A biosensor electrode and its preparation method and its application in a biosensor for electrochemical detection of ALT

Abstract

The present invention discloses a biosensor electrode and its preparation method and its application in a biosensor for electrochemical detection of alanine aminotransferase. The biosensor electrode is a MXene-GO composite material modified with pyruvate oxidase on the electrode surface, wherein the MXene-GO composite material has catalytic activity and can realize the conversion of chemical signals to electrical signals. At the same time, MXene-GO also has a large specific surface area, which can increase the load of the enzyme, promote signal amplification, and thus improve detection sensitivity, reduce detection limit, and have a wide detection range. The preparation method is simple and low cost. When the electrode is applied to a biosensor for electrochemical detection of ALT, it has the advantages of good selectivity, high sensitivity, low detection limit, and wide detection range.

Description

Technical Field

[0001] The present invention relates to a biosensor electrode, in particular to a MXene-GO / POx modified electrode, and also to a preparation method thereof and application in a biosensor for electrochemically detecting ALT, belonging to the field of biosensor technology. Background Art

[0002] Alanine aminotransferase (ALT) is an intracellular enzyme widely present in the human liver. When liver function is impaired, liver cells rupture and ALT is released into the blood, causing a sharp increase in ALT concentration. Therefore, ALT is also an important indicator for clinical assessment of liver function.

[0003] Currently, the main methods for detecting ALT include colorimetry, spectrophotometry, chemiluminescence, chromatography, and fluorescence. While these methods offer reliable results, they generally require large analytical instruments, expensive reagents, complex processing methods, and high requirements for personnel, resulting in certain limitations. Therefore, developing an ALT detection method that is simple to operate, has good selectivity, high sensitivity, and low cost is of great significance. Summary of the Invention

[0004] To address the shortcomings of existing ALT detection methods, the first objective of the present invention is to provide a biosensor electrode comprising a MXene-GO / POx-modified electrode. The MXene-GO composite material contained in the electrode exhibits catalytic activity, enabling the conversion of chemical signals into electrical signals. Furthermore, the large surface area of ​​the MXene-GO increases enzyme loading and promotes signal amplification, thereby improving detection sensitivity, lowering the detection limit, and extending the detection range. When used in a biosensor for electrochemical detection of ALT, this electrode exhibits advantages such as good selectivity, high sensitivity, a low detection limit, and a wide detection range.

[0005] The second object of the present invention is to provide a method for preparing a biosensor electrode, which is simple, low-cost, easy to operate, and can be produced on a large scale.

[0006] The third object of the present invention is to provide an application of a biosensor electrode for electrochemically detecting ALT.

[0007] To achieve the above technical objectives, the present invention provides a biosensor electrode, the electrode surface being modified with a MXene-GO composite material loaded with pyruvate oxidase. The MXene-GO composite material is obtained by ultrasonically reacting a mixture of a MXene solution and a GO solution. The MXene-GO composite material on the electrode surface is catalytic and can capture electroactive substances, achieving conversion from chemical signals to electrical signals. It also has a large specific surface area, which can increase the loading of pyruvate oxidase, promote signal amplification, and thereby improve detection sensitivity, reduce detection limits, and expand detection range.

[0008] As a preferred embodiment, the MXene-GO composite material is a composite of titanium carbide and graphite oxide. The MXene of the present invention is a titanium carbide material commonly used in the prior art and can be prepared by mixing HCl and LiF, adding Ti3AlC2 powder, stirring, and performing an etching reaction.

[0009] MXene is a transition metal compound with an accordion-shaped multilayer structure. This feature gives it good conductivity and a large specific surface area. Since active groups including hydroxyl and carboxyl groups are introduced into its surface during etching, it also has good biocompatibility and hydrophilicity. Combining it with graphite oxide is beneficial to improving the performance of biosensor electrodes.

[0010] The present invention also provides a method for preparing a biosensor electrode, which comprises the following preparation steps:

[0011] 1) Mixing MXene solution and GO solution and reacting them under ultrasonication to obtain MXene-GO composite material;

[0012] 2) coating the MXene-GO composite material on the surface of the glassy carbon electrode to obtain a MXene-GO modified electrode;

[0013] 3) The pyruvate oxidase solution is mixed with a glutaraldehyde solution, a bovine serum albumin solution, and a chitosan solution for cross-linking reaction to obtain a mixed enzyme solution, and the mixed enzyme solution is coated on the surface of the MXene-GO modified electrode.

