Method for detecting biological enzyme activity by using magnetic field enhanced photoelectrochemical sensor

Abstract

A method for detecting the activity of biological enzymes using a magnetic field-enhanced photoelectrochemical sensor includes the following steps: preparing a conductive electrode with a coupled polypeptide; preparing an MXenes / Co-TiO2 composite probe dispersion; adding a buffer solution containing the biological enzyme and adenosine triphosphate dropwise onto the conductive electrode to perform a phosphorylation reaction; adding the MXenes / Co-TiO2 composite probe dispersion dropwise onto the conductive electrode; and, after the reaction is complete, forming a magnetic field-enhanced photoelectrochemical sensor; and performing biological enzyme activity detection using a three-electrode system under light and magnetic field conditions, wherein the irradiation intensity at the magnetic field-enhanced photoelectrochemical sensor is 190 mW / cm². 2 The magnetic induction intensity at the magnetic field-enhanced photoelectrochemical sensor is 80~90mT.

Description

Technical Field

[0001] This disclosure relates to the fields of photoelectric sensing and biochemical analysis, and more specifically, to a method for detecting the activity of biological enzymes using a magnetic field-enhanced photoelectrochemical sensor. Background Technology

[0002] As a type of biological enzyme, protein kinases play a crucial role in cell signaling and regulation. By catalyzing the phosphorylation of target proteins, they regulate various cellular functions, influencing processes such as cell growth, proliferation, differentiation, and metabolism. Due to their critical role in cellular function, dysregulation of protein kinases is closely associated with a variety of diseases, including cancer, cardiovascular disease, metabolic disorders, and neurological diseases. With a deeper understanding of the mechanisms of protein kinase signaling, researchers are exploring the use of advanced technologies such as biosensors to detect and monitor kinase activity. These technological advances provide promising tools for studying the dynamic changes in cell signaling and may facilitate the development of new drugs for the treatment of related diseases.

[0003] In the detection of biological enzyme activity, traditional detection techniques such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), mass spectrometry, and fluorescent labeling have been widely used for the detection of protein kinase activity. While these techniques each have their own advantages, they also have limitations, such as high cost, experimental complexity, insufficient detection sensitivity, and the use of harmful radioactive labels. Summary of the Invention

[0004] This disclosure provides a method for detecting the activity of biological enzymes using a magnetic field-enhanced photoelectrochemical sensor, the method comprising the following steps:

[0005] Prepare a conductive electrode for coupling peptides;

[0006] Preparation of MXenes / Co-TiO2 composite probe dispersion;

[0007] A buffer solution containing biological enzymes and adenosine triphosphate is dropped onto the conductive electrode to carry out a phosphorylation reaction. The MXenes / Co-TiO2 composite probe dispersion is then dropped onto the conductive electrode. After the reaction is complete, a magnetic field-enhanced photoelectrochemical sensor is formed.

[0008] The detection of biological enzyme activity was performed using a three-electrode system under illumination and magnetic field conditions, wherein the irradiation intensity at the magnetic field-enhanced photoelectrochemical sensor was 190 mW / cm². 2 The magnetic induction intensity at the magnetic field-enhanced photoelectrochemical sensor is 80~90mT.

[0009] In some embodiments, the polypeptide includes a kemp peptide, and the preparation of the conductive electrode for the coupled polypeptide includes the following steps:

[0010] Provide indium tin oxide conductive glass and perform pretreatment on the indium tin oxide conductive glass;

[0011] Chitosan solution was dropped onto the pretreated indium tin oxide conductive glass and then dried.

[0012] Glutaraldehyde solution and Kemppeptide solution were sequentially added to indium tin oxide conductive glass with a chitosan film to modify the indium tin oxide conductive glass.

[0013] In some embodiments, the concentration of the Kemppeptide solution is 500 mM.

[0014] In some embodiments, the preparation of the MXenes / Co-TiO2 composite probe dispersion includes the following steps:

[0015] Preparation of Ti3C2 MXenes dispersion;

[0016] Preparation of Co-TiO2 nanosheet dispersion;

[0017] Mix 1.48 mg / mL MXenes dispersion and 1 mg / mL Co-TiO2 nanosheet dispersion at a volume ratio of 10:1, stir and mix well using a mixer, and let stand to form an MXenes / Co-TiO2 composite probe dispersion.

[0018] In some embodiments, the preparation of the Ti3C2 MXenes dispersion includes the following steps:

[0019] LiF was added to a 9 mol / L HCl solution, and the mixture was stirred to obtain a mixed solution.

[0020] Ti3AlC2 powder was added to the mixed solution, the mixture was stirred thoroughly, centrifuged and washed, the precipitate was collected and dispersed in deionized water and centrifuged.

[0021] The precipitate was collected again and added to deionized water, and ultrasonic treatment was performed under nitrogen protection; and

[0022] The ultrasonically treated solution was centrifuged again, and the supernatant was retained and stored at low temperature to obtain a Ti3C2MXenes dispersion.

[0023] In some embodiments, the preparation of Co-TiO2 nanosheet dispersion includes the following steps:

[0024] TiO2, CoO, K2CO3, and Li2CO3 were mixed in stoichiometric proportions, and the mixture was subjected to a series of operations including grinding, high-temperature annealing, secondary grinding, and secondary high-temperature annealing to obtain compound K.0.8 Ti (5.2-y) / 3 Li (0.8-2y） / 3 Co y O4 precursor;

[0025] The precursor was reacted thoroughly with a 9 mol / L HCl solution, followed by washing and drying to obtain the chemical formula H. (3.2-2y) / 3 Ti (5.2-y) / 3 Co y The protonation products of O4;

[0026] The protonated product was thoroughly soaked with tetrabutylammonium hydroxide (TBAOH) at a 1:1 molar ratio to produce a product with the chemical formula TBA. z H (3.2-2y) / 3-z Ti (5.2-Y) / 3 Co y The bulk compound of O4;

[0027] The bulk compound was exfoliated in deionized water by mechanical oscillation to obtain the Co-TiO2 nanosheet dispersion.

