CN110196272B

CN110196272B - Electrochemical ratio sensor for detecting protein, construction method thereof and method for detecting protein concentration

Info

Publication number: CN110196272B
Application number: CN201910582347.4A
Authority: CN
Inventors: 陈时洪; 李芹; 谭兴容
Original assignee: Chongqing ninth people's hospital; CHONGQING SHAPINGBA DISTRICT PEOPLE'S HOSPITAL; Southwest University
Current assignee: Chongqing ninth people's hospital; CHONGQING SHAPINGBA DISTRICT PEOPLE'S HOSPITAL; Southwest University
Priority date: 2019-06-28
Filing date: 2019-06-28
Publication date: 2020-07-10
Anticipated expiration: 2039-06-28
Also published as: CN110196272A

Abstract

The application provides an electrochemical ratio sensor for detecting protein, a construction method thereof and a method for detecting protein concentration, belonging to the technical field of electrochemical luminescence sensors. The construction method comprises the following steps: modifying the surface of the electrode with a first modification layer to obtain a first modified electrode, wherein the first modification layer comprises metal-g-C₃N₄. And modifying the surface of the first modified electrode with protein liquid to obtain a second modified electrode. And modifying a third modification layer on the surface of the second modification electrode to obtain the electrochemical ratio sensor, wherein the third modification layer comprises isoluminol and a metal-quencher. And placing the electrochemical ratio sensor in persulfate solution containing dissolved oxygen, and performing electrochemical detection by using the electrochemical ratio sensor as a working electrode to obtain a cathode signal value and an anode signal value. And calculating the protein concentration of the protein liquid through the cathode signal value and the anode signal value. The protein concentration value detected by the ratio sensor is more accurate.

Description

Electrochemical ratio sensor for detecting protein, construction method thereof and method for detecting protein concentration

Technical Field

The application relates to the technical field of electrochemical luminescence sensors, in particular to an electrochemical ratio sensor for detecting protein, a construction method thereof and a method for detecting protein concentration.

Background

The existing EC L ratio sensor usually uses quantum dots such as CdS or CdTe and the like as cathode luminophors, and the cathode EC L luminophors and the anode EC L luminophors usually adopt the same co-reaction reagent to simultaneously enhance two EC L signals, the toxicity of the quantum dots such as CdS and CdTe and the like limits the application of the quantum dots in biological analysis, and meanwhile, the two luminophors share the same co-reaction reagent, so that the competitive consumption of the co-reaction reagent is inevitably generated, and the accuracy of a detection result is influenced.

Disclosure of Invention

The application provides an electrochemical ratio sensor for detecting protein, a construction method thereof and a detection method of protein concentration, which can improve the accuracy of protein concentration detection.

In a first aspect, the present embodiments provide a method for constructing an electrochemical ratiometric sensor for detecting proteins, comprising the following steps: modifying the surface of the electrode with a first modification layer to obtain a first modified electrode, wherein the first modification layer comprises metal-g-C₃N₄Protein antibodies and blocking agents. And modifying the surface of the first modified electrode with a second modified layer to obtain a second modified electrode, wherein the second modified layer is protein liquid. And modifying a third modification layer on the surface of the second modification electrode to obtain the electrochemical ratio sensor, wherein the third modification layer comprises isoluminol, a protein antibody, a metal-quencher and a sealant.

The first modification layer comprises metal-g-C₃N₄Protein antibodies and blocking agents at g-C₃N₄The protein antibody can be loaded on g-C by growing metal to make the protein antibody have bonding effect with the metal₃N₄And blocking the non-specific binding sites by a blocking agent, so that the parts of the metal which are not combined with the protein antibody are covered, and the protein is prevented from being adsorbed by the metal. Correspondingly, the third modification layer comprises isoluminol, a protein antibody, a metal-quencher and a blocking agent, after the metal is combined with the quencher, the protein antibody can be bonded with the metal, so that the protein antibody is loaded on the quencher and is matched with the isoluminol to realize the combination of the isoluminol, the metal, the protein antibody and the quencher, and the blocking agent blocks a non-specific binding site. Forming protein liquid between the first modifying layer and the third modifying layer, matching with the electrode to form an electrochemical ratio sensor, and using g-C for electrochemical detection₃N₄As a cathode luminophore, isoluminol as an anode luminophore, g-C₃N₄Imparting a high cathodic signal to the electrode, the quencher quenching g-C after the third modified layer is formed on the second modified electrode₃N₄The cathode signal is reduced, and the anode signal of the isoluminol in the third modification layer is increased along with the increase of the concentration of the protein solution. By simultaneously changing the cathode signal and the anode signal, the cathode signal is reduced, and the anode signal is increased, so that the ratio detection value of the protein concentration is more accurate.

In combination with the first aspect, in another embodiment, the metal-g-C₃N₄The preparation method comprises the following steps: g to C₃N₄Mixing the dispersion, metal salt solution and reducing agent, adding stabilizer, mixing, and separating solid and liquid to obtain metal-g-C₃N₄。

Reducing metal ions in the metal salt solution into metal simple substance particles under the action of a reducing agent, so that the metal simple substance particles are uniformly distributed in g-C₃N₄And after the stabilizer is added, the metal particles can be more stabilized so as to obtain the stable metal-g-C₃N₄。

With reference to the first aspect, in another embodiment, the step of obtaining a first modified electrode after modifying the surface of the electrode with the first modification layer includes: metal-g-C₃N₄And sequentially dripping the dispersion liquid, the protein antibody solution and the sealant solution on the surface of the first electrode for incubation to obtain a first modified electrode.

By stepwise dispensing of the various solutions, the metal-g-C₃N₄The dispersion liquid, the protein antibody solution and the sealant solution are sequentially dripped on the surface of the electrode, so that the surface of the electrode is modified with a first modification layer, and the first modification layer contains metal-g-C₃N₄Protein antibodies and blocking agents.

In another embodiment, in combination with the first aspect, a method of making a metal-quencher comprises: and mixing the quencher solution, the metal salt solution and the reducing agent, adding the stabilizing agent, continuously mixing, and carrying out solid-liquid separation to obtain the metal-quencher.

Through the action of the reducing agent, metal ions in the metal salt solution are reduced into metal simple substance particles, the metal simple substance particles are uniformly combined with the quenching agent, and after the stabilizing agent is added, the metal particles can be more stable, so that the stable metal-quenching agent can be obtained, and the function exertion effect of the quenching agent is better.