[0014] The present invention uses a MXene-GO composite material as the substrate. This composite material exhibits excellent catalytic activity for hydrogen peroxide-based electroactive substances, capturing generated hydrogen peroxide and converting chemical signals into electrical signals. It also possesses a large specific surface area, which increases enzyme loading and promotes signal amplification, thereby improving detection sensitivity, lowering detection limits, and extending the detection range. Furthermore, the present invention utilizes chitosan, a biocompatible adsorbent, in combination with glutaraldehyde. The glutaraldehyde cross-links the amino groups on the enzyme with those on bovine serum albumin and chitosan, thereby immobilizing the enzyme on the electrode. Furthermore, chitosan's excellent biocompatibility and film-forming properties contribute to the preservation and immobilization of enzyme activity.

[0015] As a preferred solution, the mass ratio of the MXene solution to the GO solution is 1-2:2-1.

[0016] Controlling the mass ratio of the MXene solution to the GO solution within an appropriate range is beneficial for obtaining biosensor electrodes with superior performance. When the mass ratio of the MXene to GO solution is too large, the modified electrode's response to pyruvate is weak. As the mass ratio decreases, the modified electrode's response to pyruvate gradually increases. However, when the ratio is too small, the electrode's background current increases, affecting detection performance.

[0017] As a preferred solution, the concentration of the MXene solution is 0.5 to 1 mg / mL. The concentration of the GO solution is 0.5 to 1 mg / mL.

[0018] As a preferred solution, the reaction temperature in step 1) is 25-30° C. and the reaction time is 0.5-1.5 h.

[0019] As a preferred embodiment, the concentration of the pyruvate oxidase solution is 250-500 U / mL. The concentration of the glutaraldehyde solution is 0.25-2.5% w / w. The concentration of the bovine serum albumin solution is 10-50 mg / mL. The concentration of the chitosan solution is 1-5 mg / mL. The volume ratio of the pyruvate oxidase solution, bovine serum albumin solution, chitosan solution, and glutaraldehyde solution is 8-12:4-6:4-6:1-3.

[0020] To ensure enzyme activity and stability, the dosage and concentration of pyruvate oxidase, glutaraldehyde, bovine serum albumin, and chitosan must be controlled within an appropriate range. Too low a pyruvate oxidase concentration will affect electrode sensitivity, while too high a concentration will lead to enzyme waste. Too low a glutaraldehyde, bovine serum albumin, and chitosan concentrations will result in incomplete enzyme cross-linking, affecting enzyme stability on the electrode surface. Too high a concentration will result in an overly viscous modification layer, affecting enzyme contact and reaction with the substrate.

[0021] As a preferred solution, the cross-linking reaction temperature is 25-30° C. and the time is 10-40 minutes.

[0022] The present invention also provides an application of a biosensor electrode for electrochemically detecting ALT. The biosensor employing this electrode is based on the principle that ALT catalyzes the transamination reaction between L-alanine and α-ketoglutarate to produce pyruvate, which is then oxidized by pyruvate oxidase (POx) to produce H2O2. This H2O2 is then catalyzed by the composite material to generate a redox current. This allows electrochemical detection of ALT concentration, demonstrating advantages such as good selectivity, high sensitivity, a low detection limit, and a wide detection range.

[0023] As a preferred embodiment, the biosensor for electrochemical detection of ALT uses square wave voltammetry to detect ALT. The measurement conditions of the square wave voltammetry method are: using 100 mM phosphate buffer solution (PBS) containing thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD) and magnesium chloride as the electrolyte, in the voltage range of 0 to -0.5 V, and a frequency of 10 to 20 Hz.

[0024] Accurate measurement of ALT concentration can be achieved under the square wave voltammetry determination conditions, and the calculated recovery rate is good during the spike recovery test in normal human serum.

[0025] Compared with the prior art, the advantages of the technical solution of the present invention are:

[0026] (1) MXene-GO composite material is used as the sensor electrode substrate material. It has catalytic properties and can capture electroactive substances to achieve the conversion from chemical signals to electrical signals. It also has a large specific surface area, which can increase the enzyme loading and promote signal amplification, thereby improving detection sensitivity, reducing detection limits, and widening the detection range.

