Electrochemical test reagent card and use thereof

Abstract

The application discloses an electrochemical detection reagent card and application thereof, and belongs to the technical field of electrochemical detection reagent cards. The electrochemical detection reagent card comprises a substrate layer, a conductive area and a reaction area arranged on the surface of the substrate layer. The conductive area comprises first and second conductive tracks which are parallel to each other. The first conductive track is provided with a working electrode at one end, and the second conductive track is provided with a counter electrode at one end. The reaction area is arranged between the first and second conductive tracks. The reaction area comprises a hydroxyethyl cellulose modified carbon nanometer layer and a thrombin reaction layer which are arranged in sequence. The reaction area is in communication with the conductive area through a conductive layer. The electrochemical detection reagent card has high stability and high sensitivity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an electrochemical detection reagent card and its application. Background Technology

[0002] Coagulation time is an important indicator of normal coagulation function, and thrombin time (TT) is one of the commonly used clinical tests used to assess the function of the coagulation, anticoagulation, and fibrinolytic systems. Thrombin time reflects the common pathway of coagulation; under the action of thrombin, fibrinogen is converted into insoluble fibrin, and the time required for coagulation is measured as the plasma thrombin time. Currently, thrombin time detection is mainly based on optical methods; however, this method requires professional operation and is difficult to meet the needs of home or outdoor immediate diagnosis. Thrombin time detection systems based on electrochemical detection methods are simple to operate, do not require complex large instruments, and the prepared electrode cards are inexpensive, requiring only a small sample size, making them a promising new detection technology.

[0003] Chinese patent document CN209624464U discloses an electrochemical detection reagent card modified with carbon nanotubes. This method is based on an electrochemical current method to measure prothrombin time. The method activates the coagulation process through thrombin activation enzyme, initiating thrombin formation. Furthermore, thrombin decomposes a polypeptide substrate, generating a current change signal. While the introduction of carbon nanotubes enhances the electron transfer process, the additional introduction of the polypeptide substrate may negatively impact the stability of the reagent card. In contrast, US patent document US20210016275 discloses a detection card based on electrochemical impedance spectroscopy for measuring thrombin time. A reagent containing a quantitative amount of thrombin is uniformly distributed on a hydrophilic membrane, which is then assembled with a conductive substrate to obtain a small electrochemical test card. During the coagulation reaction, the coagulation signal is transmitted from the hydrophilic membrane to the conductive substrate and captured by the measuring device. Because this coagulation reaction involves the participation of macromolecules such as enzymes and fibrinogen, the curved three-dimensional structure of these macromolecules is not conducive to the efficient transfer of electrons. In addition, the measurement of blood coagulation signals by impedance method is affected by buffer solution, conductive substrate, etc., which will reduce the sensitivity and repeatability of the signal.

[0004] Therefore, there is an urgent need for a highly stable and sensitive test kit to detect thrombin time. Summary of the Invention

[0005] In view of this, this application provides an electrochemical detection reagent card and its application, which has high stability and high sensitivity.

[0006] To achieve the above technical objectives, this application adopts the following technical solution:

[0007] This application provides an electrochemical detection reagent card, including a substrate layer, a conductive region and a reaction region disposed on the surface of the substrate layer. The conductive region includes a first conductive track and a second conductive track that are parallel to each other. A working electrode is provided at one end of the first conductive track and a counter electrode is provided at one end of the second conductive track. The reaction region is housed between the first conductive track and the second conductive track. The reaction region includes a carbon nanolayer modified with hydroxyethyl cellulose and a thrombin reaction layer stacked sequentially. The reaction region and the conductive region are connected by a covering conductive layer.

[0008] Preferably, the first conductive orbital includes a first conductive Ag orbital layer and a first conductive carbon orbital layer stacked sequentially, and the second conductive orbital includes a second conductive Ag orbital layer and a second conductive carbon orbital layer stacked sequentially.

[0009] Preferably, the covering guide layer is a hydrophilic membrane layer.

[0010] Preferably, both the first conductive carbon orbital layer and the second conductive carbon orbital layer have an isolation layer at their ends for connecting the thrombin reaction layer and the covering flow layer.

[0011] Preferably, the method for preparing the hydroxyethyl cellulose modified carbon nanolayer includes dispersing carbon nanotube material powder in a hydroxyethyl cellulose solution to obtain a dispersion, and then coating it onto the reaction zone on the surface of the substrate layer.

[0012] Preferably, the concentration of the hydroxyethyl cellulose solution is 3-5%.

[0013] Preferably, the method for preparing the thrombin reaction layer includes coating a thrombin solution onto a carbon nanolayer modified with hydroxyethyl cellulose and then drying it. The thrombin solution comprises a mixture of bovine thrombin, buffer solution, and stabilizer.

