCl probe solution.

[0107] 3. Assembling the Photoelectric Biosensor

[0108] The blank sites of the electrode were blocked with 1 mM mercaptoethanol for 30 min to reduce nonspecific adsorption. The electrode was cleaned and a buffer solution containing PKA and ATP (0.3 U mL -1 Protein kinase, 150 μM ATP, 50 mM Tris-HCl, 20 mM MgCl2, pH 7.4) was added dropwise to the electrode. After reacting at 37°C, 20 μL of Au@MXenes / BiOCl probe solution was added dropwise and reacted for 100 minutes. After the reaction was complete, it was washed with buffer solution and dried with nitrogen to obtain the prepared photoelectric biosensor. Example 5

[0109] Preparation of photoelectric biosensors based on integrated electrodes, the specific steps include the following:

[0110] 1. Preparation of Kemptide-modified Integrated Printed Electrodes

[0111] (1) A TiO2 solution modified with chitosan (TiO2 / CS-NH2) with a mass concentration of 4 mg / mL was added dropwise to the working electrode surface of the integrated printed electrode and dried.

[0112] (2) A 5 mM glutaraldehyde solution was added to the electrode for a cross-linking reaction for 1 hour. Then, 20 μL of a 500 μM kemptide solution was added to the electrode. After the addition, the electrode was placed in a dark place at room temperature for a reaction of 10 hours to allow the kemptide to connect to the electrode.

[0113] (3) The electrode was washed with water and then dried with nitrogen to obtain a kemptide-modified electrode.

[0114] 2. Preparation of Au@MXenes / BiOCl Probes

[0115] (1) Preparation of Ti3C2 MXenes nanosheets:

[0116] 1g of Ti3AlC2 powder was added to 15mL of 48% HF and stirred at 45°C for 24h. After the reaction, the solution was centrifuged at 5000 rpm and washed. The resulting precipitate was dried at room temperature to obtain layered Ti3C2MXene nanosheets.

[0117] (2) Preparation of BiOCl:

[0118] 196 mg of bismuth nitrate and 10 mL of polydiallyldimethylammonium chloride (PDDA, MW: 300,000-400,000) were added to 160 mL of ethylene glycol, refluxed at 210°C for 2 h, cooled to room temperature, centrifuged, and washed with water several times. The resulting precipitate was dried to obtain solid BiOCl.

[0119] (3) Preparation of Ti3C2 MXenes / BiOCl probe:

[0120] 5 mL of Ti3C2 MXenes solution with a mass concentration of 0.2 mg / mL was mixed with 30 mL of BiOCl solution with a concentration of 11 μg / mL by ultrasonic vibration for 2 h.

[0121] 10 mL of the MXenes / BiOCl aqueous solution synthesized above was added to 110 mL of 0.02% w / v chloroauric acid solution and stirred for 1 min to stop the reaction to obtain the Au@MXenes / BiOCl probe solution.

[0122] 3. Assembling the Photoelectric Biosensor

[0123] The blank sites of the electrode were blocked with 1 mM mercaptoethanol for 30 min to reduce nonspecific adsorption. The electrode was cleaned and a buffer solution containing PKA and ATP (0.5 U mL -1 Protein kinase, 150 μM ATP, 50 mM Tris-HCl, 20 mM MgCl2, pH 7.4) was added dropwise to the electrode. After reacting at 37.5°C, 20 μL of Au@MXenes / BiOCl probe solution was added dropwise and reacted for 100 minutes. After the reaction was complete, it was washed with buffer solution and dried with nitrogen to obtain the prepared photoelectric biosensor. Comparative Example 1

[0124] The preparation of the photoelectric biosensor includes the following steps:

[0125] 1. Preparation of Kemptide-modified Integrated Printed Electrodes

[0126] (1) A TiO2 solution modified with chitosan (TiO2 / CS-NH2) with a mass concentration of 2 mg / mL was added dropwise to the working electrode surface of the integrated printed electrode and dried.

[0127] (2) A 5 mM glutaraldehyde solution was added to the electrode for a cross-linking reaction for 1 hour. Then, 20 μL of a 500 μM kemptide solution was added to the electrode. After the addition, the electrode was placed in a dark place at room temperature for 12 hours to allow the kemptide to connect to the electrode.

[0128] (3) The electrode was washed with water and then dried with nitrogen to obtain a kemptide-modified electrode.

[0129] 2. Preparation of Au@MXenes Probes

[0130] 1g of Ti3AlC2 powder was added to 15mL of 48% HF and stirred at 45°C for 24h. After the reaction, the solution was centrifuged at 5000 rpm and washed. The resulting precipitate was dried at room temperature to obtain layered Ti3C2MXene nanosheets.