[0027] (2) Chitosan, which has good biocompatibility and adsorption properties, is used in combination with glutaraldehyde to better immobilize the enzyme on the electrode surface and improve electrode stability.

[0028] (3) The detection of ALT is achieved based on pyruvate oxidase loaded on MXene-GO composite materials. The detection range is wide and can be extended to other sensors for detecting the concentrations of different proteins or small molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the principle of the ALT detection method of the present invention.

[0030] Figure 2This is a comparison chart of the responses of the sensor in Example 3 to a blank sample, 10 mM pyruvic acid, 10 mM hydrogen peroxide, and 200 u / LALT.

[0031] Figure 3 The square wave voltammetry curves and standard curves corresponding to different concentrations of ALT in Example 3 are shown. DETAILED DESCRIPTION

[0032] To facilitate understanding of the present invention, the present invention will be described in more comprehensive and detailed form below in conjunction with the accompanying drawings and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0033] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0034] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0035] Example 1

[0036] This embodiment provides a method for preparing a biosensor electrode, which includes the following steps:

[0037] (1) Synthesis of MXene: MXene was obtained by etching Ti3AlC2 with LiF and HCl. To 8 mL of 6 M HCl solution, 200 mg of LiF powder was added under magnetic stirring until the powder was completely dissolved, followed by the slow addition of 200 mg of Ti3AlC2 powder. The reaction mixture was then reacted at 40°C for 48 h, repeatedly washed with distilled water, and centrifuged until the pH of the supernatant after centrifugation was approximately 7. The final product was then freeze-dried to obtain MXene powder.

[0038] (2) Synthesis of MXene-GO composite material: MXene powder was dispersed in deionized water by ultrasonication for 30 min to obtain a MXene solution. A 0.5 mg / mL MXene solution and a 1 mg / mL GO solution were mixed in a mass ratio of 1:2 and ultrasonicated at 25°C for 1 h to obtain a MXene-GO composite material.

[0039] (3) Preparation of MXene-GO modified electrode: 5 μL of MXene-GO composite material was taken and suspended on the surface of glassy carbon electrode, and dried at room temperature to obtain MXene-GO modified electrode.

[0040] (4) Immobilization of POx: 10 μL of 250 U / L POx enzyme solution was mixed with 5 μL of 50 mg / mL BSA solution and 5 μL of 5 mg / mL chitosan solution, and ultrasonicated at 25°C for 10 min. Then, 2 μL of 2.5% w / w glutaraldehyde solution was added and ultrasonicated at 25°C for 10 min to obtain a cross-linked mixed enzyme solution. Then, 5 μL of the mixed enzyme solution was taken and suspended on the surface of the MXene-GO modified electrode and dried at room temperature to obtain the MXene-GO / POx modified electrode.

[0041] Example 2

[0042] This embodiment provides a method for preparing a biosensor electrode, which includes the following steps:

[0043] (1) Synthesis of MXene: MXene was obtained by etching Ti3AlC2 with LiF and HCl. To 8 mL of 6 M HCl solution, 200 mg of LiF powder was added under magnetic stirring until the powder was completely dissolved, followed by the slow addition of 200 mg of Ti3AlC2 powder. The reaction mixture was then reacted at 40°C for 48 h, repeatedly washed with distilled water, and centrifuged until the pH of the supernatant after centrifugation was approximately 7. The final product was then freeze-dried to obtain MXene powder.

[0044] (2) Synthesis of MXene-GO composite material: MXene powder was dispersed in deionized water by ultrasonication for 30 min to obtain a MXene solution. A 1 mg / mL MXene solution and a 0.5 mg / mL GO solution were mixed in a mass ratio of 2:1 and ultrasonicated at 30°C for 1 h to obtain a MXene-GO composite material.

[0045] (3) Preparation of MXene-GO modified electrode: 5 μL of MXene-GO composite material was taken and suspended on the surface of glassy carbon electrode, and dried at room temperature to obtain MXene-GO modified electrode.

