阅读说明：本技术 一种用于血液酶检测的电化学传感器 (Electrochemical sensor for detecting blood enzyme ) 是由 王龙喜 于 2020-07-20 设计创作，主要内容包括：本发明涉及一种用于血液酶检测的电化学传感器。直接使用玻碳板作为单独的一个工作电极,并且与对电极和参比电极分开,加工成本低,结构新颖,检测效果好；将对电极和参比电极制备在一片材料上,对电极和参比电极制备时充分利用铂电极,来制备参比电极,可以提高制作效率；在玻碳板上线喷银,然后在置换出金,进一步在此基础上生长金纳米枝晶,纳米星或者金纳米星等结构,大大增大了修饰的金和玻碳板的连接强度,而且增大了比表面积,提高了检测的精度。并且由于制备的纳米结构可以调节,可以进一步充分研究其制备的形貌和检测灵敏度之间的关系。(The invention relates to an electrochemical sensor for blood enzyme detection. The glassy carbon plate is directly used as a single working electrode and is separated from the counter electrode and the reference electrode, so that the processing cost is low, the structure is novel, and the detection effect is good; the counter electrode and the reference electrode are prepared on one piece of material, and the platinum electrode is fully utilized to prepare the reference electrode when the counter electrode and the reference electrode are prepared, so that the manufacturing efficiency can be improved; silver is sprayed on the glassy carbon plate, gold is replaced, and gold nano dendrites, nano stars or gold nano stars and other structures are further grown on the basis, so that the connection strength of the modified gold and the glassy carbon plate is greatly increased, the specific surface area is increased, and the detection precision is improved. And the prepared nano structure can be adjusted, so that the relationship between the prepared morphology and the detection sensitivity can be further fully researched.)

1. An electrochemical sensor for blood enzyme detection comprises a substrate layer (1), a covering layer (5), a micro-flow channel layer (3), an upper electrode layer (4) and a lower electrode layer (2); the method is characterized in that:

the electrochemical sensor is formed by sequentially overlapping a substrate layer (1), an upper electrode layer (4), a micro-channel layer (3), a lower electrode layer (2) and a covering layer (5) from bottom to top; the micro-flow channel layer (3) is provided with a first through hole (31) for liquid flowing and reacting; the upper electrode layer (4) is provided with a counter electrode (41) and a reference electrode (42), the lower electrode layer (2) is provided with a working electrode (21), and the positions of the reference electrode (42), the counter electrode (41) and the working electrode (21) correspond to the through holes of the micro-channel layer (3); the upper electrode layer (4) and the lower electrode layer (2) are provided with outgoing lines for leading out the electrodes; the covering layer (5) is provided with a sample adding port (51) for adding a sample or a reagent, and the sample adding port (51) is communicated with the through hole of the micro-flow channel layer (3) through a second through hole (43) at the upper electrode layer;

the counter electrode (41) is made of platinum, the reference electrode (42) is Ag/AgCl, and the working electrode (21) is a glutamate oxidase-modified nano-gold-modified GCE electrode.

2. The electrochemical sensor for blood enzyme detection according to claim 1, wherein:

the upper electrode layer (4) is made of PDMS, and the preparation method comprises the steps of firstly using a first mask plate to shield one side of the upper electrode layer (4), only leaking the positions of a reference electrode (42) and a counter electrode (41) to be manufactured, and isolating the two positions from each other and enabling the two positions to reach the edge of the PDMS material; then, sputtering a layer of platinum metal film with the thickness of 10-50 mu m at the position of the counter electrode (41) to be prepared by a magnetron sputtering method to obtain a platinum electrode, and shielding the reference electrode (42) during magnetron sputtering; then shielding the position of the counter electrode (41), and sputtering a layer of Ag with the thickness of 50-100 mu m on the position of the reference electrode (42) to obtain an Ag electrode; connecting the platinum electrode with a power supply cathode after sputtering, connecting the Ag electrode with a power supply anode, placing one end of the Ag electrode close to the center of the PDMS into HCl solution at normal temperature, and introducing 5mA current for a certain time to obtain an Ag/AgCl electrode at the Ag electrode; and then, leading out Ag and Pt at the edge of the PDMS, and dismantling the first mask plate.

