阅读说明：本技术 一种电化学传感器及其在人体生理生化指标检测中的应用 (Electrochemical sensor and application thereof in human body physiological and biochemical index detection ) 是由 郭振 李超 张威 姚佳 于 2021-08-26 设计创作，主要内容包括：本发明涉及一种电化学传感器及其在人体生理生化指标检测中的应用,属于电化学检测技术领域。本发明提供了一种基于丝网印刷技术的电化学传感器,所述电化学传感器包括三电极电化学传感器,所述三电极电化学传感器的工作电极上修饰有增强型材料；使用所述电化学传感器测试不同血样PT值,能够清晰的显示出区分度,且挑选三组血样进行PT重复实验,出峰时间变异系数分别为2.26％、3.22％和2.96％,实验结果与医院的临床结果线性拟合决定系数R～(2)为0.986。因此,所述电化学传感器用于PT测试具有良好的重复性和一致性,易于批量生产,大大降低了凝血测量的成本,且适合多种场合的测量,在即时检测领域具有极大潜力。(The invention relates to an electrochemical sensor and application thereof in human body physiological and biochemical index detection, belonging to the technical field of electrochemical detection. The invention provides an electrochemical sensor based on a screen printing technology, which comprises a three-electrode electrochemical sensor, wherein a working electrode of the three-electrode electrochemical sensor is modified with an enhanced material; the electrochemical sensor is used for testing PT values of different blood samples, the differentiation can be clearly displayed, three groups of blood samples are selected for PT repeated experiments, the variation coefficients of the peak time are respectively 2.26%, 3.22% and 2.96%, and the linear fitting decision coefficient R of the experimental results and the clinical results of hospitals 2 Was 0.986. Thus, it is possible to provideThe electrochemical sensor has good repeatability and consistency when used for PT test, is easy for batch production, greatly reduces the cost of blood coagulation measurement, is suitable for measurement in various occasions, and has great potential in the field of instant detection.)

1. An electrochemical sensor, wherein the electrochemical sensor comprises a three-electrode electrochemical sensor; the working electrode of the three-electrode electrochemical sensor is modified with a reinforced material.

2. The electrochemical sensor of claim 1, wherein the enhancement material is at least one of carbon nanotubes (SWCNTs) or graphene, nanogold.

3. The electrochemical sensor according to claim 1 or 2, wherein the working electrode of the three-electrode electrochemical sensor is further modified with a reagent for detecting physiological and biochemical indicators of a human body.

4. The electrochemical sensor according to any one of claims 1 to 3, wherein the substrate of the electrochemical sensor is at least one of a PET sheet, a PVC sheet, a PP sheet, an ABS sheet, or a ceramic material.

5. The electrochemical sensor according to any one of claims 1 to 4, wherein the working electrode layer of the electrochemical sensor is at least one of a conductive carbon ink material or a gold ink material.

6. The electrochemical sensor according to any one of claims 1 to 5, wherein the reference electrode layer of the electrochemical sensor is a Ag/AgCl ink material.

7. A method for detecting physiological and biochemical indexes of a human body, which is characterized in that the electrochemical sensor of any one of claims 1-6 is used for detecting the physiological and biochemical indexes of the human body on a blood sample to be detected.

8. The method of claim 7, wherein the human physiological and biochemical marker test comprises a blood coagulation test; the coagulation assay comprises at least one of a prothrombin time assay, an activated partial thromboplastin time assay, a thrombin time assay, or a plasma fibrinogen assay.

9. The use of the electrochemical sensor according to any one of claims 1 to 8 for detecting physiological and biochemical indicators of a human body.

10. The use of claim 9, wherein the human physiological and biochemical marker test comprises a blood coagulation test; the coagulation assay comprises at least one of a prothrombin time assay, an activated partial thromboplastin time assay, a thrombin time assay, or a plasma fibrinogen assay.

Technical Field

The invention relates to an electrochemical sensor and application thereof in human body physiological and biochemical index detection, belonging to the technical field of electrochemical detection.