[0028] In some embodiments, the bioenzyme includes a protein kinase, and the phosphorylation reaction is carried out by adding a buffer solution containing the bioenzyme and adenosine triphosphate to the conductive electrode:

[0029] A buffer solution containing protein kinase and adenosine triphosphate was added dropwise to the conductive electrode to carry out a phosphorylation reaction, wherein the protein kinase was used at different concentrations, the adenosine triphosphate was at a concentration of 120 μM, and the phosphorylation reaction lasted for 80 min.

[0030] In some embodiments, the detection of biological enzyme activity using a three-electrode system under light and magnetic field conditions includes:

[0031] A magnetic field-enhanced photoelectrochemical sensor in a three-electrode system is irradiated with a light source of 420 nm wavelength. At the same time, a magnetic field is set to place the magnetic field-enhanced photoelectrochemical sensor in a magnetic field environment. The magnetic field is applied using a magnetic field generator, which is placed perpendicular to the conductive electrode and 1 cm away from the conductive electrode.

[0032] In some embodiments, when the protein kinase concentration is in the range of 0.005 U / mL to 1 U / mL, the change in photocurrent intensity satisfies the following equation with respect to the protein kinase concentration:

[0033] y = 4.93 + 62.36x, where y represents the intensity of the photocurrent change and x represents the protein kinase concentration.

[0034] This disclosure provides a magnetic field-enhanced photoelectrochemical sensor for detecting biological enzyme activity, the photoelectrochemical sensor comprising:

[0035] Conductive electrodes;

[0036] A polypeptide, one end of which is coupled to the conductive electrode, and the other end of the polypeptide, away from the conductive electrode, is phosphorylated by the enzyme to be tested; and

[0037] The MXenes / Co-TiO2 composite probe binds heterotropically to the phosphorylated end of the peptide.

[0038] The magnetic field-enhanced photoelectrochemical sensor operates in a magnetic field environment to enhance the signal strength of the optical circuit.

[0039] The above-described solutions in this disclosure can have the following beneficial effects:

[0040] This disclosure employs a magnetic field-enhanced photoelectrochemical sensor constructed using a two-dimensional material-based MXenes / Co-TiO2 composite probe. Under illumination and different magnetic field conditions, a three-electrode system is used to perform biological enzyme activity detection, significantly improving the photocurrent signal and achieving ultra-high sensitivity detection of biological enzyme activity. Furthermore, the method provided in this disclosure is simple to implement and has low manufacturing cost. Attached Figure Description

[0041] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this disclosure and, together with the description, serve to explain the principles of this disclosure. It is obvious that the drawings described below are merely some embodiments of this disclosure, and those skilled in the art can obtain other drawings based on these drawings without any inventive effort. In the drawings:

[0042] Figure 1 This is a schematic diagram of the structure of a magnetic field-enhanced photoelectrochemical sensor provided in some embodiments of this disclosure;

[0043] Figure 2 Flowcharts of methods for detecting biological enzyme activity using a magnetic field-enhanced photoelectrochemical sensor provided in some embodiments of this disclosure;

[0044] Figure 3 This is a schematic flowchart of a method for detecting biological enzyme activity using a magnetic field-enhanced photoelectrochemical sensor, provided in some embodiments of this disclosure.

[0045] Figure 4 Correlation curves between different concentrations of Kemppeptide solution and the corresponding photocurrent intensity provided in some embodiments of this disclosure;

[0046] Figure 5Scanning electron microscope images of Ti3C2 MXenes provided in some embodiments of this disclosure;

[0047] Figure 6 Atomic force microscopy images of Co-TiO2 nanosheets provided in some embodiments of this disclosure;

[0048] Figure 7 Hysteresis loop curves of Co-TiO2 nanosheet dispersions provided in some embodiments of this disclosure;

[0049] Figure 8 Magnetoresistance variation curves of Co-TiO2 nanosheet dispersions provided in some embodiments of this disclosure;

[0050] Figure 9 Correlation curves between different adenosine triphosphate concentrations and corresponding photocurrent intensity provided in some embodiments of this disclosure;

[0051] Figure 10 Correlation curves between different phosphorylation times and corresponding photocurrent changes are provided for some embodiments of this disclosure;

[0052] Figure 11 This is a schematic diagram of the structure of a test system for detecting biological enzyme activity provided in some embodiments of this disclosure.

[0053] Figure 12 This disclosure provides graphs showing the relationship between the magnitude of magnetic induction intensity and the intensity of photocurrent change in some embodiments.

[0054] Figure 13 The graph shows the relationship between the concentration of protein kinase A (PKA) and the intensity of photocurrent changes provided in some embodiments of this disclosure. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of this disclosure clearer, the disclosure will be further described in detail below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. Based on the embodiments of this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0056] The terminology used in the embodiments of this disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of this disclosure. The singular forms “a,” “the,” and “the” as used in the embodiments of this disclosure and the appended claims are also intended to include the plural forms, and “multiple” generally includes at least two unless the context clearly indicates otherwise.

