CN118311121A - Ultrasensitive electrochemical device based on paper chip water channel and working method thereof - Google Patents

Abstract

The invention discloses an ultrasensitive electrochemical device based on a paper chip water channel and a working method thereof, wherein the ultrasensitive electrochemical device comprises a sample cell, a sample pad, a probe pad, a water absorption pad and an electrode chip which are arranged from top to bottom; the sample cell is used for storing a solution to be tested; the sample pad is used for filtering impurities in the solution to be detected; the probe pad is used for generating immune reaction with the filtered solution to be detected through a probe to obtain an immune complex; the water absorption pad is used for absorbing redundant immune complex and providing chromatographic power; the electrode chip is used for capturing immune complex to generate electrochemical response. According to the invention, the nano-silver flowers are modified, so that the single silver flower with smaller size can generate the same electrochemical response which is several times larger than that of the silver flower, and the smaller size is beneficial to the free flow of the silver flower in the paper chip, so that the ultra-sensitivity is realized, the real-time detection is realized, the cost is low, and the use is convenient.

Description

An ultra-sensitive electrochemical device based on paper chip water channel and its working method

Technical Field

The present invention belongs to the field of POCT technology, and in particular relates to an ultra-sensitive electrochemical device based on a paper chip water channel and a working method thereof.

Background technique

Proteins are important building blocks of human cells and tissues. All important components and physiological activities of the body require the participation of proteins, which catalyze and control all cellular processes. Protein analysis provides key information on most biological processes, cell traits, and diseases, and has various metabolic, structural, and regulatory functions in different activities and diseases. Given this background, and its importance as an indicator of various diseases and disease progression, the assessment of its levels is of particular interest.

The detection of trace protein biomarkers produced in the early stage of the disease must meet the following conditions: clinical sensitivity, specificity, and a suitable diagnostic window. However, due to the low concentration in early samples and the possibility of nonspecific interactions, the detection of specific proteins often poses certain challenges. In order to avoid the problem of concentration limitations and nonspecific interactions, several methods for determining proteins in samples have been established, including chromatography (high performance liquid chromatography-HPLC, liquid chromatography/mass spectrometry-LC/MS) and antibody-dependent methods such as enzyme-linked immunosorbent assay (ELISA), western blot or colorimetry and fluorescence assays. Although the above are visual prospects and suitable solutions for detecting specific protein biomarkers, these technologies require time-consuming and labor-intensive procedures, well-trained technicians, and sometimes additional sample preparation steps, which makes the overall detection cost expensive.

Summary of the invention

In order to solve the above technical problems, the present invention proposes an ultra-sensitive electrochemical device based on a paper chip water channel and a working method thereof to solve the problems existing in the above prior art.

To achieve the above object, the present invention provides an ultra-sensitive electrochemical device based on a paper chip water channel, comprising a sample pool, a sample pad, a probe pad, a water absorbent pad and an electrode chip arranged from top to bottom;

The sample pool is used to store the solution to be tested;

The sample pad is used to filter impurities in the solution to be tested;

The probe pad is used to generate an immune reaction between the probe and the filtered test solution to obtain an immune complex;

The absorbent pad is used to absorb excess immune complexes and provide chromatography power;

The electrode chip is used to capture the immune complex to generate an electrochemical response.

Preferably, the manufacturing process of the electrode chip includes: adhering a polyimide film tape to the surface of a phenolic resin plate to form a laser-induced graphene electrode substrate;

Using a laser engraving machine to perform laser burning on the laser-induced graphene electrode substrate to obtain a graphene electrode;

Connecting a copper tape to the end of the graphene electrode, and applying a mixed solution of Ag and AgCl to the graphene electrode to obtain a new reference electrode, and packaging the release film, the polyurethane elastomer rubber film and the new reference electrode in sequence by a laminating machine to obtain a working electrode;

The working electrode is incubated with an N-N-dimethylformamide solution containing 1-pyrenebutyric acid N-hydroxysuccinimide ester, and the non-specifically bound 1-pyrenebutyric acid N-hydroxysuccinimide ester is washed away with a dimethylformamide solution, alcohol, and polybutylene succinate in sequence;

Incubate the working electrode with a capture antibody solution of the protein to be detected and rinse the electrode with polybutylene succinate;

The working electrode was incubated with a bovine serum albumin solution, and then the electrode was rinsed with polybutylene succinate and ultrapure water to remove free bovine serum albumin, thereby obtaining an electrode chip.