Background

Whether the blood coagulation function is normal or not is related to the physiological health state of a human body, and the blood coagulation time is an important index for measuring the blood coagulation state. Prothrombin Time (PT) is an exogenous coagulation function index in the detection of coagulation Time, and the Prothrombin Time PT depends on the levels of Prothrombin, fibrinogen, coagulation factors ii, v, vii, and the like in blood, wherein the coagulation factors are mainly synthesized by the liver, so PT is of great importance for the treatment of many patients with liver diseases such as liver cirrhosis and hepatitis, and also needs to closely monitor the change of PT coagulation Time for some patients with congenital or acquired coagulation factor deficiency, patients with thrombotic diseases, patients with congenital FV increase, and the like.

Currently, large instruments in hospitals and other places often measure blood coagulation time by old methods such as thromboelastography and optical methods, wherein the PT detection precision of the instrument of the optical method is higher than that of other methods, so the optical blood coagulation instrument is regarded as a standard instrument for blood coagulation detection, but the instruments are complex to operate and require professional expertise. The sensors required by the small-scale systems for measuring coagulation parameters which are commercialized at present are expensive, and the repeatability of the test results is not excellent. With the rapid development of the instant detection technology, the traditional detection mode is difficult to meet the instant detection requirements of scenes such as sick bedside, outdoor and family bedside and the like. Therefore, the development of a new technology for detecting blood coagulation with low cost and convenience is urgently needed.

The wide application of the screen printing electrochemical sensor in the field of medical biology opens up a way for the research of the instant detection of the blood coagulation function. The electrochemical biosensor based on the screen printing technology has the characteristics of simple preparation, high detection sensitivity and short detection time, the screen printing electrode is low in cost and can be used at one time, the amount of samples required by the test is small, and the problems of pollution, corrosion and the like caused in the electrochemical analysis process can be avoided. Therefore, it is of great research value to explore an electrochemical biosensor using a screen printing technology to detect PT.

Disclosure of Invention

The invention provides an electrochemical sensor based on a screen printing technology, which has important guiding significance for the treatment of anticoagulant drug users and hepatopaths, and the like, and solves the problems of high cost, complex operation and the like of the existing human body physiological and biochemical index detection instrument, wherein the electrochemical sensor comprises a three-electrode electrochemical sensor; the working electrode of the three-electrode electrochemical sensor is modified with a reinforced material.

In one embodiment of the present invention, the enhancement material is carbon nanotubes (SWCNTs) or at least one of graphene and gold nanoparticles.

In an embodiment of the invention, a reagent for detecting physiological and biochemical indexes of a human body is further modified on the working electrode of the three-electrode electrochemical sensor.

In one embodiment of the invention, the reagent for detecting the physiological and biochemical indexes of the human body is a blood coagulation reaction reagent; the components of the coagulation reaction reagent comprise a thrombin substrate solution and a PT reagent.

In one embodiment of the invention, the carbon nanotubes and the reagent for detecting the physiological and biochemical indexes of the human body are covered with a hydrophilic film.

In one embodiment of the present invention, the substrate of the electrochemical sensor is at least one of a PET sheet, a PVC sheet, a PP sheet, an ABS sheet, or a ceramic material.

In one embodiment of the present invention, the thickness of the substrate of the electrochemical sensor is 0.2 to 0.5 mm.

In one embodiment of the present invention, the working electrode layer of the electrochemical sensor is at least one of a conductive carbon ink material or a gold ink material.

In one embodiment of the invention, the reference electrode layer of the electrochemical sensor is an Ag/AgCl ink material.

In one embodiment of the present invention, the conductive line layer of the electrochemical sensor is an Ag/AgCl ink material or an Ag ink material.

In one embodiment of the present invention, the protective layer of the electrochemical sensor is an insulating ink material.

The invention also provides a method for detecting the human body physiological and biochemical indexes, which is used for detecting the human body physiological and biochemical indexes of the blood sample to be detected by using the electrochemical sensor.

In one embodiment of the invention, the human physiological and biochemical indicator test comprises a blood coagulation test; the coagulation assay comprises at least one of a Prothrombin Time (PT) assay, an Activated Partial Thromboplastin Time (APTT) assay, a Thrombin Time (TT) assay, or a plasma Fibrinogen (FIB) assay.

The invention also provides the application of the electrochemical sensor in the detection of the physiological and biochemical indexes of the human body.

In one embodiment of the invention, the human physiological and biochemical indicator test comprises a blood coagulation test; the coagulation assay comprises at least one of a Prothrombin Time (PT) assay, an Activated Partial Thromboplastin Time (APTT) assay, a Thrombin Time (TT) assay, or a plasma Fibrinogen (FIB) assay.