[0057] It should be understood that the term "and / or" used in this article is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. Additionally, the character " / " in this article generally indicates that the preceding and following related objects have an "or" relationship.

[0058] It should be understood that although the terms first, second, third, etc. may be used to describe the embodiments in this disclosure, it should not be limited to these terms.

[0059] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that an article or device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such an article or device. Without further limitation, an element defined by the phrase "comprising one" does not exclude the presence of other identical elements in the article or device that includes said element.

[0060] With the advancement of science and technology, new biosensors and detection technologies, such as photoelectrochemical biosensors, have emerged, providing more flexible and efficient detection solutions for the detection of biological enzyme activity. Photoelectrochemical methods are based on photoexcitation and electrochemical reactions for detection. Photoelectrochemical sensors possess high sensitivity, simple operation, and good cost-effectiveness, making them valuable tools in the field of biosensing and representing a promising research direction in the field of biological enzyme activity detection.

[0061] This disclosure provides a method for detecting the activity of biological enzymes using a magnetic field-enhanced photoelectrochemical sensor. The method includes the following steps: preparing a conductive electrode with coupled peptides; preparing an MXenes / Co-TiO2 composite probe dispersion; adding a buffer solution containing the biological enzyme and adenosine triphosphate to the conductive electrode for phosphorylation; adding the MXenes / Co-TiO2 composite probe dispersion to the conductive electrode; and forming a magnetic field-enhanced photoelectrochemical sensor after the reaction is complete; and performing biological enzyme activity detection using a three-electrode system under light and magnetic field conditions.

[0062] This disclosure employs a magnetic field-enhanced photoelectrochemical sensor constructed using a two-dimensional material-based MXenes / Co-TiO2 composite probe. Under illumination and magnetic field conditions, a three-electrode system is used to perform biological enzyme activity detection, significantly improving the photocurrent signal and achieving ultra-high sensitivity detection of biological enzyme activity. Furthermore, the method provided in this disclosure is simple to implement and has low manufacturing costs.

[0063] The optional embodiments of this disclosure are described in detail below with reference to the accompanying drawings.

[0064] Figure 1 This is a schematic diagram of the structure of a magnetic field-enhanced photoelectrochemical sensor provided in some embodiments of this disclosure. Figure 1 As shown, some embodiments of this disclosure provide a magnetic field enhanced photoelectrochemical sensor 100 for detecting biological enzyme activity. The photoelectrochemical sensor 100 includes: a conductive electrode 10, a polypeptide 20, and an MXenes / Co-TiO2 composite probe 30.

[0065] The conductive electrode 10 is, for example, indium tin oxide conductive glass, i.e., glass plated with indium tin oxide (ITO); one end of the polypeptide 20 is coupled to the conductive electrode 10, and the other end of the polypeptide 20 away from the conductive electrode 10 is phosphorylated by the biological enzyme to be tested; the MXenes / Co-TiO2 composite probe 30 binds heterotropically to the other end of the polypeptide phosphorylated.

[0066] The magnetic field-enhanced photoelectrochemical sensor operates in a magnetic field environment to enhance the signal strength of the optical circuit.

[0067] MXenes are a class of two-dimensional materials renowned for their excellent electrical conductivity, mechanical properties, and surface chemical characteristics. Typically composed of transition metals and carbon or nitrogen, MXenes exhibit high surface activity and good electrochemical performance, making them ideal electrode materials in photoelectrochemical sensors. TiO2, as an n-type semiconductor, primarily absorbs ultraviolet light (accounting for only 4% of sunlight) due to its wide bandgap, resulting in low utilization of visible light. Co doping can introduce intermediate energy levels into the bandgap of TiO2, effectively reducing the bandgap and enabling it to absorb visible light (400-700 nm), significantly improving light energy utilization. Co doping can also form trapped states, reducing the recombination of photogenerated electrons and holes, thereby improving photoelectric conversion efficiency. By introducing the Co doping strategy, the bandgap of the material is significantly reduced, thus extending the light absorption range into the visible light region (400-700 nm). In some embodiments of this disclosure, the two-dimensional MXene material, for example, Ti3C2 MXenes, is selected.

[0068] A two-dimensional MXenes / Co-TiO2 composite probe was employed. The positively valence Ti ions in Ti3C2 MXenes and Co-TiO2 can complex with phosphate groups, allowing the MXenes / Co-TiO2 composite probe to be attached to a conductive electrode via a peptide. Ti3C2 MXenes exhibits metal-like conductivity and a large specific surface area, which is beneficial for the immobilization and efficient interaction of biomolecules. Cobalt doping reduces the band gap of TiO2, enhancing its visible light absorption. The composite of MXenes and Co-TiO2 further synergistically optimizes light-harvesting performance. Simultaneously, an external magnetic field can affect the spin-polarized electrons in the Co-TiO2 material, suppressing electron spin recombination and enhancing the concentration of photogenerated carriers. The magnetoresistance effect of Co-TiO2 in the magnetic field optimizes carrier mobility and improves charge separation efficiency, thereby significantly enhancing the photocurrent signal and achieving ultra-sensitive detection of biological enzyme activity.

[0069] In some embodiments, the polypeptide includes kemp peptide, and the bioenzyme includes a protein kinase, such as protein kinase A (PKA).

[0070] Figure 2 This is a flowchart illustrating a method for detecting biological enzyme activity using a magnetic field-enhanced photoelectrochemical sensor, provided in some embodiments of this disclosure. Figure 3 This is a schematic flowchart illustrating a method for detecting biological enzyme activity using a magnetic field-enhanced photoelectrochemical sensor, provided in some embodiments of this disclosure.