[0131] 10 mL of 0.2 mg / mL Ti3C2 MXenes solution was added to 110 mL of 0.02% w / v chloroauric acid solution and stirred for 1 min to stop the reaction to obtain the Au@MXenes probe solution.

[0132] 3. Assembling the Photoelectric Biosensor

[0133] The blank sites of the electrode were blocked with 1 mM mercaptoethanol for 30 min to reduce nonspecific adsorption. The electrode was cleaned and a buffer solution containing PKA and ATP (0.5 U mL-1 Protein kinase, 150 μM ATP, 50 mM Tris-HCl, 20 mM MgCl2, pH 7.4) was added dropwise to the electrode, and after reacting at 37°C, 20 μL of MXenes probe solution was added dropwise for 100 minutes. After the reaction was complete, it was washed with buffer solution and dried with nitrogen to obtain the prepared photoelectric biosensor.

[0134] Next, the performance of the photoelectric biosensor prepared by the present invention was tested.

[0135] Test Example 1

[0136] According to the preparation method of Example 1, buffer solutions containing different concentrations of PKA kinase were added dropwise to the electrodes to prepare biosensors, and detection was performed under visible light to obtain photocurrents corresponding to different concentrations of PKA kinase. Figure 3 As shown in Figure 2, with the increase of PKA concentration, the photocurrent gradually increased. When the concentration of PKA was 0.005-0.5 U mL -1 The detected photocurrent is linearly related to the activity of PKA, and the linear equation is I=238.98×logc PKA +755.28, correlation coefficient R=0.9976, where I is the photocurrent intensity and c is the activity of protein kinase PKA.

[0137] according to Figure 1 It can be seen that the Au@MXenes / BiOCl probe of the present invention is modified to the phosphorylated kemptide on the electrode through the complexation of a large amount of Ti on its surface with the phosphate group on the kemptide. Under the excitation of visible light, the Au in the Au@MXenes / BiOCl probe generates free electrons due to localized plasma, and at the same time, the MXenes and BiOCl semiconductors generate photoexcited electrons. These two parts of electrons transition to the electrode surface to generate photocurrent, further increasing the current generated by the probe under visible light, improving the sensitivity and linear range of detection. When the activity of protein kinase is high, the degree of phosphorylation of kemptide is high, thereby increasing the number of Au@MXenes / BiOCl probes, and then increasing the photocurrent. Conversely, when the activity of protein kinase is low, the number of probe links decreases, thereby reducing the photocurrent generated under visible light. In this way, the size of protein kinase activity can be judged by the change in photocurrent, indicating that the photoelectric biosensor of the present invention has good detection sensitivity for PKA kinase.

[0138] Test Example 2

[0139] Visible light response photocurrent performance test

[0140] The currents of the photoelectric biosensors prepared in Example 1 of the present invention and Comparative Example 1 were respectively detected under visible light. The results are as follows Figure 5 As shown in the figure, a is a current change diagram generated by the Au@MXenes / BiOCl probe prepared in Example 1 of the present invention, and b is a current change diagram generated by the Au@MXenes probe prepared in Comparative Example 1. Since the concentrations of PKA and ATP in the buffer solution used in Comparative Example 1 and Example 1 of the present invention are the same, according to the detected current diagram, it can be seen that the Au@MXenes / BiOCl probe prepared by the present invention generates a large photoelectric flux and has a higher detection sensitivity. Under the excitation of visible light, the Au in the probe of the photoelectric biosensor of the present invention generates free electrons due to localized plasma, and at the same time, MXenes and BiOCl semiconductors work synergistically to generate photoexcited electrons. These two parts of electrons jointly jump to the electrode surface to generate a larger photocurrent, making the detection sensitivity stronger.

[0141] Test Example 3

[0142] The buffer solution of PKA kinase in SW480, SW620 and RKO tumor cells was added dropwise to the photoelectric biosensor electrode of the present invention for detection. The results are as follows: Figure 4 As shown, the photoelectric biosensor of the present invention can effectively detect the activity of PKA kinase in different tumor cells, and has a wide range of applications and strong practicality.

[0143] In summary, the photoelectric biosensor based on integrated electrodes and the preparation method thereof of the present invention have a simple preparation and assembly process, strong operability, a wide range of applications, ensure the sensitivity of detection, and at the same time simplify the experimental process and time, thereby improving work efficiency.