Preferably, the probe pad comprises a water channel pad and a probe;

The production process of the water channel pad includes: using a wax jet printer to print annular paraffin wax on the surface of filter paper, placing the filter paper printed with the annular paraffin wax on a hot plate at 180° C. for heating, and obtaining a water channel pad with a three-dimensional water channel structure inside the filter paper.

Preferably, the production process of the probe includes:

Add ultrapure water, silver nitrate solution and citric acid solution to the centrifuge tube in sequence, stir with a magnetic stirrer in an ice-water bath and in the dark, then add ascorbic acid solution and continue stirring, and after stirring, centrifuge, wash and dry to obtain nano silver flowers;

Adding cysteine solution to the nano silver flower, mixing in an ultrasonic grinder, and reacting under magnetic stirring after the mixing to obtain a first mixed solution;

After centrifugal washing, the first mixed solution is added with a glutaraldehyde solution, then mixed in an ultrasonic grinder, and then placed in a magnetic stirrer for reaction to obtain a second mixed solution;

The second mixed solution is centrifuged and washed, and then mixed in an ultrasonic grinder, and a detection antibody solution and a bovine serum albumin solution are added at the same time, and reacted at 4° C. with magnetic stirring to obtain a third mixed solution;

The third mixed solution is centrifuged and washed to obtain a bottom precipitate, a polybutylene succinate solution is added to the bottom precipitate and ultrasonic dispersion is performed to obtain a probe.

Preferably, the production process of the probe includes:

The water channel was soaked with Tris-HCL buffer containing 3% sucrose, 1% bovine serum albumin and 0.5% Tween, and after drying, the probe was dropped into the water channel to obtain a probe pad.

Preferably, the sample pad preparation process comprises: soaking the water channel with Tris-HCL buffer containing 1% bovine serum albumin and 0.5% Tween, and obtaining the sample pad after drying.

Preferably, the material of the sample pool includes an acrylic plate, and a hole is punched at the center where the acrylic plate contacts the sample pad to obtain the sample pool.

The present invention also provides a method for operating an ultrasensitive electrochemical device based on a paper chip water channel, comprising the following steps:

The test solution is dropped into the sample pool, and the test solution filtered by the sample pad flows to the probe pad. The probe reacts with the filtered test solution to obtain an immune complex, which passes through the absorbent pad to reach the electrode chip. The probes captured on the surface of the electrode chip will produce different current peak changes depending on the number, and the content of the test protein in the test solution can be obtained based on the current peak change.

Compared with the prior art, the present invention has the following advantages and technical effects:

The present invention combines the paper chip technology that can realize real-time detection with the highly sensitive electrochemical protein detection technology, and proposes a micro electrochemical detection device that is easy to operate, low in cost and extremely sensitive and is applied to specific protein detection in various complex environments to meet the market demand at home and abroad, and selects an appropriate amount of cysteine to be connected to the surface of the nano silver flower through a silver sulfur bond, while giving it the ability to exist stably in water and exposing as much surface area of the nano silver flower as possible. A larger surface area means more active area, which produces a stronger electrochemical response after contacting with an electrolyte, making the detection result more accurate.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

FIG1 is a structural diagram of an ultrasensitive electrochemical device according to an embodiment of the present invention;

FIG2 is a differential pulse voltammogram of the gradient concentration of the protein to be tested according to an embodiment of the present invention;

FIG. 3 is a diagram of device selective verification data according to an embodiment of the present invention.

Detailed ways

It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

It should be noted that the steps shown in the flowcharts of the accompanying drawings can be executed in a computer system such as a set of computer executable instructions, and that, although a logical order is shown in the flowcharts, in some cases, the steps shown or described can be executed in an order different from that shown here.