[0071] like Figure 2 and Figure 3 As shown, some embodiments of this disclosure provide a method for detecting the activity of biological enzymes using a magnetic field-enhanced photoelectrochemical sensor, the method comprising the following steps:

[0072] S100: Conductive electrode for preparing coupled peptides;

[0073] S200: Preparation of MXenes / Co-TiO2 composite probe dispersion;

[0074] S300: A buffer solution containing biological enzymes and adenosine triphosphate is dropped onto the conductive electrode to carry out a phosphorylation reaction. The MXenes / Co-TiO2 composite probe dispersion is dropped onto the conductive electrode. After the reaction is complete, a magnetic field enhanced photoelectrochemical sensor is formed.

[0075] S400: Performs biological enzyme activity detection using a three-electrode system under light and magnetic field conditions.

[0076] In some embodiments, the two-dimensional structural material MXenes are selected, for example, Ti3C2 MXenes, which are a common type of MXenes. Peptides include kemp peptides, and biological enzymes include protein kinases, such as protein kinase A (PKA).

[0077] This disclosure utilizes the ability of positively valent Ti ions in Ti3C2 MXenes / Co-TiO2 to complex with phosphate groups to construct a magnetic field-enhanced photoelectrochemical sensor, which is used to detect the activity of protein kinases.

[0078] MXenes have metal-like conductivity. Cobalt doping introduces intermediate energy levels into the band gap of TiO2, thereby reducing the band gap and enhancing its visible light absorption and photoelectric sensing performance. MXenes / Co-TiO2 composite probes can effectively enhance the sensitivity of biosensors.

[0079] An external magnetic field can manipulate the spin-polarized electrons in the magnetic material Co-TiO2, suppressing electron-hole recombination and thus enhancing the concentration of photogenerated carriers. The magnetoresistance effect of Co-TiO2 in a magnetic field can optimize carrier mobility and improve charge separation efficiency, thereby significantly enhancing the photocurrent signal.

[0080] This biosensor has a simple preparation method, low manufacturing cost, and low detection sensitivity, achieving the goal of efficient and sensitive detection of biological enzymes, such as protein kinase A activity.

[0081] In some embodiments, the polypeptide includes a kemp peptide, and step S100: preparing a conductive electrode coupled with the polypeptide includes the following steps:

[0082] S110 provides indium tin oxide conductive glass and performs pretreatment on the indium tin oxide conductive glass.

[0083] Specifically, the indium tin oxide conductive glass was ultrasonically cleaned in acetone, isopropanol, and ethanol for 15 minutes in sequence, and then dried with nitrogen gas for later use.

[0084] S120: Chitosan solution is dropped onto the pretreated indium tin oxide conductive glass and then dried.

[0085] Specifically, 15-20 μL of a 0.5% (w / w) chitosan solution is dropped onto a predetermined area of ​​the indium tin oxide conductive glass, for example, 0.5 cm². 2 The material is dried at room temperature to form a uniform chitosan film on the indium tin oxide conductive glass.

[0086] S130: Glutaraldehyde solution and Kemppeptide solution are sequentially added to indium tin oxide conductive glass on which a chitosan film has been formed to modify Kemppeptide onto the indium tin oxide conductive glass.

[0087] Specifically, 15-20 μL of a 2.5% glutaraldehyde solution was added dropwise to an indium tin oxide conductive glass layer with a chitosan film, and the mixture was allowed to stand at room temperature for 1 h. Then, 15-20 μL of a 50-500 mM Kemppeptide solution was added dropwise, and the glass was placed in the dark and allowed to stand for 6-8 h. Glutaraldehyde acts as a cross-linking agent; its two aldehyde groups (-CHO) can connect to the amino groups (-NH2) of chitosan and Kemppeptide, respectively, thereby modifying the surface of the indium tin oxide conductive glass with Kemppeptide.

[0088] In some embodiments, a 1 mM 6-aminohexanoic acid solution can be added dropwise to block blank sites for 2 h to reduce nonspecific adsorption. Due to the condensation reaction between the carboxyl group (-COOH) of 6-aminohexanoic acid (EACA) and the amino group (-NH2) of chitosan, an amide bond (-CONH-) is formed, which stabilizes the binding.

[0089] In some embodiments, the concentration of the Kempeptide solution is 500 mM. Figure 4 The figures show the correlation curves between different concentrations of Kempeptide solution and the corresponding photocurrent changes in some embodiments of this disclosure. Figure 4 As shown, Kempeptide, as the "gold standard" substrate for protein kinase A (PKA) research, can monitor PKA activity in real time through phosphorylation-induced signal changes. The concentration of the Kempeptide solution used was optimized experimentally, such as... Figure 4 As shown, the photocurrent intensity of the modified electrode increases with increasing Kempeptide concentration, reaching a maximum at a concentration of 500 mM. Beyond this concentration, the photocurrent intensity remains stable, indicating that the phosphorylation reaction has reached saturation. Therefore, the optimal concentration of Kempeptide was determined to be 500 mM.

[0090] In some embodiments, step S200: preparing the MXenes / Co-TiO2 composite probe dispersion includes the following steps:

[0091] S210: Preparation of Ti3C2 MXenes dispersion;

[0092] S220: Preparation of Co-TiO2 nanosheet dispersion;

[0093] S230: Mix 1.48 mg / mL MXenes dispersion and 1 mg / mL Co-TiO2 nanosheet dispersion at a volume ratio of 10:1, stir and shake well using a mixer, and let stand to form MXenes / Co-TiO2 composite probe dispersion.