[0144] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A method for preparing a photoelectric biosensor based on an integrated electrode, characterized in that: The following steps are involved: S1. Preparation of kemptide-modified integrated printed electrodes; S2. A buffer solution containing protein kinase and ATP is added dropwise to the electrode for reaction. Then, an Au@MXenes / BiOCl probe solution is added dropwise to the electrode. After the reaction is complete, a photoelectric biosensor based on an integrated electrode is prepared. In step S1, the steps of preparing the kemptide-modified integrated printed electrode include the following: S101. The chitosan-modified TiO2 solution is added dropwise to the working electrode surface of the integrated printed electrode and dried; S102. Adding a glutaraldehyde solution dropwise to the electrode prepared in step S101 to carry out a cross-linking reaction, and then continuing to add a kemptide solution dropwise to the electrode to carry out a reaction so that the kemptide is connected to the electrode; S103. After the electrode after the reaction in step S102 is cleaned and dried, an integrated printed electrode modified with kemptide is obtained; The mass concentration of the chitosan-modified TiO2 solution is 0.2 mg / mL-4 mg / mL, the molar concentration of the glutaraldehyde solution is 4 mM-6 mM, and the molar concentration of the kemptide solution is 400 μM-600 μM. The preparation method of the Au@MXenes / BiOCl probe solution in step S2 includes the following: S201. Preparation of Ti3C2 MXenes nanosheets: Ti3AlC2 was added to a 48% HF solution, the reaction was stirred thoroughly, the solution was centrifuged and washed, and the resulting precipitate was dried to obtain layered Ti3C2 MXenes nanosheets. The mass / volume ratio of Ti3AlC2 to HF was 0.1 g-1 g:5 mL-15 mL. S202. Preparation of BiOCl: Bismuth nitrate and polydiallyldimethylammonium chloride were added to ethylene glycol, heated for reaction, cooled, centrifuged, washed, and the resulting precipitate was dried to obtain BiOCl, wherein the addition ratio of bismuth nitrate, polydiallyldimethylammonium chloride, and ethylene glycol was 0.196 g:9.6 mL:160 mL; S203. Ultrasonic mixing of a Ti3C2 MXenes solution having a mass concentration of 0.15 mg / mL-0.25 mg / mL and a BiOCl solution having a mass concentration of 10 μg / mL-12 μg / mL is performed to prepare Ti3C2 MXenes / BiOCl, wherein the volume ratio of the Ti3C2 MXenes solution to the BiOCl solution is 1:5-7; S204. The Ti3C2 MXenes / BiOCl solution synthesized above is added to a 0.01%-0.02% w / v chloroauric acid solution, and the mixture is stirred for reaction to obtain Au@MXenes / BiOCl. The volume ratio of the Ti3C2 MXenes / BiOCl solution to the chloroauric acid solution is 1:10-12.

2. The method for preparing a photoelectric biosensor based on an integrated electrode according to claim 1, wherein: The reaction temperature when the buffer solution and the Au@MXenes / BiOCl probe solution are added dropwise to the electrode is 36° C.-37.5° C., and the reaction time is 60 min-120 min.

3. The method for preparing a photoelectric biosensor based on an integrated electrode according to claim 1, wherein: The volume ratio of the kemptide solution and the Au@MXenes / BiOCl probe solution added dropwise onto the electrode was 1:

1.

4. The method for preparing a photoelectric biosensor based on an integrated electrode according to claim 1, wherein: In S102, the glutaraldehyde solution is added dropwise to the electrode for a cross-linking reaction for 1 hour to 1.2 hours. After the kemptide solution is added dropwise to the electrode, the electrode is placed in a dark place for a reaction of 8 hours to 14 hours to allow the kemptide to connect to the electrode.

5. The method for preparing a photoelectric biosensor based on an integrated electrode according to claim 1, wherein: The reaction conditions in S202 are: heating under reflux at 190°C-210°C for 1.5h-2h.

6. The method for preparing a photoelectric biosensor based on an integrated electrode according to claim 1, wherein: Before adding the buffer solution to the electrode, mercaptoethanol with a molar concentration of 0.8 mM-1.2 mM was added to the electrode for 25 min-35 min to block the blank sites. After cleaning the electrode, the Au@MXenes / BiOCl probe solution was added.

7. The method for preparing a photoelectric biosensor based on an integrated electrode according to claim 1, wherein: The buffer solution has a molar concentration of 0.005 U mL -1 -0.5U mL -1 The invention relates to a novel lysine-based protein kinase, 145 μM-155 μM ATP, 48 mM-53 mM Tris-HCl and 19 mM-21 mM MgCl2, wherein the pH value of the buffer solution is 7.3-7.

5.

8. The photoelectric biosensor based on an integrated electrode prepared according to the method for preparing a photoelectric biosensor based on an integrated electrode according to any one of claims 1 to 7.

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