Embodiment 1

As shown in FIG1 , an ultrasensitive electrochemical device based on a paper chip water channel is provided in this embodiment, comprising a sample pool, a sample pad, a probe pad, a water absorbent pad and an electrode chip arranged from top to bottom;

The sample pool is used to store the solution to be tested;

The sample pad is used to filter impurities in the solution to be tested;

The probe pad is used to generate immune reaction between the probe and the filtered test solution to obtain immune complexes;

The absorbent pad is used to absorb excess immune complexes and provide chromatographic power;

The electrode chip is used to capture the immune complex and generate an electrochemical response.

The preparation method is as follows:

Electrode chip preparation:

A 150μm thick polyimide film (PI) tape is adhered to the surface of a phenolic resin plate to form a laser-induced graphene electrode (LIG) substrate. The PI film with a surface carbon content of more than 70% is used as a carbon precursor to engrave the graphene electrode. The bottom phenolic resin plate provides a certain mechanical strength for the entire electrode, making it less likely to bend or wrinkle under high-temperature preparation conditions.

According to a certain electrode size, a laser engraving machine (5W) was used to laser burn the substrate, and the parameters of the laser engraving machine were set as follows: power 14%, depth 14%. The size of the obtained working electrode was 3 μm, and the size of the electrode chip was about 2 cm*1.2 cm.

Double-sided conductive copper tape was used as a wire to connect the end of the LIG electrode, and a mixed solution of Ag/AgCl was applied to the LIG reference electrode as a new reference electrode. The release film, polyurethane elastomer rubber film (TPU), and electrode were stacked in order, and the TPU was melted under the action of a laminating machine (120°C) to encapsulate the electrode so that only the working area was exposed.

The working electrode was incubated with a DMF (N-N-dimethylformamide) solution (5 mM, 2 μL) of 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBASE) for 1 h to connect the capture antibody to the working electrode surface. The incubation process was carried out in an environment with a humidity of 90% and a temperature of 20°C. After 1 h, the non-specifically bound PBASE was washed away with DMF, alcohol, and PBS in sequence.

The working electrode was incubated with 5 μL of a capture antibody solution with a concentration of 20 μg/mL against the protein to be detected. After 1 hour, the electrode was rinsed with PBS to remove the antibody that failed to specifically bind.

The working electrode was incubated with 1% BSA solution for 1 h to block the nonspecific binding sites of PBASE, and then the electrode was rinsed with PBS ultrapure water to remove free BSA. The electrode chip was placed in a 4°C refrigerator for later use.

Sample pad preparation:

A wax jet printer was used to print a circular paraffin ring with an outer diameter of 3 mm and an inner diameter of 2 mm on the surface of the filter paper, which was then placed on a hot plate at 180° C. and heated for 1 min to obtain a water channel pad having a three-dimensional water channel structure inside the filter paper.

The water channel was soaked with Tris-HCL buffer containing 1% BSA and 0.5% Tween 20 and used as a sample pad after drying.

Probe pad preparation:

The water channel was soaked with Tris-HCL buffer containing 3% sucrose, 1% BSA, and 0.5% Tween 20, and after drying, 10 μL of the probe was dropped into the water channel of the water channel pad to serve as a probe pad.

Probe preparation:

10 mL of ultrapure water, 1 mL of 0.05 M silver nitrate solution, and 1 mL of 0.25 M citric acid solution were added to the centrifuge tube in sequence, and stirred for 15 min using a magnetic stirrer in an ice-water bath and protected from light to allow them to be fully mixed. Subsequently, 50 μL of 1 M ascorbic acid solution was added, and stirring continued for 15 min in an ice-water bath and protected from light to allow them to react fully.

The solution after the reaction is centrifuged, washed and dried to obtain nano silver flowers with rich surface structures, the diameter of which is about 600nm.

Take 10 mg of the above-mentioned nanosilver flower, add 1 mL of cysteine solution (2 mg/mL), and mix in an ultrasonic grinder for 30 min, with the power set to 1%, the ultrasonic working time 9 s, and the interval time 3 s. Then, react for 4 h under magnetic stirring.