[0046] (4) Immobilization of POx: 10 μL of 500 U / L POx enzyme solution was mixed with 5 μL of 25 mg / mL BSA solution and 5 μL of 2 mg / mL chitosan solution, and ultrasonicated at 30°C for 10 min. Then, 2 μL of 0.25% w / w glutaraldehyde solution was added and ultrasonicated at 30°C for 10 min to obtain a cross-linked mixed enzyme solution. Then, 5 μL of the mixed enzyme solution was taken and suspended on the surface of the MXene-GO modified electrode and dried at room temperature to obtain the MXene-GO / POx modified electrode.

[0047] Example 3

[0048] The MXene-GO / POx modified electrode obtained in Example 1 was used to prepare a biosensor for electrochemical detection of alanine aminotransferase (ALT) in human serum. The following is an ALT detection method, which includes the following steps:

[0049] (1) Preparation of ALT test solution: 50 μM TPP, 5 μM FAD, 1 mM magnesium chloride, 10 mM α-ketoglutarate, and 100 mM L-alanine were added to 100 mM PBS at pH 7.

[0050] (2) Incubation with different concentrations of ALT: Add ALT at concentrations of 0.5u / L, 1u / L, 50u / L, 100u / L, 200u / L, 300u / L, and 400u / L to the ALT test solution, respectively, and incubate in a 37°C water bath for 40 minutes.

[0051] (3) Square wave voltammetry: The incubated ALT test solution was used as the electrolyte, and the voltage range was 0 to -0.5 V at a frequency of 15 Hz to obtain the peak current value of each electrode. The peak current value of each electrode was then compared with the corresponding ALT concentration to form a standard curve to obtain the standard curve for measuring ALT concentration.

[0052] (4) Spiked recovery test in normal human serum: 0.5 mL of normal human serum was added to 4.5 mL of ALT test solution, and different amounts of ALT were added: 0 u / L, 2 u / L, 20 u / L, and 200 u / L. The mixture was then incubated in a 37°C water bath for 40 min. The peak current value was measured by square wave voltammetry and compared with the standard curve to obtain the measured concentration value. The recovery rate was calculated to be between 103.93% and 97.35%, which was a good recovery rate.

[0053] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An application of a biosensor electrode, characterized in that: A biosensor for electrochemically detecting ALT, wherein the surface of the biosensor electrode is modified with a pyruvate oxidase-loaded MXene-GO composite material, wherein the MXene-GO composite material is a composite of titanium carbide and graphite oxide; The preparation method of the biosensor electrode comprises the following preparation steps: 1) Mixing MXene solution and GO solution and reacting them under ultrasonication to obtain MXene-GO composite material; 2) Coating the MXene-GO composite material on the electrode surface to obtain a MXene-GO modified electrode; 3) mixing the pyruvate oxidase solution with the glutaraldehyde solution, the bovine serum albumin solution, and the chitosan solution for cross-linking reaction to obtain a mixed enzyme solution, and coating the mixed enzyme solution on the surface of the MXene-GO modified electrode; The mass ratio of the MXene solution to the GO solution is 1~2:2~1; The concentration of the MXene solution is 0.5-1 mg / mL; The GO solution concentration is 0.5-1 mg / mL; The concentration of the pyruvate oxidase solution is 250-500 U / L; The concentration of the glutaraldehyde solution is 0.25-2.5% w / w; The concentration of the bovine serum albumin solution is 10-50 mg / mL; The concentration of the chitosan solution is 1-5 mg / mL; The volume ratio of pyruvate oxidase solution, bovine serum albumin solution, chitosan solution and glutaraldehyde solution is 8-12:4-6:4-6:1-3.

2. The use of a biosensor electrode according to claim 1, characterized in that: In step 1), the reaction temperature is 25-30° C. and the reaction time is 0.5-1.5 h.

3. The use of a biosensor electrode according to claim 1, characterized in that: The cross-linking reaction temperature is 25-30° C., and the time is 10-40 minutes.

4. The use of a biosensor electrode according to claim 1, wherein: The biosensor for electrochemically detecting ALT uses square wave voltammetry to detect ALT. The measurement conditions of the square wave voltammetry method are: using 100 mM phosphate buffer solution (PBS) containing thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD) and magnesium chloride as the electrolyte, in the voltage range of 0~-0.5 V, and the frequency of 10~20 Hz.

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