In combination with the first aspect, in another embodiment, the reducing agent includes sodium borohydride and/or hydroxylamine hydrochloride. So as to reduce the metal salt solution into elemental metal particles, and the elemental metal particles are uniformly combined with the quenching agent.

With reference to the first aspect, in another embodiment, the step of obtaining the electrochemical ratio sensor after modifying the surface of the second modified electrode with the third modification layer includes: and mixing the metal-quencher dispersion liquid, the protein antibody solution, the isoluminol solution and the sealant solution to obtain a secondary antibody compound solution, and dropwise coating the secondary antibody compound solution on the surface of the second modified electrode.

The second antibody complex solution is formed first, so that the metal-quencher, the protein antibody and the isoluminol can be well combined, and the protein concentration can be conveniently detected.

In combination with the first aspect, in another embodiment, the metal-g-C₃N₄And the metal in the metal-quencher is a metal nanoparticle. Optionally, the metal includes gold, silver and platinumOne or more of (a).

Formation of metal nanoparticles, may be from g to C₃N₄Better binding with protein antibody, so that the protein antibody is loaded on metal-g-C₃N₄The above. The metal nanoparticles grow on the surface of the quencher in situ, the combination is firm, and after the metal nanoparticles are combined with isoluminol and the protein antibody, the combination of all components of the obtained third modification layer is firmer, and the detection of the protein concentration is more accurate.

In combination with the first aspect, in another embodiment, the blocking agent solution includes one or both of a hexitol solution and a BSA solution.

The blocking agent solution described above was used to block non-specific binding sites and prevent non-specific adsorption of the protein fluid.

In another embodiment in combination with the first aspect, the quencher is polyaniline.

The quencher can effectively quench the cathode signal, reduce the cathode signal, enhance the anode signal along with the increase of the concentration of the protein liquid, weaken the cathode signal and enhance the anode signal, thereby realizing the high-sensitivity ratio detection of the protein liquid.

In a second aspect, embodiments of the present application provide an electrochemical ratiometric sensor for detecting a protein, comprising: the electrode, the first modification layer, the second modification layer and the third modification layer. The first modification layer is modified on the surface of the electrode, wherein the first modification layer comprises metal-g-C₃N₄Protein antibodies and blocking agents. The second modification layer is modified on the surface of the first modification layer, wherein the second modification layer is protein liquid. The third modification layer is modified on the surface of the second modification layer, wherein the third modification layer comprises isoluminol, a protein antibody, a metal-quencher and a sealant.

The first modification layer comprises metal-g-C₃N₄Protein antibodies and blocking agents at g-C₃N₄The protein antibody can be loaded on g-C by growing metal to make the protein antibody have bonding effect with the metal₃N₄And blocking non-specific binding sites by blocking agents to exclude eggs from metalsThe binding site of the white antibody is covered, and the protein is prevented from being adsorbed by metal. Correspondingly, the third modification layer comprises isoluminol, a protein antibody, a metal-quencher and a blocking agent, after the metal is combined with the quencher, the protein antibody can be bonded with the metal, so that the protein antibody is loaded on the quencher and is matched with the isoluminol to realize the combination of the isoluminol, the metal, the protein antibody and the quencher, and the blocking agent blocks a non-specific binding site. Forming protein liquid between the first modifying layer and the third modifying layer, matching with the electrode to form an electrochemical ratio sensor, and using g-C for electrochemical detection₃N₄As a cathode luminophore, isoluminol as an anode luminophore, g-C₃N₄Imparting a high cathodic signal to the electrode, the quencher quenching g-C after the third modified layer is modified on the second modified electrode₃N₄The cathode signal is reduced, and the anode signal of the isoluminol in the third modification layer is enhanced along with the increase of the concentration of the protein solution. By simultaneously changing the cathode signal and the anode signal, the cathode signal is reduced, and the anode signal is increased, so that the ratio detection value of the protein concentration is more accurate.

In a third aspect, an embodiment of the present application provides a method for detecting a protein concentration, including the following steps: and placing the electrochemical ratio sensor in a persulfate solution containing dissolved oxygen, and performing electrochemical detection by using the electrochemical ratio sensor as a working electrode to obtain a cathode signal value and an anode signal value, wherein the protein solution is to-be-detected protein solution. And calculating the concentration of the protein solution to be detected according to the cathode signal value and the anode signal value.

Using persulfate and dissolved oxygen as co-reactant for electrochemical ratiometric sensors, g-C₃N₄The luminophores take persulfate as a co-reaction reagent, the isoluminol luminophores take dissolved oxygen as a co-reaction reagent, g-C₃N₄The luminous potential of the luminophor and the isoluminol luminophor can be well distinguished without mutual interference, and the two luminophors do not share the same co-reaction reagent, so that the competition of the co-reaction reagent is avoided, and the ratio of the protein concentration is detectedThe measurement is more accurate.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.

Fig. 1 is a diagram of a method for constructing a secondary antibody complex and an electrochemical ratiometric sensor provided in example 1 of the present application;

FIG. 2 is a graph of the characteristics of PANI and Au @ PANI provided in example 1 of the present application;

FIG. 3 shows g-C provided in example 1 of the present application₃N₄And Au-g-C₃N₄A characteristic diagram of (1);

FIG. 4 is a representation of an electrochemical ratiometric sensor provided in example 1 of the present application;

FIG. 5 is a graph showing the measurement of the concentration of calcitonin solution provided in example 1 of the present application;

FIG. 6 is a graph showing stability and selectivity of the electrochemical ratiometric sensor provided in example 1 of the present application.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The construction method of the electrochemical ratio sensor for detecting the protein comprises the following steps:

s110, preparation g-C₃N₄：

Synthesis of g-C Using high temperature calcination polymerization₃N₄. Firstly, 10-20g of melamine weighed in weight is placed inAnd (3) placing the crucible in a porcelain crucible, and heating the crucible in a muffle furnace for 2-3h at the temperature of 500-700 ℃, wherein the heating rate in the muffle furnace is 4-6 ℃/min. After cooling, the obtained yellow block-shaped solid is block-shaped g-C₃N₄Grinding into powder for later use. Weighing g-C₃N₄Dispersing the powder of 100-200mg in water of 100-200m L, continuously performing ultrasonic treatment for more than 10h, centrifuging the dispersion liquid after ultrasonic treatment at 5000rpm, collecting the upper suspension, and drying the upper suspension to obtain a solid which is g-C₃N₄Nanosheets.