The technical scheme of the invention has the following advantages:

the invention provides an electrochemical sensor based on a screen printing technology, which comprises a three-electrode electrochemical sensor; a working electrode of the three-electrode electrochemical sensor is modified with multi-walled carbon nanotubes (MWCNTs) and a coagulation reaction reagent, and the components of the coagulation reaction reagent comprise a thrombin substrate solution and a PT reagent. The blood coagulation process is an extremely complex physiological reaction, a plurality of substances in blood can interfere the detection process, a three-electrode system is selected, MWCNTs are modified on the surface of a silk-screen electrode, and the electrode can obtain a larger specific surface areaThe electrochemical sensor can measure the current change caused by electron transfer in the reaction process of the electrode surface in real time. The electrochemical sensor is used for testing PT values of different blood samples, the differentiation can be clearly displayed, three groups of blood samples are selected for PT repeated experiments, the variation coefficients of the peak time are respectively 2.26%, 3.22% and 2.96%, and the linear fitting decision coefficient R of the experimental results and the clinical results of hospitals²Was 0.986. Therefore, the electrochemical sensor has good repeatability and consistency when used for PT test, is easy for batch production, greatly reduces the cost of blood coagulation measurement, is suitable for measurement on various occasions, and has great potential in the field of instant detection.

Drawings

FIG. 1: schematic structure of three-electrode electrochemical sensor. In fig. 1, a base layer; 2. a conductive track; 3. an Ag/AgCl layer; 4. a carbon layer; 5. an insulating layer.

FIG. 2: PT test results and negative control results of the blood samples of the 3 groups.

FIG. 3: the PT results for MWCNTs type sensors were compared to SYSMEX CS 5100.

Detailed Description

The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.

The following examples do not show specific experimental procedures or conditions, and can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

Example 1: multi-walled carbon nanotube (MWCNTs) electrochemical sensor

The present embodiments provide a multi-walled carbon nanotube (MWCNTs) electrochemical sensor comprising a three-electrode electrochemical sensor; the working electrode of the three-electrode electrochemical sensor is modified with a carbon nano tube and a coagulation reaction reagent, and the components of the coagulation reaction reagent comprise a thrombin substrate solution and a PT reagent.

Preferably, the carbon nanotubes and the coagulation reaction reagent are covered with a hydrophilic film.

Preferably, the substrate of the electrochemical sensor is at least one of a PET sheet, a PVC sheet, a PP sheet, an ABS sheet, or a ceramic material.

Preferably, the thickness of the base material of the electrochemical sensor is 0.2-0.5 mm.

Preferably, the working electrode layer of the electrochemical sensor is at least one of a conductive carbon ink material or a gold ink material.

Preferably, the reference electrode layer of the electrochemical sensor is an Ag/AgCl ink material.

Preferably, the conductive wire layer of the electrochemical sensor is an Ag/AgCl ink material or an Ag ink material.

Preferably, the protective layer of the electrochemical sensor is an insulating ink material.

Preferably, the carbon nanotubes are at least one of multi-walled carbon nanotubes or single-walled carbon nanotubes. .

Experimental example 1: functional verification of multi-walled carbon nanotube (MWCNTs) electrochemical sensor

1. Experimental methods

1.1 Experimental materials and instruments

The required parameters were measured using a CHI660E electrochemical workstation (shanghai chenhua instruments ltd) connected to the sensor and verified for consistency with clinical results using a SYSMEX CS 5100 optical coagulometer.

1.2 electrochemical sensor preparation

A general three-electrode electrochemical sensor is manufactured by a full-automatic screen printer, and its structure is shown in fig. 1. Selecting the PET as a substrate, washing the PET substrate with deionized water to remove surface impurities before printing, and drying with nitrogen. Firstly, a conductive silver layer is printed to improve the conductivity of the screen printing electrode and is used as a reference electrode and a conductive track of the screen printing electrode. And printing silver chloride slurry to cover the reference electrode, and then printing conductive carbon ink slurry as a working electrode and a counter electrode, wherein the working electrode is disc-shaped. And after each step is finished, placing the silk-screen electrode in a specially-dried constant temperature box, and baking at the temperature of 70-90 ℃ until the silk-screen slurry is completely dried. And after the drying step is finished, fixing the insulating layer on the electrode layer by adopting insulating printing ink, leaving a working area for fixing a reagent and reacting, and baking again in a drying constant temperature box at the temperature of 80-100 ℃ until the insulating slurry is completely cured. The prepared sensor was aged at room temperature for several days, during which the electrodes were kept in a dry environment protected from light.