[0094] Specifically, 1.48 mg / mL of MXenes dispersion and 1 mg / mL of Co-TiO2 nanosheet dispersion were mixed at a volume ratio of 10:1, stirred and shaken with a mixer, and allowed to stand for several hours before being placed in an environment at 4°C for use.

[0095] In some embodiments, step S210: preparing the Ti3C2 MXenes dispersion includes the following steps:

[0096] S211: LiF is added to a 9 mol / L HCl solution, and the mixture is stirred to obtain a mixed solution;

[0097] Specifically, 0.8 g LiF was added to 10 mL of 9 mol / L HCl, and the resulting mixture was gently stirred to obtain a mixed solution.

[0098] S212: Add Ti3AlC2 powder to the mixed solution, stir the reaction thoroughly, centrifuge and wash, collect the obtained precipitate, disperse the precipitate in deionized water, and centrifuge it.

[0099] Specifically, 0.5 g of Ti3AlC2 powder was added to the mixed solution, and then stirred at 35°C for 24 hours. The mixed solution was centrifuged several times until the pH of the washing supernatant reached 6. The precipitate was collected and redispersed in deionized water. The resulting mixed solution was then centrifuged at 3500 rpm for 60 minutes.

[0100] S213: Collect the precipitate again and add it to deionized water, then perform ultrasonic treatment under nitrogen protection;

[0101] Specifically, the precipitate was collected again and added to deionized water, and then ultrasonically treated for 1 hour in a nitrogen-protected environment.

[0102] S214: The ultrasonically treated solution is centrifuged again, the supernatant is retained and stored at low temperature to obtain Ti3C2 MXenes dispersion.

[0103] Specifically, the solution was centrifuged at 3500 rpm for 60 minutes to retain the supernatant and stored at 4°C.

[0104] Figure 5Scanning electron microscope (SEM) images of Ti3C2 MXenes provided in some embodiments of this disclosure. Specifically, these are SEM images of the two-dimensional Ti3C2 MXenes material after solvent evaporation from the Ti3C2 MXenes dispersion. Figure 5 As shown, the scanning electron microscope image reveals an ultrathin and nearly transparent Ti3C2 MXenes film with excellent dispersibility.

[0105] In some embodiments, step S220: preparing the Co-TiO2 nanosheet dispersion includes the following steps:

[0106] S221: TiO2, CoO, K2CO3 and Li2CO3 are mixed in stoichiometric ratio, and the mixture is subjected to high-temperature annealing, grinding and secondary high-temperature annealing in sequence to obtain compound K. 0.8 Ti (5.2-y) / 3 Li (0.8-2y） / 3 Co y O4 precursor;

[0107] Specifically, in the precursor synthesis stage: TiO2 (0.25 mol, 20 g), CoO (0.03 mol, 2.25 g), K2CO3 (0.06 mol, 5.94 g), and Li2CO3 (0.01 mol, 0.67 g) were mixed in stoichiometric ratios and ground in a corundum crucible, then annealed at 1000 °C for 5 hours. The resulting mixture was ground again at room temperature and then annealed a second time at 1000 °C for 20 hours, yielding compound K. 0.8 Ti (5.2-y) / 3 Li (0.8-2y） / 3 Co y O4.

[0108] S222: The precursor is reacted thoroughly with a 9 mol / L HCl solution, followed by washing and drying to obtain the chemical formula H. (3.2−2y) / 3 Ti (5.2−y) / 3 Co y The protonation products of O4;

[0109] Specifically, in the protonation stage: K... 0.8 Ti (5.2-y) / 3 Li (0.8-2y) / 3 Co y O4 (1 g) was mixed with 200 mL HCl (1 M) and stirred for 4 days, using a magnetic stirrer to allow Li to flow. + and K + Ions and H + Complete ion exchange. Collect H+ after allowing the solution to settle. (3.2-2y) / 3 Ti (5.2-y) / 3 Co yThe protonated precipitate of O4 was washed with deionized water to remove acid residues and dried in an oven at 80°C for 12 hours.

[0110] S223: The protonated product is thoroughly soaked with tetrabutylammonium hydroxide (TBAOH) at a 1:1 molar ratio to produce a product with the chemical formula TBA. z H (3.2-2y) / 3-z Ti (5.2-y) / 3 Co y The bulk compound of O4;

[0111] Specifically, the organic intercalation stage: by using H (3.2-2y) / 3 Ti (5.2-y) / 3 Co y O4 powder was soaked in a 10% (w / v) TBAOH aqueous solution (H + TBA + In a 1:1 molar ratio, H was added for 5 hours. (3.2-2y) / 3 Ti (5.2-y) / 3 Co y Protons in O4 and TBA + Exchange to produce TBA z H (3.2-2y) / 3-z Ti (5.2-y) / 3 Co y O4.

[0112] S224: The bulk compound is exfoliated in deionized water by mechanical oscillation to obtain the Co-TiO2 nanosheet dispersion.

[0113] Specifically, the stripping stage involves mechanically oscillating the bulk compound TBA for 48 hours. z H (3.2-2y) / 3-z Ti (5.2-y) / 3Co y O4 was stripped in deionized water to obtain a two-dimensional cobalt-doped titanium dioxide stable suspension.