S120, preparation of Metal-g-C₃N₄：

G to C₃N₄Mixing the dispersion, metal salt solution and reducing agent, adding stabilizer, mixing, and separating solid and liquid to obtain metal-g-C₃N₄. Wherein g-C₃N₄g-C in the Dispersion₃N₄Is in flake structure, and gold ions are reduced by the action of a reducing agent to be in g-C₃N₄Metal particles grow on the flakes and the loading of the metal particles is made more stable by the action of the stabilizer for subsequent binding to protein antibodies.

Optionally, the metal is a nano-metal ion, and the metal includes one or more of gold, silver, and platinum. For example: the metal is gold nanoparticles; the metal is silver nano-particles; the metal is platinum nanoparticles, and the metal is gold nanoparticles and silver nanoparticles; the metal is gold nanoparticles and platinum nanoparticles; the metal is silver nano-particle and platinum nano-particle; the metal is gold nanoparticles, silver nanoparticles and platinum nanoparticles. The following description will be given taking gold nanoparticles as an example.

The reducing agent comprises sodium borohydride and/or hydroxylamine hydrochloride. The following description will be given taking sodium borohydride as an example of the reducing agent. The stabilizer comprises potassium citrate and/or sodium citrate. The stabilizer is sodium citrate as an example.

In detail, 2-4mg of g-C is weighed₃N₄Dispersing the nano-sheets in 2-4m L water, adding HAuCl with the mass concentration of 20-40 mu L of 1-4% under stirring₄The solution is kept stirring for 6-8 h. However, the device is not suitable for use in a kitchenThen 50-100 mu L is prepared in situ with the concentration of 10-20 mmol/L sodium borohydride (NaBH)₄) Adding the solution into the mixed solution, stirring for 20-40min to reduce AuCl₄ ^—Continuously adding sodium citrate solution with concentration of 20-40 mu L of 10-20 mmol/L, stirring for 30-60min, centrifuging the mixed solution, washing for several times, and collecting solid Au-g-C₃N₄Disperse in 2m L water for further use to obtain Au-g-C₃N₄And (3) dispersing the mixture.

S130, preparing a quenching agent, wherein the quenching agent is Polyaniline (PANI).

The Polyaniline (PANI) is synthesized by dissolving 0.8-1g ammonium persulfate and 150 plus 200 mu L aniline monomer in 4-10m L hydrochloric acid solution with concentration of 0.8-1.5 mol/L, respectively, performing ultrasonic treatment for 10-15min, dropping the ammonium persulfate hydrochloric acid solution into the aniline solution under stirring, adding 250 plus 300 mu L absolute ethyl alcohol to obtain a mixture, stirring the mixture for more than 12h in ice bath, performing centrifugal separation on the mixture, washing for several times, and dispersing in 8-10m L water to obtain PANI solution.

S140, preparation of metal-quencher:

and mixing the quencher solution, the metal salt solution and the reducing agent, adding the stabilizing agent, continuously mixing, and carrying out solid-liquid separation to obtain the metal-quencher. The metal particles can grow on the quencher in situ, the metal particles grow on the surface of the polyaniline, the combination of the metal particles and the polyaniline is firmer, and all components in the finally obtained secondary antibody compound solution have stable combination force.

The reducing agent comprises sodium borohydride and/or hydroxylamine hydrochloride. The following description will be given taking sodium borohydride as an example of the reducing agent. The stabilizer comprises potassium citrate and/or sodium citrate. The stabilizer is sodium citrate as an example. The quencher is illustrated by Polyaniline (PANI).

In detail, the PANI solution of 50-100 μ L in the above step S130 is added to 2-4m L water, and HAuCl of 20-40 μ L mass concentration of 1-4% is added with stirring₄Stirring the solution for 6-8h, and then preparing sodium borohydride (NaBH) with the concentration of 10-20 mmol/L in situ by 50-100 mu L₄) Adding the solution into the mixed solution, stirring for 20-40min to reduce AuCl₄ ^—Continuously adding sodium citrate solution with the concentration of 20-40 mu L of 10-20 mmol/L, stirring for 30-60min, centrifugally separating and washing the mixed solution for several times to obtain Au-PANI, and dispersing in 2-4m L water to obtain Au-PANI dispersion for later use.

S150, electrode pretreatment:

the electrode may be a glassy carbon electrode or a platinum electrode, and the electrode is described below as a glassy carbon electrode as an example. And respectively polishing a Glassy Carbon Electrode (GCE) with the diameter of 4mm by using alumina powder with the particle size of 3 microns and alumina powder with the particle size of 0.05 microns, and respectively performing ultrasonic treatment on the polished glassy carbon electrode and the absolute ethyl alcohol for three times to obtain a clean glassy carbon electrode surface. Wherein, the surface of the electrode refers to the polished surface of the electrode.

S160, preparing a first modified electrode:

modifying the surface of the glassy carbon electrode with a first modification layer to obtain a first modified electrode, wherein the first modification layer comprises metal-g-C₃N₄Protein antibodies and blocking agents. Wherein the blocking agent solution comprises one or two of hexitol solution and BSA solution, and the blocking agent solution is hexitol solution, and metal-g-C₃N₄Is Au-g-C₃N₄The dispersion is described as an example.

In the first modifying layer, in g-C₃N₄Gold nanoparticles are grown on the surface of the carrier, so that the protein antibody can be bonded with the gold nanoparticles, and the protein antibody is loaded in g-C₃N₄And blocking the non-specific binding sites by hexanol, covering the parts of the gold nanoparticles which are not combined with the protein antibodyAnd the protein solution is prevented from being adsorbed by the gold nanoparticles, so that the detection value of the protein concentration is more accurate.

In this embodiment, the first modified electrode can be obtained by dispensing for a plurality of times, specifically, Au-g-C₃N₄The dispersion liquid, the protein antibody solution and the hexanehexol are sequentially dripped on the surface of the electrode for incubation.