[0014] Preferably, the concentration of the thrombin solution is 0.1-0.3 mg / ml.

[0015] Preferably, the base layer is made of polyterephthalic acid plastic.

[0016] Secondly, this application provides an application of an electrochemical detection reagent card in the detection of thrombin time.

[0017] The beneficial effects of this application are as follows:

[0018] The hydroxyethyl cellulose-modified carbon nanolayer in this scheme facilitates the dispersion of carbon nanotubes, allowing the originally easily aggregated carbon nanoparticles to be uniformly dispersed within the hydroxyethyl cellulose network structure. This further increases the specific surface area of ​​the reaction, enabling rapid electron transfer, enhancing the ion mass transfer process, and thus improving and amplifying the resistance signal. Furthermore, the modified carbon nanotubes exhibit good stability, which helps reduce signal disturbances caused by the complex components of blood during coagulation, thereby improving the stability and repeatability of the detection.

[0019] The electrochemical detection reagent card of this scheme is simple in construction, easy to operate, rapid in detection, and requires a small sample volume. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the structure of the substrate layer and the first conductive Ag orbital layer;

[0021] Figure 2 This is a schematic diagram of the reaction region and the conductive region;

[0022] Figure 3 This is a schematic diagram of the isolation layer structure;

[0023] Figure 4 This is a schematic diagram of the finished electrochemical detection reagent card for this scheme.

[0024] Figure 5 A comparison graph of thrombin time impedance test results for the electrochemical detection reagent cards of Example 1 and Comparative Example 1;

[0025] Figure 6 The image shows the thrombin time impedance test result of the electrochemical detection reagent card in Example 1.

[0026] In the figure: 1. Substrate layer; 2. First conductive Ag orbital layer; 3. First conductive carbon orbital layer; 4. Working electrode; 5. Counter electrode; 6. Hydroxyethyl cellulose modified carbon nanolayer; 7. Isolation layer; 8. Covering flow-conducting layer; 9. Inlet; 10. Outlet. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0028] Example 1

[0029] This application provides an electrochemical detection reagent card, including a base layer 1, a conductive region and a reaction region disposed on the surface of the base layer 1. The conductive region includes a first conductive track and a second conductive track that are parallel to each other. A working electrode 4 is provided at one end of the first conductive track and is attached to the upper end of the first conductive track for conduction. A counter electrode 5 is provided at the upper end of the second conductive track and is attached to one end of the second conductive track for conduction. The reaction region is housed between the first conductive track and the second conductive track and is disposed between the lower end of the first conductive track and the lower end of the second conductive track. The reaction region includes a hydroxyethyl cellulose-modified carbon nanolayer 6 and a thrombin reaction layer stacked sequentially. The reaction region and the conductive region are connected by a covering conductive layer 8. The thrombin reaction layer is used to generate a resistance signal of blood coagulation, and the hydroxyethyl cellulose-reinforced carbon nanolayer is used to transmit and amplify the resistance signal. The covering conductive layer 8 is used to guide the sample to be tested into the reaction region for reaction.

[0030] Both the counter electrode 5 and the working electrode 4 are screen-printed electrodes. The preparation process of screen-printed electrodes is simple, the detection sensitivity is high, and the two-electrode system has a simple structure and is easy to operate.

[0031] Preferably, the first conductive track includes a first conductive Ag track layer 2 and a first conductive carbon track layer 3 stacked sequentially, and the second conductive track includes a second conductive Ag track layer and a second conductive carbon track layer stacked sequentially; the first conductive Ag track layer 2 and the second conductive Ag track layer are used to correspondingly improve the conductivity of the counter electrode 5 and the working electrode 4, and the first conductive carbon track layer 3 and the second conductive carbon track layer correspondingly improve the friction resistance and oxidation resistance of the first conductive Ag track layer 2 and the second conductive Ag track layer.

[0032] The covering flow layer 8 is a hydrophilic film layer. In order to connect the conductive area and the reaction area through the covering flow layer 8, the lower ends of the first conductive carbon track layer 3 and the second conductive carbon track layer are provided with an isolation layer 7 for connecting the thrombin reaction layer and the covering flow layer 8. In this embodiment, the isolation layer 7 is a double-sided adhesive that is intermittently arranged. The covering flow layer 8 covers and adheres to the reaction area, and the end of the covering flow layer 8 is connected to the conductive area through the isolation layer 7.