[0114] Figure 6 The images shown are atomic force microscopy (AFM) images of Co-TiO2 nanosheets provided in some embodiments of this disclosure. These are the Co-TiO2 nanosheets remaining after the solvent in the Co-TiO2 nanosheet dispersion has evaporated; they also exhibit a two-dimensional structure. Figure 6 As shown, the average thickness of the layered Co-TiO2 nanosheets is 4.2 nm and the lateral dimension is 395.22 nm, which was confirmed by atomic force microscopy (AFM).

[0115] Figure 7 Here are some hysteresis loop curves of Co-TiO2 nanosheet dispersions provided in some embodiments of this disclosure, such as... Figure 7As shown, the hysteresis loop of the Co-TiO2 dispersion indicates that Co-TiO2 has excellent magnetic effects.

[0116] Figure 8 The image shows magnetoresistance (MR) curves of Co-TiO2 nanosheet dispersions provided in some embodiments of this disclosure. Magnetoresistance (MR) is the change in resistance caused by electron spin polarization in a magnetic field. It directly reflects the charge transfer induced by electron spin polarization, and is expressed by the formula MR% = [R(H) - R(0)] / R(0), where R(H) and R(0) represent the resistance under magnetic and no magnetic fields, respectively. The magnetoresistance (MR) of the Co-TiO2 nanosheet dispersion samples was recorded at room temperature in magnetic fields ranging from -3T to 3T. Figure 8 As shown, a negative magnetoresistance (MR) effect of approximately 5.5% exists in the Co-TiO2 samples at 3T and -3T. Negative MR implies high conductivity, resulting in the generation and transfer of more photoinduced charges to the electrode surface.

[0117] In some embodiments, the bioenzyme includes a protein kinase, such as protein kinase A (KPA). In step S300, adding a buffer solution containing the bioenzyme and adenosine triphosphate (ATP) to the conductive electrode to perform a phosphorylation reaction includes:

[0118] A buffer solution containing protein kinase and adenosine triphosphate was added dropwise to the conductive electrode to carry out a phosphorylation reaction, wherein the protein kinase was used at different concentrations, the adenosine triphosphate was at a concentration of 120 μM, and the phosphorylation reaction lasted for 80 min.

[0119] Specifically, 15-20 μL of a buffer solution (50 mM Tris-HCl and 20 mM MgCl2, pH 7.4) containing different concentrations of protein kinase A (PKA) and adenosine triphosphate ATP was added dropwise to the electrode for 2-3 hours. The phosphorylation principle of Kemp peptide is to remove the phosphate group (PO4) of ATP through an enzymatic reaction. 3- The phosphorylated Kempeptide and adenosine diphosphate (ADP) are transferred to the amino acid residues of Kempeptide. Finally, the MXenes / Co-TiO2 composite probe mixture is dropped onto the electrode surface. After the solvent evaporates, the prepared photoelectric biosensor is obtained and stored in a 4°C refrigerator for later use.

[0120] Figure 9 This is a correlation curve showing the relationship between different adenosine triphosphate concentrations and corresponding photocurrent changes in some embodiments of this disclosure. Figure 10The figures show correlation curves between different phosphorylation times and corresponding photocurrent changes in some embodiments of this disclosure. Phosphorylation of Kempide typically occurs in the presence of adenosine triphosphate (ATP), which not only provides a phosphate group for the phosphorylation reaction but also provides the energy required for the enzymatic activity of the kinase. Figure 9 As shown, the photocurrent intensity of the electrode increases with increasing adenosine triphosphate (ATP) concentration, reaching a maximum at 120 μM, indicating that 120 μM ATP is sufficient to catalyze the PKA-catalyzed phosphorylation reaction. Therefore, the optimal concentration of ATP was determined to be 120 μM. Furthermore, phosphorylation time is an important parameter in electrode preparation. Figure 10 It can be seen that the intensity of photocurrent change gradually increases with the increase of phosphorylation reaction time, and reaches a plateau at 80 minutes, indicating that the phosphorylation reaction of Kemp peptide is close to completion. Therefore, the optimal phosphorylation time is selected as 80 minutes.

[0121] In some embodiments, step S400: the detection of biological enzyme activity using a three-electrode system under light and magnetic field conditions includes:

[0122] A magnetic field-enhanced photoelectrochemical sensor in a three-electrode system was irradiated with a light source of 420 nm wavelength. Simultaneously, a magnetic field was established to place the sensor in a magnetic field environment. The irradiation intensity at the sensor was 190 mW / cm². 2 The magnetic induction intensity at the magnetic field-enhanced photoelectrochemical sensor is 80~90mT.

[0123] Specifically, the light source used in step S400 is a xenon lamp, employing a visible light filter with a wavelength of 420nm, and an irradiance of 190mW / cm². 2Step 400 is performed, for example, on a CHI802B electrochemical workstation, using a three-electrode system for detection. The reference electrode is an Ag / AgCl electrode, the counter electrode is a platinum wire electrode, and the working electrode is an ITO electrode. The detection process is performed under open-circuit voltage, using a PBS phosphate solution containing 0.1M ascorbic acid as an electron donor as the reaction solution. An external magnetic field is generated by a magnetic field generator by adjusting different current magnitudes. The magnetic induction intensity at the working electrode is measured using a magnetic response probe, and the magnetic induction intensity is 80~90mT, for example, 85 mT. Under visible light irradiation, the bare ITO electrode and the ITO electrode with a chitosan film layer showed almost no photoresponse. When the ITO electrode with a chitosan film layer was modified with Kempeptide and phosphorylated by PKA, the electrode still showed no photocurrent response. Finally, after modifying the Kempeptide with positively valence Ti ions, a significant enhancement of the photocurrent signal could be detected. This is because under visible light excitation, photogenerated electrons of Co-TiO2 are transferred to the electrode to form a photocurrent. MXenes, due to its high conductivity, accelerates the electron transfer ability and also significantly enhances the photoelectric conversion efficiency of Co-TiO2. At this time, the working electrode is the conductive electrode of the magnetic field enhanced photoelectrochemical sensor.