In detail, 10-20 mu L of Au-g-C₃N₄Dropping the dispersion on the surface of a glassy carbon electrode, air-drying at room temperature, and adding 10-20 μ L protein antibody (Ab)₁) Dropping the solution on the surface of the electrode, incubating overnight at 3-6 deg.C, dropping 10-20 μ L Hexitol (HT) on the surface of the electrode, and incubating for 40-60min to block non-specific binding sites to obtain a first modified electrode, and obtaining the first modified electrode (HT/Ab)₁/Au-g-C₃N₄/GCE) is stored at 3-6 ℃ for further use.

By stepwise dispensing of the various solutions, the metal-g-C₃N₄The dispersion liquid, the protein antibody solution and the sealant solution are sequentially dripped on the surface of the electrode, so that the surface of the electrode is modified with a first modification layer, and the first modification layer contains Au-g-C₃N₄Protein antibodies and blocking agents.

S170, preparing a second modified electrode:

and modifying the surface of the first modified electrode with a second modified layer to obtain a second modified electrode, wherein the second modified layer is protein liquid. Wherein, the protein type of the detection protein liquid is related to the protein type of the protein antibody. For example: the protein liquid comprises ferritin, alpha-fetoprotein, oncofetal protein, calcitonin and the like; if the protein antibody is a ferritin antibody, the ferritin antibody can only capture ferritin on the electrode in order to detect the concentration of ferritin; if the protein antibody is an alpha-fetoprotein antibody, the alpha-fetoprotein antibody can only capture the alpha-fetoprotein on the electrode so as to detect the concentration of the alpha-fetoprotein; if the protein antibody is a calcitonin antibody, the calcitonin antibody can only capture the calcitonin protein on the electrode in order to detect the concentration of the calcitonin protein. The detection value of the protein concentration is more accurate.

S180, preparing an electrochemical ratio sensor:

and modifying a third modification layer on the surface of the second modification electrode to obtain the electrochemical ratio sensor, wherein the third modification layer comprises isoluminol, a protein antibody, a metal-quencher and a sealant. The blocking agent solution includes one or two of a hexitol solution and a BSA solution, and the blocking agent solution is a BSA solution, and the metal-quencher is an Au-PANI dispersion liquid as an example.

In the third modification layer, after the gold nanoparticles are combined with the PANI, the protein antibody can be bonded with the gold nanoparticles, so that the protein antibody is loaded on the PANI and is matched with isoluminol (ABEI, (N- (aminobutyl) -N- (ethyl isoluminol))), the combination of the isoluminol, the gold nanoparticles, the protein antibody and the PANI is realized, and the nonspecific combination sites are sealed by a BSA solution. Forming protein liquid between the first modifying layer and the third modifying layer, matching with glassy carbon electrode to form electrochemical ratio sensor, and performing electrochemical detection with g-C₃N₄As a cathode luminophore, isoluminol as an anode luminophore, g-C₃N₄Imparting a high cathodic signal to the electrode, the quencher quenching g-C after the third modified layer is formed on the second modified electrode₃N₄The cathode signal is reduced, and the anode signal of the isoluminol in the third modification layer is increased along with the increase of the concentration of the protein solution. By simultaneously changing the cathode signal and the anode signal, the cathode signal is reduced, and the anode signal is increased, so that the ratio detection value of the protein concentration is more accurate.

In this embodiment, the secondary antibody composite solution may be prepared first, and then the electrochemical ratio sensor may be prepared. Specifically, Au-PANI dispersion, protein antibody ((Ab)₂) Wherein: ab₁And Ab₂Protein of the same type, Ab₁Within the first modification layer, Ab₂In the third modification layer)), the isoluminol solution and the BSA solution are mixed to obtain a secondary antibody compound solution, the secondary antibody compound solution is dripped on the surface of the second modification electrode and is incubated for 1-2h to obtain the electrochemical ratio of the sandwich immune modeA sensor.

In detail, 500-protein antibody (Ab) of 1000. mu. L was added to 1-2m L Au-PANI dispersion₂) Adding 500-1000 mu L ABEI solution with concentration of 8-10 mmol/L, stirring the obtained mixed solution at 3-6 ℃ overnight, adding 100-200 mu L BSA solution with mass concentration of 1-2%, stirring for 1-2h, and centrifuging the mixed solution to collect secondary antibody complex solution BSA-Ab₂ABEI-Au-PANI, dispersed in 1-2m L water, stored at 3-6 ℃ for further use, the prepared secondary antibody complex solution of 10-20 mu L was applied dropwise on the surface of the second modified electrode, and incubated at room temperature for 1-2h, to obtain the electrochemical ratiometric sensor of sandwich immuno-mode.

The second antibody complex solution is formed first, and Au-PANI or protein antibody (Ab) can be added₂) And isoluminol ABEI can be well combined together to form a stable secondary antibody compound solution so as to facilitate the detection of protein concentration.

The electrochemical ratio sensor can be obtained by the preparation method, and the obtained electrochemical ratio sensor can accurately detect the concentration of the protein.

The electrochemical ratio sensor obtained by the above preparation method can be used to detect the concentration of protein. The specific mode comprises the following steps:

s210, using an Ag/AgCl electrode as a reference electrode, a platinum wire electrode as a counter electrode, using the electrochemical ratio sensor prepared by the preparation method as a working electrode, connecting the electrochemical ratio sensor in a cassette of an electrochemiluminescence analyzer, closing the cassette, and setting parameters of the electrochemiluminescence analyzer, wherein the parameters of the electrochemiluminescence analyzer comprise that the scanning voltage range is-1.3-0.6V, the photomultiplier voltage is 800V, the scanning speed is 300mV/S, using persulfate solution containing dissolved oxygen as a test base solution, optionally using PBS solution with the concentration of 0.1-0.2 mol/L and the pH value of 7.4-8 as a base solution, adding K with the concentration of 300-400 nmol/L into the base solution₂S₂O₈And (c) dissolving to obtain a test base solution, wherein the test base solution is not sealed, thereby enabling dissolved oxygen to enter the test base solution.

S220, obtaining a linear curve:

the linear curve is obtained by first configuring standard protein liquids with different concentrations, for example, six standard protein liquids with the concentrations of the protein liquids being 0.1pg/m L, 0.5pg/m L, 1pg/m L, 5pg/m L, 10pg/m L and 40pg/m L, and respectively dropping the standard protein liquids with different concentrations to obtain six electrochemical ratio sensors when preparing the electrochemical ratio sensors, that is, the protein liquid in the step S170 is selected to be one of the six standard protein liquids to obtain one electrochemical ratio sensor, and each standard protein liquid is selected once to obtain six electrochemical ratio sensors.