[0033] The preparation method of hydroxyethyl cellulose modified carbon nanolayer 6 includes dispersing carbon nanotube material powder in a hydroxyethyl cellulose solution to obtain a dispersion, and then coating it in the reaction zone on the surface of the substrate layer 1. The concentration of the hydroxyethyl cellulose solution is 3-5%.

[0034] The preparation method of the thrombin reaction layer includes coating a thrombin solution onto a carbon nanolayer 6 modified with hydroxyethyl cellulose and then drying it. The thrombin solution includes a mixture of bovine thrombin, buffer, and stabilizer, and the concentration of the thrombin solution is 0.1-0.3 mg / ml.

[0035] The material of the base layer 1 is polyterephthalic acid plastic.

[0036] Specifically, the preparation method of the electrochemical detection reagent card is as follows:

[0037] Polyterephthalic acid plastic was selected as the substrate layer 1 material. Before printing, the surface of the substrate layer 1 was rinsed with ethanol and deionized water respectively to remove surface impurities and then dried with nitrogen.

[0038] Preparation of conductive regions:

[0039] like Figure 1 As shown, a conductive silver layer is first printed on the substrate layer 1, serving as the first conductive Ag orbital layer 2 and the second conductive Ag orbital layer, as follows. Figure 2 As shown, conductive carbon ink material paste is then printed on the surfaces of the first conductive Ag orbital layer 2 and the second conductive Ag orbital layer, corresponding to the first conductive carbon orbital layer 3 and the second conductive carbon orbital layer.

[0040] like Figure 2 As shown, the counter electrode 5 is screen-printed onto the upper end of the first conductive track, and the working electrode 4 is screen-printed onto the upper end of the second conductive track. The counter electrode 5 and the working electrode 4 are positioned facing each other between the first conductive track and the second conductive track; thus, the preparation of the conductive region is completed.

[0041] Preparation of the reaction zone:

[0042] like Figure 2 As shown, the carbon nanolayer modified with hydroxyethyl cellulose 6 was prepared by weighing 7 mg of purified carbon nanotube powder, dispersing it in a 3% hydroxyethyl cellulose solution, and ultrasonically treating it until the powder was evenly dispersed. Then, the dispersion was evenly coated between the lower ends of the first and second conductive tracks in small amounts and multiple times, and placed in a constant temperature oven at 60°C for one hour to dry. The resulting carbon nanolayer was then obtained.

[0043] Thrombin reaction layer: Weigh 5 mg of bovine thrombin and dissolve it in buffer solution to prepare thrombin stock solution. Further dilute the thrombin stock solution to prepare a 0.1 mg / ml thrombin solution. Then, coat 0.5 μL of the thrombin solution onto the carbon nanolayer 6 modified with hydroxyethyl cellulose. After that, place it in a constant temperature oven at 37°C for 15 min to dry, so that the thrombin solution completely covers the surface of the carbon nanolayer 6 modified with hydroxyethyl cellulose, thus obtaining the thrombin reaction layer.

[0044] Covering the flow guide layer 8:

[0045] like Figure 3 As shown, an insulating layer 7 is attached to the lower end of the first conductive track and the lower end of the second conductive track. The insulating layer 7 can be double-sided adhesive, such as... Figure 4As shown, a hydrophilic membrane layer is then covered on the isolation layer 7 to form a reaction pool. The inlet 9 and outlet 10 are located on the left and right sides of the hydrophilic membrane layer. Under the action of the hydrophilic membrane, the sample will fill the reaction zone within 1-2 seconds.

[0046] Comparative Example 1

[0047] An electrochemical detection reagent card is identical to that in Example 1, except that the hydroxyethyl cellulose-reinforced carbon nanolayer is replaced with an unmodified carbon nanolayer 6. The preparation steps are as follows: 7 mg of purified carbon nanotube powder is weighed and dispersed in a 3% perfluorosulfonic acid polymer solution. The powder is ultrasonically treated until it is evenly dispersed. Then, the dispersion is applied in small amounts multiple times and evenly between the lower ends of the first and second conductive tracks. The card is then placed in a constant temperature oven and dried at 60°C for one hour. The card is then obtained after drying.

[0048] Testing and Analysis

[0049] Sensitivity

[0050] The sensitivity of the electrochemical detection reagent cards in Example 1 and Comparative Example 1 was tested using the following method: Freshly collected human blood samples from a hospital were used, and the thrombin time in the blood samples was detected using the electrochemical reagent cards prepared in Example 1 and Comparative Example 1, respectively. An electrochemical workstation was connected to the counter electrode 5 and the working electrode 4 for testing. The impedance-time test mode was selected, with the working voltage set to 500mV, the frequency set to 1000Hz, and the test time set to 250s. The reaction electrode card device was placed in a constant temperature oven at 37℃ for reaction. 6µL of blood sample was added from the side injection port. Under the action of the hydrophilic membrane, the sample filled the reaction chamber within 1-2s. The coagulation reaction process of the sample was recorded using AC impedance spectroscopy.