1.3 modification of MWCNTs

And cleaning the surface electrode of the sensor by using deionized water, and drying by using nitrogen. And then 3 mu L of MWCNTs uniform dispersion liquid is weighed and coated on the surface of the working electrode of the manufactured silk-screen electrode, the silk-screen electrode is placed in a thermostat and dried at 70-90 ℃, and the MWCNTs type electrochemical sensor is manufactured after drying.

1.4 measurement of blood coagulation parameters PT

(1) Sensor preparation

And coating a modified coagulation PT reaction reagent on the surface of the working electrode of the manufactured MWCNTs type sensor. And then placing the sensor in a thermostat for reagent curing treatment, then packaging a layer of hydrophilic material on the surface of the electrode to form a 5-10 mu L reaction tank, preparing the MWCNTs type electrochemical sensor for PT detection, and placing the sensor in a vacuum sealing bag to be required by an experiment.

(2) Procedure of experiment

The electrode is placed on a stable experiment table, a certain amount of plasma is firstly extracted from a blood sample before a coagulation experiment is carried out, the plasma sample is placed in a 37 ℃ water bath for 3min, a pipettor is used for injecting 5-10 mu L of the sample into a reaction cavity through a liquid inlet of a sensor, and the reaction process of sample solidification is recorded by a timing current method.

2. Results and analysis

2.1 test results

Plasma samples of 3 patients were taken from hospitals in the experiment, wherein the results in fig. 2 and table 1 correspond to hospital test PT standard values of blood samples of 10.4s, 15.6s and 19.9s respectively, and the experimental results show that the signal time of the blood samples with different PT values tested by using the MWCNTs electrochemical sensor has obvious discrimination, and the test results of 3 groups of samples are 42.75s, 58.94s and 82.86s respectively.

2.2 results of sensor repeatability experiments

To verify the reproducibility of the measurement of PT by the MWCNTs type electrochemical sensor, 5 replicates of 3 plasma samples were used herein. The 3 sets of samples are designated as set 1, set 2 and set 3 in this order. As shown in table 1, the CV values of the 3 experimental results were 2.26%, 3.22%, and 2.96%, respectively, so that it can be judged that the sensor herein has good consistency and repeatability for measuring PT.

TABLE 1 three groups of blood samples 5 replicates (time: s)

2.3 clinical comparative test results

Clinical application value of the MWCNTs enhanced electrochemical sensor herein was verified using a SYSMEX CS 5100 optical coagulometer. 8 blood samples were selected from the hospital in the experiment and compared with the standard thrombometer results using the sensor of the present invention, the PT results are shown in FIG. 3, the straight line is the linear fitting curve of the measurement results, and the coefficient R is determined²Was 0.986. The result shows that the electrochemical sensor can be better applied to clinical detection of the blood coagulation parameter PT.

3. Conclusion

The MWCNTs enhanced electrochemical sensor with good repeatability and low cost is designed and prepared, and is used for measuring the blood coagulation parameter PT. In the PT parameter experiment, the blood samples with different PT values can clearly display the discrimination, and each group of blood samples is subjected to repeated PT measurement experiment, the variation coefficients of the test results of 3 groups are respectively 2.26%, 3.22% and 2.96%, and are controlled within 5%,therefore, the sensor is judged to have good repeatability. The experimental result is linearly fitted with a hospital SYSMEX CS 5100 optical coagulation detector to determine a coefficient R²Was 0.986. The MWCNTs type electrochemical sensor can effectively measure the blood coagulation parameter PT, the sensor can be produced in batch, the blood coagulation detection cost is greatly reduced, the test time is short, the required sample amount is small, and the MWCNTs type electrochemical sensor can be suitable for instant measurement in various occasions. MWCNTs type sensors also have the potential to measure the measurement of other coagulation parameters and the analysis of other types of reagents.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.