And respectively placing the six electrochemical ratio sensors in a test base solution, and detecting by using an electrochemiluminescence analyzer to obtain six different cathode signal values and anode signal values of the electrochemical ratio sensors. And calculating the ratio of the cathode signal value to the anode signal value, calculating the logarithm value of the ratio to obtain 6 logarithm values of the ratio, calculating the logarithm value of the concentration to obtain 6 corresponding logarithm values of the concentration. And taking the logarithm value of the concentration as an abscissa and the logarithm value of the ratio as an ordinate to obtain a linear curve.

S230, detecting the concentration of the protein solution:

and (3) selecting the protein solution to be detected in the step (S170) to obtain an electrochemical ratio sensor, placing the electrochemical ratio sensor in persulfate solution containing dissolved oxygen, and performing electrochemical detection by using the electrochemical ratio sensor as a working electrode to obtain a cathode signal value and an anode signal value. And calculating the ratio of the cathode signal value to the anode signal value, calculating the logarithm value of the ratio, putting the logarithm value into a linear curve to obtain the logarithm value of the concentration of the protein to be detected, and calculating the concentration of the protein to be detected.

Wherein, when electrochemical detection is carried out, g-C is used₃N₄As a cathode luminophore, isoluminol as an anode luminophore, g-C₃N₄Imparting a high cathodic signal to the electrode, the quencher quenching g-C after the third modified layer is formed on the second modified electrode₃N₄The signal of (a) is received,the cathode signal is reduced, and the anode signal of isoluminol in the third modification layer is increased along with the increase of the concentration of the protein solution. By simultaneously changing the cathode signal and the anode signal, the cathode signal is reduced, and the anode signal is increased, so that the ratio detection value of the protein concentration is more accurate.

And using persulfate and dissolved oxygen as co-reactant for the electrochemical ratiometric sensor, g-C₃N₄The luminophores take persulfate as a co-reaction reagent, the isoluminol luminophores take dissolved oxygen as a co-reaction reagent, g-C₃N₄The luminescence potential of the luminophor and the isoluminol luminophor can be well distinguished without mutual interference, and the two luminophors do not share the same co-reaction reagent, so that the competition of the co-reaction reagent is avoided, and the ratio detection of the protein concentration is more accurate.

Example 1

FIG. 1 is a diagram of a method of constructing a secondary antibody complex and an electrochemical ratiometric sensor. Referring to fig. 1 of the drawings, a drawing,

the method for detecting the concentration of calcitonin comprises the following steps:

(1) preparation of g-C₃N₄：

Firstly, 10g of weighed melamine is placed in a ceramic crucible, and then the crucible is placed in a muffle furnace to be heated for 2 hours at the temperature of 600 ℃, wherein the heating rate in the muffle furnace is 5 ℃/min. After cooling, the obtained yellow block-shaped solid is block-shaped g-C₃N₄Grinding into powder for later use. Weighing g-C₃N₄Dispersing 100mg of the powder in 100m L of water, continuously performing ultrasonic treatment for more than 10m, centrifuging the ultrasonic dispersion at 5000rpm, collecting the upper suspension, and drying to obtain solid g-C₃N₄Nanosheets.

(2) Preparation of Au-g-C₃N₄：

Weighing 2mg of g-C₃N₄Dispersing the nano-sheets in 2m L water, adding HAuCl with the mass concentration of 20 mu L of 1% under stirring₄The solution was kept under stirring for 6h, then 50. mu. L freshly prepared 10 mmol/L sodium borohydride (NaBH)₄) Adding the solution into the mixtureMixing the solution, stirring for 20min to reduce AuCl₄ ^—Adding sodium citrate solution with concentration of 20 mu L of 10 mmol/L, stirring for 30min, centrifuging and washing the mixed solution for several times, and collecting solid Au-g-C₃N₄Disperse in 2m L water for further use to obtain Au-g-C₃N₄And (3) dispersing the mixture.

(3) Synthetic Polyaniline (PANI): referring to FIG. 1(A), the preparation process of the secondary antibody complex,

respectively dissolving 0.91g of ammonium persulfate and 184 mu L of aniline monomer in 4m of hydrochloric acid solution with L concentration of 1 mol/L, carrying out ultrasonic treatment on both solutions for 10min, dropwise adding the ammonium persulfate hydrochloric acid solution into the aniline solution under the stirring condition, then adding 250 mu L of absolute ethyl alcohol to obtain a mixture, stirring the mixture for more than 12h in an ice bath, carrying out centrifugal separation on the finally obtained mixture, washing for several times, and dispersing in 8m of L water to obtain the PANI solution.

(4) Preparation of Au @ PANI: referring to FIG. 1(A),

the PANI solution 50. mu. L was added to 2m L water, and HAuCl was added to the solution at a concentration of 1% by mass of 20. mu. L with stirring₄The solution was kept under stirring for 6h, then 50. mu. L freshly prepared sodium borohydride (NaBH) with a concentration of 10 mmol/L₄) Adding the solution into the mixed solution, and stirring for 30min to reduce AuCl₄ ^—Adding sodium citrate solution with the concentration of 20 mu L being 10 mmol/L continuously, stirring for 30min, centrifuging and washing the mixed solution for several times to obtain Au @ PANI, and dispersing in 2m L water to obtain Au @ PANI dispersion for later use.

(5) Preparing a secondary antibody complex solution: referring to FIG. 1(A),

add 500. mu. L calcitonin antibody (Ab) to 1m L of Au @ PANI dispersion₂) The solution was added with 500. mu. L isoluminol ABEI solution at 8 mmol/L and the resulting mixed solution was stirred overnight at 4. mu.g.then 100. mu. L BSA solution at 1% by mass was added and stirring was continued for 1h₂-ABEI-Au @ PANI, dispersed in 1m L water and stored at 4 ℃ until use.

(6) Electrode pretreatment:

and respectively polishing a Glassy Carbon Electrode (GCE) with the diameter of 4mm by using alumina powder with the particle size of 3 microns and alumina powder with the particle size of 0.05 microns, and respectively performing ultrasonic treatment on the polished glassy carbon electrode and the absolute ethyl alcohol for three times to obtain the clean surface of the glassy carbon electrode.