[0051] The results are as follows Figure 5 As shown, the resistance change intensity of the electrochemical reagent card without hydroxyethyl cellulose-reinforced carbon nanotube modification increased by about 350 ohms during the time from the start to the end of the coagulation reaction, while the resistance change intensity of the electrochemical reagent card with hydroxyethyl cellulose-reinforced carbon nanotube modification increased by about 1760 ohms during the time from the start to the end of the coagulation reaction. The resistance change signal was 5 times that of the original, and the detection sensitivity was significantly improved. It can be seen that the electrochemical reagent card modified with carbon nanotubes has a larger change in electrochemical resistance and a slightly shorter coagulation time.

[0052] Validity

[0053] Three freshly collected blood samples from patients were selected from the hospital to test the effectiveness of the electrochemical detection reagent card in Example 1 for detecting thrombin time. The results are as follows: Figure 6As shown, the plasma test values ​​for the three patients on the CS-2400 were 12s, 19s, and 38s, respectively, while the normal plasma test range is 14-21s. Correspondingly, the plasma test times using the electrochemical reagent card in Example 1 were 36s, 60s, and 140s, respectively. After the blood sample enters the reaction zone of the electrochemical reagent card, soluble fibrinogen in the plasma is converted into insoluble fibrin under the action of thrombin. Since the fibrinogen content varies in different samples, the amount of fibrin produced during the coagulation reaction also varies, resulting in different resistance changes. Therefore, the electrochemical reagent card prepared in this method has good sample discrimination.

[0054] stability

[0055] The stability of the electrochemical reagent card in Example 1 was evaluated by repeatability testing. The thrombin time of the same sample was repeatedly tested, and its mean value and coefficient of variation (CV) were calculated. Table 1 shows the repeatability test results of the three groups of blood samples. The CV values ​​of the experimental results of the three groups of blood samples were 2.86%, 5.13%, and 6.45%, respectively. It can be seen that the electrochemical detection reagent card of this scheme has good repeatability for measuring thrombin time.

[0056] Table 1 Repeatability Test Results

[0057]

[0058] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. An electrochemical detection reagent card, characterized in that, The system includes a substrate layer (1), a conductive region and a reaction region disposed on the surface of the substrate layer (1). The conductive region includes a first conductive track and a second conductive track that are parallel to each other. A working electrode (4) is provided at one end of the first conductive track and a counter electrode (5) is provided at one end of the second conductive track. The reaction region is disposed between the first conductive track and the second conductive track. The reaction region includes a carbon nanolayer (6) modified with hydroxyethyl cellulose and a thrombin reaction layer stacked sequentially. The reaction region and the conductive region are connected by a covering conductive layer (8). The method for preparing the hydroxyethyl cellulose modified carbon nanolayer (6) includes dispersing carbon nanotube material powder in a hydroxyethyl cellulose solution to obtain a dispersion, and then coating it onto the reaction zone on the surface of the substrate layer (1).

2. The electrochemical detection reagent card according to claim 1, characterized in that, The first conductive orbital includes a first conductive Ag orbital layer (2) and a first conductive carbon orbital layer (3) stacked sequentially, and the second conductive orbital includes a second conductive Ag orbital layer and a second conductive carbon orbital layer stacked sequentially.

3. The electrochemical detection reagent card according to claim 1, characterized in that, The covering guide layer (8) is a hydrophilic membrane layer.

4. The electrochemical detection reagent card according to claim 2, characterized in that, The ends of the first conductive carbon orbital layer (3) and the second conductive carbon orbital layer are provided with an isolation layer (7) for connecting the thrombin reaction layer and the covering flow layer (8).

5. The electrochemical detection reagent card according to claim 1, characterized in that, The concentration of the hydroxyethyl cellulose solution is 3-5%.

6. The electrochemical detection reagent card according to claim 1, characterized in that, The preparation method of the thrombin reaction layer includes coating the thrombin solution onto the hydroxyethyl cellulose modified carbon nanolayer (6) and then drying it. The thrombin solution includes a mixture of bovine thrombin, buffer solution and stabilizer.

7. The electrochemical detection reagent card according to claim 6, characterized in that, The concentration of the thrombin solution is 0.1-0.3 mg / ml.

8. The electrochemical detection reagent card according to claim 1, characterized in that, The base layer (1) is made of polyterephthalic acid plastic.

9. The use of an electrochemical detection reagent card as described in any one of claims 1-8 in the detection of thrombin time.

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