[0124] A magnetic field generator, such as an electromagnet, is placed near the working electrode to apply a constant magnetic field, thereby manipulating spin-polarized electrons and promoting the effective separation of photoexcited electron-hole pairs, thus improving light conversion efficiency. Under the influence of the magnetic field (MF), electron spin polarization is realized and enhanced, resulting in the spin state of photoexcited electrons in the conduction band (CB) being opposite to the spin state of holes in the valence band (VB). Due to hyperfine interactions and spin-orbit coupling, the spin state of the excited electrons is flipped in a specific ratio. Meanwhile, the remaining holes in the valence band (VB) maintain their original spin orientation in the highly spin-polarized environment, effectively and maximally hindering the recombination of photoexcited charge carriers.

[0125] Figure 11 This disclosure provides schematic diagrams of the structure of a testing system for detecting biological enzyme activity in some embodiments, such as... Figure 11 As shown, the testing system 100 includes an electrochemical workstation 200, a light source 300, and a magnetic field generator 400. In the electrochemical workstation 200, a three-electrode system is used for detection. The reference electrode is an Ag / AgCl electrode, the counter electrode is a platinum wire electrode, and the working electrode is an ITO electrode. The detection process is performed under open-circuit voltage, using a PBS phosphate solution containing 0.1M ascorbic acid as an electron donor as the reaction solution. The light source 300 is, for example, a xenon lamp, using a 420nm wavelength visible light filter, with an irradiance of 190 mW / cm². 2The magnetic field generator 400 is, for example, an electromagnet, and the magnetic induction intensity is controlled by adjusting the current of the electromagnet. The light source 300 and the magnetic field generator 400 are located on both sides of the electrochemical workstation 200, and the light source 300 and the magnetic field generator 400 are continuously perpendicular to the working electrode 210 of the electrochemical workstation 200.

[0126] In some embodiments, the magnetic field generator 400 is placed vertically relative to the working electrode 210 and as close as possible to the working electrode 210 to provide it with a sufficiently strong magnetic field, for example, the distance between the magnetic field generator 400 and the working electrode 210 is 1 cm.

[0127] use Figure 11 The testing system in the document can detect the effect of the magnetic field on the photocurrent when a magnetic field-enhanced photoelectrochemical sensor performs detection. Figure 12 The graphs showing the relationship between the magnitude of magnetic induction intensity and the intensity of photocurrent change are provided for some embodiments of this disclosure. Figure 12 As shown, this illustrates the changes in photocurrent signal of a magnetic field-enhanced photoelectrochemical sensor under different magnetic induction intensities. The results indicate that the intensity of the photocurrent change increases with increasing magnetic induction intensity and tends to stabilize at a magnetic induction intensity of 80 mT. Figure 12 As shown in the inset, within the range of 0 to 60 mT, the intensity of the photocurrent change is directly proportional to the magnitude of the magnetic flux density, which can be described by the correlation equation y = 5.379 + 0.446x, where y represents the intensity of the photocurrent change, x represents the magnitude of the magnetic flux density, and the correlation coefficient R0 is... 2 = 0.994. Therefore, as mentioned above, in step S400, the magnetic induction intensity at the magnetic field-enhanced photoelectrochemical sensor is designed to be 80~90mT, for example 85mT, which can achieve sufficiently good testing results with low energy consumption.

[0128] In some embodiments, the foregoing Figure 9 Correlation curves between different concentrations of adenosine triphosphate and the corresponding photocurrent intensity. Figure 10 The correlation curves between different phosphorylation times and the corresponding photocurrent changes are used, for example, by... Figure 11 The test system was used to obtain the data, for example, an irradiation intensity of 190 mW / cm² was used. 2 The test environment is designed with visible light illumination at a wavelength of 420nm and a magnetic field intensity of 80~90mT at the working electrode, for example, 85mT.

[0129] Figure 13This disclosure presents a graph showing the relationship between the concentration of protein kinase A (PKA) and the intensity of photocurrent changes in some embodiments. In the following embodiments, different concentrations of protein kinase A (PKA) were used to evaluate the activity of protein kinase A. Figure 13 As shown, the intensity of the photocurrent change increases with increasing PKA concentration, reaching a plateau at 80 U / mL. Figure 13 As shown in the inset, within the range of 0.005 U / mL to 1 U / mL, the intensity of the photocurrent change is substantially proportional to the protein kinase A (PKA) concentration, which can be described by the correlation equation y = 4.93 + 62.36x, where y represents the photocurrent change intensity, x represents the protein kinase A (PKA) concentration, and the correlation coefficient R0 is 1. 2 = 0.998. The detection limit for protein kinase A (PKA) is calculated to be 0.00016 U / mL. For protein kinase A, the magnetic field-enhanced photoelectrochemical sensor provided in some embodiments of this disclosure has a low detection limit.

[0130] Finally, it should be noted that the various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the systems or apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the descriptions are relatively simple, and relevant parts can be referred to the method section.

[0131] The above embodiments are only used to illustrate the technical solutions of this disclosure, and are not intended to limit it. Although this disclosure has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this disclosure.