(7) And preparing an electrochemical ratio sensor: referring to fig. 1(B), construction and detection of an electrochemical ratiometric sensor,

mixing 10 mu of L Au-g-C₃N₄Dropping the dispersion on the surface of a glassy carbon electrode, air-drying at room temperature to obtain a first electrode, and adding 10 mu L calcitonin antibody (Ab)₁) The solution was dropped onto the surface of the first electrode and incubated overnight at 4 ℃ to give a second electrode, then 10 μ L Hexanehexol (HT) was dropped onto the surface of the second electrode and incubated for 40min to block non-specific binding sites₁/Au-g-C₃N₄/GCE) was stored at 4 ℃ until use.

Respectively dripping standard calcitonin liquid with the concentration of 0.1pg/m L, standard calcitonin liquid with the concentration of 0.5pg/m L, standard calcitonin liquid with the concentration of 1pg/m L, standard calcitonin liquid with the concentration of 5pg/m L, standard calcitonin liquid with the concentration of 10pg/m L, standard calcitonin liquid with the concentration of 40pg/m L, first calcitonin liquid to be detected, second calcitonin liquid to be detected and third calcitonin liquid to be detected on the surface of the first modified electrode, and incubating for 1h at room temperature to obtain a second modified electrode.

And dripping 10 mu L of the prepared secondary antibody compound solution on the surface of the second modified electrode, and incubating for 1h at room temperature to obtain the electrochemical ratio sensor in the sandwich immune mode, wherein 5 standard electrochemical ratio sensors and 3 electrochemical ratio sensors to be detected are obtained in total.

(8) And obtaining a standard curve:

connecting a standard electrochemical ratio sensor prepared by the preparation method as a working electrode with an Ag/AgCl electrode as a reference electrode and a platinum wire electrode as a counter electrode in a cassette of an electrochemiluminescence analyzer, adding PBS solution with the concentration of 0.1-0.2 mol/L and the pH value of 7.4-8 as base solution into the base solutionK at a concentration of 300-400 nmol/L₂S₂O₈The solution is used as a test base solution, and the test base solution contains dissolved oxygen. And (3) closing the cassette, setting the parameters of the electrochemiluminescence analyzer, and detecting the standard calcitonin liquid under the conditions that the scanning voltage range is-1.3-0.6V, the voltage of the photomultiplier is set to 800V, and the scanning speed is 300 mV/S.

Six different cathodic signal values and anodic signal values were detected using the 6 standard electrochemical ratio sensors described above. And calculating the ratio of the cathode signal value to the anode signal value, calculating the logarithm value of the ratio to obtain 6 logarithm values of the ratio, calculating the logarithm value of the concentration to obtain 6 corresponding logarithm values of the concentration. Taking the logarithm of the concentration as the abscissa and the logarithm of the ratio as the ordinate, a standard curve relating to the calcitonin concentration is obtained.

(9) And obtaining the concentration of the calcitonin solution to be detected:

and (3) taking the 3 electrochemical rate sensors to be detected obtained in the step (7) as working electrodes to perform electrochemical detection in the step (8) to obtain 3 different cathode signal values and anode signal values. And calculating the ratio of the cathode signal value to the anode signal value, calculating the logarithm value of the ratio, putting the logarithm value into a standard curve to obtain the logarithm value of the concentration of the calcitonin liquid to be detected, and calculating the concentration of the calcitonin liquid to be detected.

Experimental example 1

FIG. 2 is a graph of the characteristics of PANI and Au @ PANI, wherein the microscopic features of PANI and Au @ PANI provided in example 1 above were observed using a Scanning Electron Microscope (SEM), SEM images of PANI (A) and PANI-Au (B), and the elements of Au @ PANI were characterized using X-ray photoelectron spectroscopy (XPS), and (C) XPS spectra of PANI-Au; (D) XPS spectra of Au, (E) C, (F) N. In fig. 2(a), a scanning electron micrograph of PANI is shown, and it can be seen that PANI has a nanorod structure that is staggered with each other. Fig. 2(B) shows a scanning electron micrograph of Au @ PANI, and it can be seen that a large number of Au nanoparticles are generated on the PANI surface. The elemental composition of Au-PANI was characterized using X-ray photoelectron spectroscopy (XPS). Fig. 2(C) shows an XPS survey of Au @ PANI, and characteristic peaks of Au 4f, C1 s and N1 s can be clearly observed. The characteristic peaks such as 88.05eV and 84.35eV shown in FIG. 2(D), 284.55eV shown in FIG. 2(E), and 399.45eV shown in FIG. 2(F) correspond to the characteristic peaks of Au 4F, C1 s, and N1 s, respectively. Illustrating the successful preparation of Au @ PANI.

FIG. 3 is g-C₃N₄And Au-g-C₃N₄Using a Transmission Electron Microscope (TEM) on the g-C provided in example 1 above₃N₄And Au-g-C₃N₄The microscopic morphology of the particles was observed, g-C₃N₄(A) And Au-g-C₃N₄(B) SEM picture of (g) of Au-g-C using X-ray photoelectron spectroscopy (XPS)₃N₄Is characterized by (C) Au-g-C₃N₄XPS spectra of (a); (D) XPS spectra of C, (E) N, (F) Au. Wherein FIG. 3(A) shows g-C₃N₄As seen in the projection electron micrograph, g-C₃N₄Presenting a lamellar structure. FIG. 3(B) shows Au-g-C₃N₄The projection electron microscope shows that the Au nano-particles grow uniformly in g-C₃N₄The surface of the nanoplatelets. Characterization of Au-g-C using X-ray photoelectron spectroscopy (XPS)₃N₄The composition of elements (A) and (B). FIG. 3(C) shows Au-g-C₃N₄The XPS full spectrum of the sample shows that characteristic peaks of C1 s, N1 s and Au 4f can be observed. Among them, 288.3eV shown in FIG. 3(D), 398.85eV shown in FIG. 3(E), and 87.35eV and 83.65eV shown in FIG. 3(F) correspond to the characteristic peaks of C1 s, N1 s, and Au 4F, respectively. Illustrates Au-g-C₃N₄The successful preparation.