After the above solution was washed by centrifugation, 2 mL of 10% glutaraldehyde solution was added, and the mixture was first mixed in an ultrasonic grinder for 30 min, and then placed in a magnetic stirrer for reaction for 4 h.

After the above solution was washed by centrifugation, it was first mixed in an ultrasonic grinder for 10 minutes, and then 1 μL of detection antibody solution (1 mg/mL) and 1 mL of BSA solution (10 mg/mL) were added simultaneously, and the reaction was carried out at 4° C. with magnetic stirring for 4 hours.

After centrifugal washing, 2 mL of PBS solution was added to the bottom precipitate, and after ultrasonic dispersion, the nanosilver flower probe was obtained. The probe was stored in a 4°C refrigerator away from light for future use.

The acrylic plate, the electrode chip, the water absorbent pad, the probe pad, the sample pad and the acrylic plate are stacked in sequence and extruded and packaged to obtain a paper chip electrochemical device.

The detection process is as follows:

The function of the sample pool is that when a certain amount of the test solution is added to the detection device, the test solution cannot immediately enter the liquid flow channel of the device, so the excess test solution is stored in the sample pool to prevent the loss of the test sample during the detection process.

The sample pad treated with the treatment liquid can separate and filter impurities in the sample, pre-treat the sample, balance the pH of the sample to be tested and adjust the salt ion strength, so that the sample maintains a certain uniformity and controllability during the chromatography process.

The probe pad is mainly loaded with a large number of probes and serves as the main site for immune response when the test fluid passes through it.

The absorbent pad is used to absorb excess reactants and provide upward chromatographic power for the entire reaction process.

In addition, the vertical water flow channel can provide additional chromatographic power for the movement of reactants.

When the test solution containing the target protein enters the sample pool, it first passes through the sample pad, which can pre-treat the sample in the test solution and absorb the impurities therein. Subsequently, the test solution enters the probe pad, and the probe in the probe pad combines with the target protein to form an immune complex (protein-probe complex). The immune complex continues to move downward under the action of chromatography, reaches the absorption pad, and is captured by the capture probe on the electrode surface to form a sandwich structure.

Verify as follows:

40 μL of a series of gradient concentrations of the protein solution to be tested was added to the sample pool. After 10 minutes, the sensor was connected to the electrochemical workstation and tested using differential pulse voltammetry with a scanning voltage of -0.2V to 0.2V. The higher the concentration of the test solution, the more protein-probe complexes are formed with the probes on the probe pad, and the more probes are captured by the electrode surface, which can produce a stronger electrochemical response. As the concentration of the test solution changes, when the concentration of the test solution is between 1fg and 10pg, there is a certain logarithmic relationship between the peak current change of the sensor and the logarithm of the concentration of the test solution, that is, Y = AlgX + B, where A = 0.1445, B = 0.2845 (as shown in Figure 2).

After PBS, fetal bovine serum (FBS), and BSA were added to the sample cell, the peak current of the sensor did not change significantly (as shown in FIG3 ), indicating that the sensor has good selectivity.

The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the technical field within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims (8)

1. An ultrasensitive electrochemical device based on a paper chip water channel is characterized by comprising a sample cell, a sample pad, a probe pad, a water absorption pad and an electrode chip which are arranged from top to bottom;

the sample cell is used for storing a solution to be tested;

The sample pad is used for filtering impurities in the solution to be detected;

The probe pad is used for generating immune reaction with the filtered solution to be detected through a probe to obtain an immune complex;

The water absorption pad is used for absorbing redundant immune complex and providing chromatographic power;

The electrode chip is used for capturing immune complex to generate electrochemical response.