3. The electrochemical sensor for blood enzyme detection according to claim 1, wherein:

the lower electrode layer (2) is made of a glassy carbon sheet, and the preparation method comprises the following steps:

shielding one side of the lower electrode layer (2) by using a second mask plate, only exposing the position of the working electrode (21) to be manufactured, and then sputtering a layer of Ag under the condition of the mask by using magnetron sputtering, wherein the Ag cannot completely cover the glassy carbon sheet, and the sputtering heating time is less than 100 s;

then placing the glassy carbon sheet in 2.8mmol/L HAuCl₄In the sulfuric acid ethanol aqueous solution; growing for 10-20 min under a constant potential of-0.2V to-0.3V, wherein HAuCl is generated in the growth process₄The Au in the glass-carbon plate forms ions, and is tightly connected to the glass-carbon plate after being replaced by the sputtered Ag; then Au further grows to form nano star or nano flower on the glassy carbon plate; thereby obtaining the Au modified glassy carbon electrode;

then placing the Au modified glassy carbon electrode into a solution containing 50mg/L of glutamate oxidase, 2mg/L of bovine serum albumin, 20mg/L of glutaraldehyde and 0.5% of Nafion and having the pH =7.3 for 30-60min, thereby modifying the glutamate oxidase on the electrode; thereby obtaining the GCE electrode modified by the glutamate oxidase and modified by the nanogold.

4. The electrochemical sensor for blood enzyme detection according to claim 2 or 3, characterized in that:

the thickness of the substrate layer (1), the upper electrode layer (4), the micro-flow channel layer (3), the lower electrode layer (2) and the covering layer (5) is 500-1000 microns; wherein the basal layer (1), the upper electrode layer (4), the micro-flow channel layer (3) and the covering layer (5) are all made of PDMS materials.

5. The electrochemical sensor for blood enzyme detection according to claim 2 or 3, characterized in that:

the specific surface area of the nano Au formed on the glassy carbon sheet is more than 400m²/g。

6. The electrochemical sensor for blood enzyme detection according to claim 2 or 3, characterized in that:

the blood enzyme tested was alanine Aminotransferase (ALT).

7. The electrochemical sensor for blood enzyme detection according to claim 3, wherein:

the method comprises the following steps of cleaning the glass carbon sheet by using ultrapure water, polishing by using 400, 800 and 1000-mesh aluminum oxide abrasive paper in sequence, ultrasonically cleaning by using ethanol, soaking in a sulfuric acid solution, and activating by using a cyclic voltammetry.

Technical Field

The invention relates to the field of nano material sensors, in particular to an electrochemical sensor for detecting blood enzyme.

Background

Glutamate pyruvate transaminase (ALT) is mainly present in various cells, most preferably hepatocytes, and has an intracellular transaminase content of about 100 times that of blood. In normal conditions, the activity of the enzyme in the serum can be significantly increased by only a small amount of release into the blood. ALT is released into blood in large quantity in acute stage of various viral hepatitis and when toxic liver cells in medicine are destroyed, so that it is an important index for diagnosing viral hepatitis and toxic hepatitis. The concentration of glutamic-pyruvic transaminase in the liver cells is 1000-3000 times higher than that of serum. As long as 1% of hepatocytes are necrotic, the enzyme activity in blood is increased by 1-fold, and transaminases (especially ALT) are therefore sensitive markers of acute hepatocyte damage.

The existing main methods for detecting ALT comprise a color development method, a chemical titration method, a chromatography or mass spectrometry method, an electrochemical method and the like; especially, the electrochemical method has high detection speed and high precision, and can be combined with the microfluidic technology. However, in the current detection, because the concentration of ALT in the actual detection is generally low and the sample amount is small, the detection accuracy is not good because the combination of the electrode and the sample is poor in the actual detection.

The electrode modification technology developed in recent years modifies the nano material on the electrode, so that the detection precision is improved, and the detection effect is influenced.

Disclosure of Invention

In view of the above, in order to solve the above problems, an electrochemical sensor for blood enzyme detection is provided, which includes a substrate layer, a covering layer, a micro flow channel layer, an upper electrode layer and a lower electrode layer;

the electrochemical sensor is formed by sequentially overlapping a substrate layer, an upper electrode layer, a micro-channel layer, a lower electrode layer and a covering layer from bottom to top; the micro-flow channel layer is provided with a first through hole for liquid flowing and reacting; the upper electrode layer is provided with a counter electrode and a reference electrode, the lower electrode layer is provided with a working electrode, and the positions of the reference electrode, the counter electrode and the working electrode correspond to the through holes of the micro-channel layer; the upper electrode layer and the lower electrode layer are provided with outgoing lines for leading out the electrodes; the covering layer is provided with a sample adding port for adding a sample or a reagent, and the sample adding port is communicated with the through hole of the micro-flow channel layer through a second through hole at the upper electrode layer;

the counter electrode is made of platinum, the reference electrode is made of Ag/AgCl, and the working electrode is a glutamate oxidase-modified nano-gold-modified GCE electrode.