FIG. 4 is a graph representing the electrochemical ratiometric sensor, and the Cyclic Voltammetry (CV) results for the modified electrode are shown in FIG. 4(A), a, bare electrode, b, bare electrode/Au-g-C₃N₄C, bare electrode/Au-g-C₃N₄Ab1, d, bare electrode/g-C₃N₄Ab1/HT, e, bare electrode/g-C₃N₄CV characterization in the presence of 5.0 mmol/L [ Fe (CN) ]₆]^4–/3–In PBS (pH7.4), modified Au-g-C compared to a bare electrode (glassy carbon electrode) (curve a)₃N₄The peak current of the electrode of (1) is reduced (curve b). Due to protein molecules andinhibition of electron transport by Hexanol (HT) molecules when Ab is incubated sequentially at the electrodes₁The redox peak currents of the CV curves decrease sequentially after HT and Calcitonin (CT) (curves c, d, e).

The results of the alternating impedance (EIS) of the modified electrodes are shown in FIG. 4(B), a, bare electrode, B, bare electrode/Au-g-C₃N₄C, bare electrode/Au-g-C₃N₄Ab1, d, bare electrode/g-C₃N₄Ab1/HT, e, bare electrode/g-C₃N₄EIS assay at 5.0 mmol/L [ Fe (CN) ]₆]^4–/3–In PBS solution (ph7.4), the semi-circle diameter of the bare electrode (glassy carbon electrode) is small (curve a), corresponding to relatively low ac impedance values. Au-g-C₃N₄Compared with the bare electrode, the semi-circle diameter of the modified electrode in the impedance spectrum is increased (curve b) because of the modification layer Au-g-C₃N₄Thereby inhibiting electron transfer. When the electrodes were incubated with antibody, HT and CT sequentially, the semi-circle diameters of EIS profiles increased sequentially (curves, d, e), indicating that the impedance values increased sequentially due to the blocking effect of antibody, HT and CT on electron transport. CV and EIS characterize successful modification of the electrode.

As shown in FIG. 4(C), a, non-incubated BSA-Ab₂-ABEI-Au @ PANI and b, incubation of BSA-Ab₂EC L response after ABEI-Au @ PANI, the anode had almost no EC L signal, while g-C before no incubation of the secondary antibody complex (curve a)₃N₄At S₂O₈ ^2-In the presence of the cathode, a strong EC L signal was generated at-1.3V Au-g-C₃N₄At S₂O₈ ^2-In the presence, the mechanism of light emission is:

g-C₃N₄+e^-→g-C₃N₄ ^-(1)

S₂O₈ ^2-+e^-→SO₄ ^2-+SO₄ ^·-(2)

g-C₃N₄ ^·-+SO₄ ^·-→g-C₃N₄*+SO₄ ^2-(3)

and/or

g-C₃N₄+SO₄ ^·-→g-C₃N₄ ⁺+SO₄ ^2-(4)

g-C₃N₄ ⁺+g-C₃N₄ ^·-→g-C₃N₄*+g-C₃N₄(5)

Finally, the process is carried out in a batch,

g-C₃N₄*→g-C₃N₄+hν (6)

when the secondary antibody complexes were incubated on the electrodes (curve b), the ABEI produced an EC L signal at +0.6V, while Au-g-C₃N₄The signal at-1.3V decreases. The mechanism of light emission by the ABEI in the presence of dissolved oxygen is:

ABEI－e^-→ABEI^·+(1)

O₂+e^-→O₂ ^·-(2)

ABEI^·++O₂ ^·-→ABEI* (3)

ABEI*→ABEI+hv (4)

after loading of the secondary antibody complex, g-C₃N₄Cathode signal reduction of cathode luminophores, g-C₃N₄Energy transfer takes place with the anode emitter PANI. To verify g-C₃N₄Mechanism of energy transfer with PANI for g-C₃N₄The EC L spectrum and the UV-vis absorption spectrum of PANI were scanned, as shown in FIG. 4(D), the UV-vis absorption spectra of a, PANI, and b, g-C₃N₄The spectrum of EC L, PANI, has a broad absorption band (curve a), g-C, around 300 to 500nm₃N₄The maximum radiation wavelength of the spectrum of EC L is around 460nm, g-C₃N₄The EC L spectrum of (A) and the UV-vis absorption spectrum of PANI have a large degree of overlap, indicating that energy transfer between them is likely to occur, PANI for g-C₃N₄Is due to energy transfer and can reduce g-C by PANI₃N₄The cathode signal and the anode signal are prevented from interfering with each other, so that an accurate calcitonin detection value is obtained.

FIG. 5 is a graph showing the detection of the concentration of calcitonin solution, as shown in FIG. 5(A), the EC L response of the sensor to different concentrations of CT, a-f are respectively 0.1pg/m L, 0.5pg/m L, 1pg/m L, 5pg/m L, 10pg/m L and 40pg/m L, when different concentrations of target CT are incubated on the prepared sensor, the ABEI signal of the anode is gradually increased along with the increase of the concentration, and at the same time, the Au-g-C of the cathode is increased along with the increase of the concentration₃N₄The signal gradually decreases. As shown in FIG. 5(B), the log of the ratio of cathode signal to anode signal and the log of the concentration of the target CT in the standard curve show a good linear relationship, and the linear regression equation is lg (I)_Au-g-C3N4/I_ABEI) Table 1 was obtained comparing the concentration of the target CT with-0.43 lgc-5.23, the correlation coefficient R with 0.993, and the detection limit with 0.033pg/m L.

TABLE 1 comparison of the Performance of calcitonin detection by different methods

Method of producing a composite material	Linear range (ng/m L)	Detection limit (mol/L)	Reference to the literature
				Radioimmunoassay	0.02-3.0	——	Comparative example 1
Electrochemistry method	10-8.0×10⁶	3.5	Comparative example 2
				Electrochemistry method	1.0×10^-3～1.0	0.7×10^-3	Comparative example 3
Electrochemical ratiometric sensor	1.0×10^-4～4.0×10^-2	0.33×10^-4	This application

Among them, comparative example 1 is described in Radioimmunoassay of calcium in Normal HumanUrine, analytical Chemistry, Snider, R.h., Moore, C.F, Silva, O. L, Becker, K. L, 1978,50,449 454, comparative example 2 is described in Detection of calcium in medium carbon by electrochemical sensor, Sha, D.S., Zhuge, b.z., L in, F.Int.J.Electrochem.Sci.2017, 12,10129, 10139. and comparative example 3 is described in Albel-free electrochemical sensor, 1109. J.Electrochem.Sci.2017,12,10129, 11035. orange-carbon in 1109. J..