Claims

1. A method for detecting the activity of biological enzymes using a magnetic field-enhanced photoelectrochemical sensor, characterized in that, The method includes the following steps: Prepare a conductive electrode for coupling peptides; Preparation of MXenes / Co-TiO2 composite probe dispersion; A buffer solution containing biological enzymes and adenosine triphosphate is dropped onto the conductive electrode to carry out a phosphorylation reaction. The MXenes / Co-TiO2 composite probe dispersion is then dropped onto the conductive electrode. After the reaction is complete, a magnetic field-enhanced photoelectrochemical sensor is formed. The detection of biological enzyme activity was performed using a three-electrode system under illumination and magnetic field conditions, wherein the irradiation intensity at the magnetic field-enhanced photoelectrochemical sensor was 190 mW / cm². 2 The magnetic induction intensity at the magnetic field-enhanced photoelectrochemical sensor is 80~90mT.

2. The method according to claim 1, characterized in that, The polypeptide includes Kemp peptide, and the preparation of the conductive electrode for the coupled polypeptide includes the following steps: Provide indium tin oxide conductive glass and perform pretreatment on the indium tin oxide conductive glass; Chitosan solution was dropped onto the pretreated indium tin oxide conductive glass and then dried. Glutaraldehyde solution and Kemppeptide solution were sequentially added to indium tin oxide conductive glass with a chitosan film to modify the indium tin oxide conductive glass.

3. The method according to claim 2, characterized in that, The concentration of the Kempeptide solution was 500 mM.

4. The method according to claim 1, characterized in that, The preparation of the MXenes / Co-TiO2 composite probe dispersion includes the following steps: Preparation of Ti3C2 MXenes dispersion; Preparation of Co-TiO2 nanosheet dispersion; Mix 1.48 mg / mL MXenes dispersion and 1 mg / mL Co-TiO2 nanosheet dispersion at a volume ratio of 10:1, stir and mix well using a mixer, and let stand to form an MXenes / Co-TiO2 composite probe dispersion.

5. The method according to claim 4, characterized in that, The preparation of the Ti3C2 MXenes dispersion includes the following steps: LiF was added to a 9 mol / L HCl solution, and the mixture was stirred to obtain a mixed solution. Ti3AlC2 powder was added to the mixed solution, the mixture was stirred thoroughly, centrifuged and washed, the precipitate was collected and dispersed in deionized water and centrifuged. The precipitate was collected again and added to deionized water, and ultrasonic treatment was performed under nitrogen protection; and The ultrasonically treated solution was centrifuged again, and the supernatant was retained and stored at low temperature to obtain a Ti3C2MXenes dispersion.

6. The method according to claim 4, characterized in that, The preparation of Co-TiO2 nanosheet dispersion includes the following steps: TiO2, CoO, K2CO3, and Li2CO3 were mixed in stoichiometric proportions, and the mixture was subjected to a series of operations including grinding, high-temperature annealing, secondary grinding, and secondary high-temperature annealing to obtain compound K. 0.8 Ti (5.2-y) / 3 Li (0.8-2y） / 3 Co y O4 precursor; The precursor was reacted thoroughly with a 9 mol / L HCl solution, followed by washing and drying to obtain the chemical formula H. (3.2−2y) / 3 Ti (5.2−y) / 3 Co y The protonation products of O4; The protonated product was thoroughly soaked with tetrabutylammonium hydroxide (TBAOH) at a 1:1 molar ratio to produce a product with the chemical formula TBA. z H (3.2-2y) / 3-z Ti (5.2-y) / 3 Co y The bulk compound of O4; The bulk compound was exfoliated in deionized water by mechanical oscillation to obtain the Co-TiO2 nanosheet dispersion.

7. The method according to any one of claims 1 to 6, characterized in that, The bioenzyme includes a protein kinase, and the step of adding a buffer solution containing the bioenzyme and adenosine triphosphate to the conductive electrode to carry out a phosphorylation reaction includes: A buffer solution containing protein kinase and adenosine triphosphate was added dropwise to the conductive electrode to carry out a phosphorylation reaction, wherein the protein kinase was used at different concentrations, the adenosine triphosphate was at a concentration of 120 μM, and the phosphorylation reaction lasted for 80 min.

8. The method according to any one of claims 1 to 6, characterized in that, The method of performing bioenzyme activity detection using a three-electrode system under light and magnetic field conditions includes: A magnetic field-enhanced photoelectrochemical sensor in a three-electrode system is irradiated with a light source of 420 nm wavelength. At the same time, a magnetic field is set to place the magnetic field-enhanced photoelectrochemical sensor in a magnetic field environment. The magnetic field is applied using a magnetic field generator, and the distance between the electrode of the magnetic field generator and the conductive electrode is 1 cm.

9. The method according to claim 7, characterized in that, When the protein kinase concentration is in the range of 0.005 U / mL to 1 U / mL, the change in photocurrent intensity and the protein kinase concentration satisfy the following equation: y = 4.93 + 62.36x, where y represents the intensity of the photocurrent change and x represents the protein kinase concentration.

10. A magnetic field-enhanced photoelectrochemical sensor for detecting biological enzyme activity, characterized in that, The photoelectrochemical sensor includes: Conductive electrodes; A polypeptide, one end of which is coupled to the conductive electrode, and the other end of the polypeptide, away from the conductive electrode, is phosphorylated by the enzyme to be tested; and The MXenes / Co-TiO2 composite probe binds heterotropically to the phosphorylated end of the peptide. The magnetic field-enhanced photoelectrochemical sensor described herein operates in a magnetic field environment to enhance the signal strength of the optical circuit.

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