The upper electrode layer is made of PDMS, the preparation method comprises the steps that a first mask plate is used for shielding one side of the upper electrode layer, only the positions of the reference electrode and the counter electrode to be manufactured are leaked, and the two positions are mutually isolated and reach the edge of the PDMS material; then, a layer of platinum metal film is sputtered to a thickness of 10-50 microns at the position of the counter electrode to be prepared by a magnetron sputtering method to obtain a platinum electrode, and the reference electrode is shielded during magnetron sputtering; then shielding the position of the counter electrode, and sputtering a layer of Ag with the thickness of 50-100 mu m on the position of the reference electrode to obtain an Ag electrode; connecting the platinum electrode with a power supply cathode after sputtering, connecting the Ag electrode with a power supply anode, placing one end of the Ag electrode close to the center of the PDMS into HCl solution at normal temperature, and introducing 5mA current for a certain time to obtain an Ag/AgCl electrode at the Ag electrode; and then, leading out Ag and Pt at the edge of the PDMS, and dismantling the first mask plate.

The lower electrode layer is made of a glassy carbon sheet, and the preparation method comprises the following steps:

shielding one side of the lower electrode layer by using a second mask plate, only exposing the position of the working electrode to be manufactured, and then sputtering a layer of Ag under the condition of the mask by using magnetron sputtering, wherein the Ag cannot completely cover the glassy carbon sheet, and the sputtering heating time is less than 100 s;

then placing the glassy carbon sheet in 2.8mmol/L HAuCl₄In the sulfuric acid ethanol aqueous solution; growing for 10-20 min under a constant potential of-0.2V to-0.3V, wherein HAuCl is generated in the growth process₄The Au in the glass-carbon plate forms ions, and is tightly connected to the glass-carbon plate after being replaced by the sputtered Ag; then Au further grows to form nano star or nano flower on the glassy carbon plate; thereby obtaining the Au modified glassy carbon electrode;

then placing the Au modified glassy carbon electrode into a solution containing 50mg/L of glutamate oxidase, 2mg/L of bovine serum albumin, 20mg/L of glutaraldehyde and 0.5% of Nafion, wherein the pH value is 7.3 for 30-60min, so that the glutamate oxidase is modified on the electrode; thereby obtaining the GCE electrode modified by the glutamate oxidase and modified by the nanogold.

The thickness of the substrate layer, the upper electrode layer, the micro-flow channel layer, the lower electrode layer and the covering layer is 500-1000 microns; the basal layer, the upper electrode layer, the micro-flow channel layer and the covering layer are all made of PDMS materials.

The specific surface area of the nano Au formed on the glassy carbon sheet is more than 400m²/g。

The blood enzyme tested was alanine Aminotransferase (ALT).

The method comprises the following steps of cleaning the glass carbon sheet by using ultrapure water, polishing by using 400, 800 and 1000-mesh aluminum oxide abrasive paper in sequence, ultrasonically cleaning by using ethanol, soaking in a sulfuric acid solution, and activating by using a cyclic voltammetry.

The invention has the beneficial effects that:

1) the glassy carbon plate is directly used as a single working electrode and is separated from the counter electrode and the reference electrode, so that the processing cost is low, the structure is novel, and the detection effect is good;

2) the counter electrode and the reference electrode are prepared on one piece of material, and the platinum electrode is fully utilized to prepare the reference electrode when the counter electrode and the reference electrode are prepared, so that the manufacturing efficiency can be improved;

3) silver is sprayed on the glassy carbon plate, gold is replaced, and gold nano dendrites, nano stars or gold nano stars and other structures are further grown on the basis, so that the connection strength of the modified gold and the glassy carbon plate is greatly increased, the specific surface area is increased, and the detection precision is improved. And the prepared nano structure can be adjusted, so that the relationship between the prepared morphology and the detection sensitivity can be further fully researched.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosed subject matter, are incorporated in and constitute a part of this specification. The drawings illustrate the implementations of the disclosed subject matter and, together with the detailed description, serve to explain the principles of implementations of the disclosed subject matter. No attempt is made to show structural details of the disclosed subject matter in more detail than is necessary for a fundamental understanding of the disclosed subject matter and various modes of practicing the same.

FIG. 1 is a schematic front view of the present invention;

FIG. 2 is an exploded schematic view of the present invention;

FIG. 3 is a schematic cross-sectional view of the present invention;

FIG. 4 is a standard graph of the test of the present invention.

Detailed Description

The advantages, features and methods of accomplishing the same will become apparent from the drawings and the detailed description that follows.