2. The paper chip water channel-based ultrasensitive electrochemical device of claim 1, wherein,

The manufacturing process of the electrode chip comprises the following steps: adhering a polyimide film adhesive tape to the surface of a phenolic resin plate to form a laser-induced graphene electrode substrate;

Performing laser cauterization on the laser-induced graphene electrode substrate by using a laser carving machine to obtain a graphene electrode;

connecting a copper tape to the tail end of a graphene electrode, coating a mixed solution of Ag and AgCl on the graphene electrode to obtain a new reference electrode, and sequentially packaging a release film, a polyurethane elastomer rubber film and the new reference electrode by a label pressing machine to obtain a working electrode;

Incubating the working electrode by using an N-N-dimethylformamide solution containing 1-pyrene butyric acid N-hydroxysuccinimide ester, and washing the non-specifically bound 1-pyrene butyric acid N-hydroxysuccinimide ester by using the dimethylformamide solution, alcohol and polybutylene succinate in sequence;

incubating the working electrode with a capture antibody solution of the protein to be detected, and flushing the electrode with polybutylene succinate;

and incubating the working electrode by using a bovine serum albumin solution, and then flushing the electrode by using polybutylene succinate and ultrapure water to remove free bovine serum albumin, so as to obtain the electrode chip.

3. The paper chip water channel-based ultrasensitive electrochemical device of claim 1, wherein,

The probe pad includes a water channel pad and a probe;

The manufacturing process of the water channel pad comprises the following steps: and printing the circular paraffin on the surface of the filter paper by using a wax spraying printer, and heating the filter paper printed with the circular paraffin on a hot plate at 180 ℃ to obtain the water channel pad with the three-dimensional structure water channel inside the filter paper.

4. The ultrasensitive electrochemical device based on paper chip water channel according to claim 3, characterized in that,

The manufacturing process of the probe comprises the following steps:

sequentially adding ultrapure water, silver nitrate solution and citric acid solution into a centrifuge tube, stirring by using a magnetic stirrer under the conditions of ice-water bath and light shielding, then adding ascorbic acid solution, continuing stirring, and obtaining nano-silver flowers through centrifugation, washing and drying operations after stirring is completed;

adding cysteine solution into the nano-silver flowers, mixing in an ultrasonic pulverizer, and reacting under the action of magnetic stirring after the mixing is finished to obtain a first mixed solution;

The first mixed solution is centrifugally washed, glutaraldehyde solution is added, then the glutaraldehyde solution is mixed in an ultrasonic pulverizer, and then the obtained mixture is placed in a magnetic stirrer for reaction to obtain a second mixed solution;

Centrifugally washing the second mixed solution, mixing in an ultrasonic pulverizer, adding a detection antibody solution and a bovine serum albumin solution, and reacting at 4 ℃ under the magnetic stirring condition to obtain a third mixed solution;

and (3) centrifugally washing the third mixed solution to obtain a bottom precipitate, adding a polybutylene succinate solution into the bottom precipitate, and performing ultrasonic dispersion to obtain the probe.

5. The ultrasensitive electrochemical device based on paper chip water channel according to claim 3, characterized in that,

The manufacturing process of the probe comprises the following steps:

the water channel was soaked with Tris-HCl buffer containing 3% sucrose, 1% bovine serum albumin and 0.5% Tween, and after air-drying, probes were added dropwise to the water channel to obtain a probe pad.

6. The ultrasensitive electrochemical device based on paper chip water channel according to claim 3, characterized in that,

The manufacturing process of the sample pad comprises the following steps: the water channel was soaked with Tris-HCL buffer containing 1% bovine serum albumin and 0.5% tween, and dried to obtain a sample pad.

7. The paper chip water channel-based ultrasensitive electrochemical device of claim 1, wherein,

The material of the sample cell comprises an acrylic plate, and a hole is punched in the center of the acrylic plate, which is in contact with the sample pad, so that the sample cell is obtained.

8. The working method of the ultrasensitive electrochemical device based on the paper chip water channel is characterized by comprising the following steps of:

The solution to be measured is dripped into a sample pool, the solution to be measured filtered by the sample pad flows to a probe pad, the probe and the filtered solution to be measured react in an immune mode to obtain an immune complex, the immune complex passes through a water absorption pad to reach an electrode chip, different current peak changes are generated by probes captured on the surface of the electrode chip according to different quantities, and the content of the protein to be measured in the solution to be measured is obtained according to the current peak changes.