As can be seen from Table 1, the electrochemical ratio sensor provided by the application has a lower detection limit, and the detection value of the concentration of the calcitonin liquid is more accurate.

FIG. 6 is a graph of stability and selectivity of an electrochemical ratiometric sensor, wherein FIG. 6(A) shows the stability of the sensor and FIG. 6(B) shows the selectivity of the sensor, the sensor incubated at 1pg/m L CT was continuously scanned in cycles for 5 cycles to test the stability of the sensor, as shown in FIG. 6(A), the EC L intensity changes were small, indicating better stability, for the purpose of assessing the selectivity of the prepared EC L sensor, ferritin (Fer), Alpha Fetoprotein (AFP), carcinoembryonic antigen (CEA) were selected for testing, as shown in FIG. 6(B), the above interferents did not cause significant signal changes compared to the blank experiment, however, the log of the EC L intensity ratio was significantly reduced in the presence of CT, as CT antibodies are specific for CT, other proteins cannot be captured on the electrodes, and no corresponding signal changes could be generated, the above results indicate that the resulting ratiometric EC L sensor has high selectivity to CT.

The results of the three calcitonin solutions to be detected provided in example 1 are shown in Table 2. Wherein, the three calcitonin liquids to be detected are 3 serum samples obtained from the ninth people hospital in Chongqing, the constructed sensors are respectively used for testing the content of calcitonin, and are compared with the detection values provided by the hospital, and the relative error (E) is calculated_r)。

TABLE 2 measurement values of calcitonin solutions to be detected

Sample (I)	Hospital test value (pg/m L)	Detection value (pg/m L)^a	Er(％)
				The first calcitonin solution to be detected	3.25	3.35±0.176	+3.1
Second calcitonin solution to be detected	1.62	1.55±0.125	-4.3
				Third calcitonin solution to be detected	0.67	0.743±0.069	+10.1

As can be seen from Table 2, the calcitonin concentration value obtained by the method for detecting calcitonin concentration provided by the application has smaller error compared with the detection value provided in hospitals, and has wide application prospect.

In summary, the electrochemical ratio sensor provided by the present application has the following main advantages:

(1) g-C being non-toxic₃N₄As a cathode emitter, ABEI as an anode emitter, S₂O₈ ^2-And dissolving O₂The EC L rate type sensor is respectively constructed as the co-reaction reagent, so that the toxicity problem caused by using the traditional quantum dot containing heavy metal as a cathode luminophor is avoided, the limitation that two luminophors share the same co-reaction reagent in the common EC L rate strategy is broken, and the inaccuracy of the detection result caused by competitive consumption when two luminophors use one co-reaction reagent is effectively avoided.

(2) Polyaniline (PANI) as g-C₃N₄The efficient quencher for the EC L signal of (1), can make g-C₃N₄So as to obtain a ratio detection value.

(3) The electrochemical ratio sensor has the advantages of being rapid, sensitive, low in cost and the like, the cathode signal and the anode signal-double signal are used for detecting the concentration of the calcitonin, the defect that the existing single-signal-based calcitonin detection method is easily interfered by environmental and instrument factors can be effectively overcome (if the cathode signal is reduced and the anode signal is increased in the detection process, the concentration of protein liquid is caused by different concentrations, and if the cathode signal is not reduced and the anode signal is increased, the external environmental factor is interfered), and the accuracy and the reliability of the detection result are remarkably improved.

The foregoing is illustrative of the present application and is not to be construed as limiting thereof, as numerous modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing an electrochemical ratio sensor for detecting proteins is characterized by comprising the following steps:

mixing Au-g-C₃N₄The dispersion liquid, the protein antibody solution and the sealant solution are sequentially dripped on the surface of the first electrode to be incubated to obtain a first modified electrode;

modifying a second modification layer on the surface of the first modification electrode to obtain a second modification electrode, wherein the second modification layer is protein liquid;

mixing the Au-polyaniline dispersion solution, the protein antibody solution, the isoluminol solution and the sealant solution to obtain a secondary antibody compound solution, and dropwise coating the secondary antibody compound solution on the surface of the second modified electrode for incubation to obtain the electrochemical ratio sensor;

wherein the co-reactant of the electrochemical ratiometric sensor is a persulfate solution containing dissolved oxygen.

2. The method for constructing an electrochemical ratiometric sensor for detecting proteins according to claim 1, wherein the Au-g-C is₃N₄The preparation method comprises the following steps:

g to C₃N₄Dispersion liquid, HAuCl₄Mixing the solution with a reducing agent, adding a stabilizer, continuously mixing, and carrying out solid-liquid separation to obtain Au-g-C₃N₄。

3. The method for constructing an electrochemical ratiometric sensor for detecting proteins according to claim 1, wherein the preparation method of Au-polyaniline comprises:

mixing polyaniline solution and HAuCl₄And mixing the solution and a reducing agent, adding a stabilizing agent, continuously mixing, and carrying out solid-liquid separation to obtain the Au-polyaniline.

4. The method for constructing an electrochemical ratio sensor for detecting proteins of claim 3, wherein the reducing agent comprises sodium borohydride and/or hydroxylamine hydrochloride.

5. The method for constructing an electrochemical ratio sensor for detecting proteins of any one of claims 1 to 4, wherein the blocking agent solution includes one or both of a hexitol solution and a BSA solution.

6. An electrochemical rate sensor for detecting a protein, constructed by the construction method according to any one of claims 1 to 5, comprising:

an electrode is arranged on the base plate and is provided with a plurality of electrodes,

a first modification layer which modifies the surface of the electrode, wherein the first modification layer comprises Au-g-C₃N₄Protein antibodies and blocking agents;

the second modification layer is modified on the surface of the first modification layer, wherein the second modification layer is protein liquid;

the third modification layer is modified on the surface of the second modification layer, and comprises isoluminol, a protein antibody, Au-polyaniline and a sealant.

7. A method for detecting a protein concentration, comprising the steps of:

placing the electrochemical ratio sensor constructed by the construction method of the electrochemical ratio sensor for detecting protein according to any one of claims 1 to 5 in persulfate solution containing dissolved oxygen, and performing electrochemical detection by using the electrochemical ratio sensor as a working electrode to obtain a cathode signal value and an anode signal value, wherein the protein solution is a protein solution to be detected;

and calculating the concentration of the protein solution to be detected according to the cathode signal value and the anode signal value.