CN111638256B - Electrochemical sensor for detecting blood enzyme - Google Patents

Electrochemical sensor for detecting blood enzyme Download PDF

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CN111638256B
CN111638256B CN202010697924.7A CN202010697924A CN111638256B CN 111638256 B CN111638256 B CN 111638256B CN 202010697924 A CN202010697924 A CN 202010697924A CN 111638256 B CN111638256 B CN 111638256B
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CN111638256A (en
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王龙喜
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels

Abstract

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 material, and the reference electrode is prepared by fully utilizing the platinum 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.

Description

Electrochemical sensor for blood enzyme detection
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 the acute stage of various viral hepatitis and when toxic liver cells in the medicine die, 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.
In the electrode modification technology developed in recent years, the electrode is modified with the nano material, 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 detecting blood enzyme is provided, which includes a substrate layer, a covering layer, a micro 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 lead-out wires 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, and 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 a reference electrode and a counter electrode to be manufactured are leaked, and the two positions are mutually isolated and reach the edge of 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 glass 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 HAuCl 4 In 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 process 4 The 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 400m 2 /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 beneficial effects of the invention are as follows:
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.
Example 1:
the blood enzyme tested was alanine Aminotransferase (ALT).
An electrochemical sensor for blood enzyme detection comprises a substrate layer 1, a covering layer 5, a micro-channel layer 3, an upper electrode layer 4 and a lower electrode layer 2;
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-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 lead-out wires 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.
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 the reference electrode 42 and the 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, a layer of platinum metal film is sputtered by a magnetron sputtering method at the position of the counter electrode 41 to be prepared, the sputtering thickness is 10-50 mu m, a platinum electrode is obtained, and the reference electrode 42 is shielded 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, connecting the Ag electrode with a power supply anode after sputtering, placing one end of the Ag electrode close to the center of 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 2 is made of a glassy carbon sheet, and the preparation method comprises the following steps:
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.
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 sputtering a layer of Ag under the mask condition 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 HAuCl 4 In the sulfuric acid ethanol aqueous solution; growing for 10-20 min under a constant potential of-0.2V to-0.3VLong in-process HAuCl 4 The 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 1, the upper electrode layer 4, the micro-channel layer 3, the lower electrode layer 2 and the covering layer 5 is 500-1000 microns; wherein the substrate layer 1, the upper electrode layer 4, the micro-channel layer 3 and the cover layer 5 are all made of PDMS materials.
The specific surface area of the nano Au formed on the glassy carbon sheet is more than 400m 2 /g。
Example 2:
ALT detection was performed using the sensor of example 1.
The principle of the reaction is as follows:
Figure BDA0002591930020000041
l-glutamic acid + H 2 O + glutamate oxidase (FAD) → alpha-ketoglutarate + NH 3 + glutamate oxidase (FADH) 2 )
Glutamate oxidase (FADH) 2 )+O 2 → glutamate oxidase (FAD) + H 2 O 2
H 2 O 2 →2H + +O 2 +4e -
The detection method comprises the steps of adding a reaction reagent into the sample adding hole, wherein the reaction reagent is human serum, and the ALT content is respectively 10g/L, 20g/L, 100g/L, 200g/L and 1000 g/L; the reaction reagent also contains L-alanine and alpha-ketoglutaric acid
Then 300mv voltage is selected, the voltage of the working electrode relative to the reference electrode is positive 300mv, and the content of glutamic-pyruvic transaminase in the sample is measured under the condition of room temperature. The measurements were averaged 10 times at each concentration and a standard regression curve was drawn.
The measurement results were as follows:
Figure BDA0002591930020000042
curve C-0.9282I +95.71, R 2 ＝0.9995。
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims (4)

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;
wherein 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;
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; then, leading out Ag and Pt at the edge of PDMS, and dismantling the first mask plate;
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 HAuCl 4 In 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 process 4 The 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 a GCE electrode modified by glutamate oxidase and modified by nanogold;
the specific surface area of the nano Au formed on the glassy carbon sheet is more than 400m 2 /g。
2. The electrochemical sensor for blood enzyme detection according to claim 1, wherein:
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; the basal layer (1), the upper electrode layer (4), the micro-channel layer (3) and the covering layer (5) are all made of PDMS materials.
3. The electrochemical sensor for blood enzyme detection according to claim 1, wherein:
the blood enzyme tested was alanine Aminotransferase (ALT).
4. The electrochemical sensor for blood enzyme detection according to claim 1, 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.
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JP2007187491A (en) * 2006-01-12 2007-07-26 Fujifilm Corp Electrode for electrochemical detection and method of manufacturing same
CN110618179A (en) * 2019-09-23 2019-12-27 正仁（北京）医疗仪器有限公司 Glucose electrochemical microelectrode sensor based on nano porous metal film

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