CN100415896C - Preparation method of creatine kinase biosensor and reagent thereof - Google Patents

Abstract

The preparation method of the creatine kinase biosensor and the reagent thereof of the present invention relates to the technical field of biosensors, and can rapidly detect the concentration of total creatine kinase. The creatine kinase biosensor of the present invention has a nanomaterial layer on the surface of the two electrodes of the capillary action channel, and a reaction reagent is fixed on the upper surface of the nanomaterial layer; the nanomaterial layer is one of platinum black particles or polymer nanomaterials; In order to obtain a highly active electrode surface and a nanomaterial layer with a high specific surface area. The preparation method of the reagent is that two kinds of solutions are first prepared, and then mixed with the electron acceptor solution. When the analyte - the sample containing creatine kinase - is dropped onto the reaction zone of the test strip through capillary action, the concentration of total creatine kinase can be quickly detected by the electrochemical biosensor detection system.

Description

Preparation method of creatine kinase biosensor and reagent thereof

technical field

The invention relates to the technical field of biosensors, and relates to a current biosensor test strip for rapid detection of total creatine kinase and a manufacturing method for the reagent used. The test strip can quickly detect the concentration of total creatine kinase.

technical background

Creatine Kinase (Creatine Kinase) exists in the cytoplasm and mitochondria of muscle tissue and brain tissue throughout the body. It is an important kinase that is directly related to intracellular energy metabolism, muscle contraction, and ATP regeneration. Reversibly catalyzes the following reactions:

Note: CK: creatine kinase, Creatinephosphate: creatine phosphate, ADP: adenosine diphosphate, Creatine: creatine, ATP: adenosine triphosphate, Mg: magnesium ion necessary for creatine kinase reactivity.

The determination of creatine kinase activity is one of the basic items in routine examinations for clinical diagnosis of muscle diseases, neurological diseases, encephalopathy and heart disease. Cardiovascular disease, especially myocardial infarction is the leading cause of death among cardiovascular diseases. The patient has an acute onset and a high risk of death, and creatine kinase increases within a few hours after myocardial infarction, so it is very necessary to quickly diagnose the patient's creatine kinase. In addition, creatine kinase levels in patients with prostate cancer, lung cancer, and neurological true tumors are higher than normal. However, hyperglycemia in diabetic patients inhibits the expression of kinase activity in muscle tissue, and the decrease in creatine kinase activity is the result of the development of diabetes or the cause of the further development of diabetes. And the detection of creatine kinase is one of the important indicators of athletes' physical training intensity. Under normal circumstances, the muscle cell membrane has a complete structure and normal function, and creatine kinase (CK) rarely leaks out of the cell membrane. Training and training-induced micro-injuries increase creatine kinase activity in serum. Serum creatine kinase is an important sensitive indicator for assessing muscle stimulation and skeletal muscle damage and its adaptation and recovery.

At present, the commonly used methods for measuring serum creatine kinase are enzyme-coupled kit method, dry biochemical analysis method and immunoassay method, all of which require expensive supporting detection instruments. The instrument itself does not have the design function of being on call at any time for emergency needs. Since the detection instrument adopts the principle of optical colorimetry, etc., it is easily interfered by other substances in the serum, and there are errors in the detection, and it cannot detect whole blood.

Contents of the invention

The purpose of the present invention is to provide a fast, accurate and convenient portable biosensor creatine kinase test strip for whole blood detection that can be matched with a portable small instrument for applications such as myocardial infarction emergency patients and athletes going out for training.

In order to achieve the above object, the technical solution of the present invention is to provide a creatine kinase biosensor, comprising the following structure:

There are a pair of parallel electrodes and electrode leads on the upper surface of the insulating substrate;

The two electrodes are respectively the working electrode and the counter electrode, and the top of the two electrodes is covered with an insulating layer;

There is a groove in the lateral direction of the insulating layer;

The top of the groove is covered with an encapsulation layer;

The space surrounded by the insulating substrate, the groove and the encapsulation layer forms a capillary action channel, and the two ends of the capillary action channel are capillary inlets, and the capillary action channel is an electrode reaction area. There is a nanomaterial layer on the surface, and a reaction reagent layer is fixed on the upper surface of the nanomaterial layer; the nanomaterial layer is one of platinum black particles or polymer nanomaterials; to obtain a highly active electrode surface and a nanomaterial layer with a high specific surface area .

In the creatine kinase biosensor, the reaction reagents include electron acceptors, coupling reaction combination reagents, buffers, enzyme activators, enzyme reducers, anticoagulants and surfactants.

In the creatine kinase biosensor, the macromolecule nanomaterial is high molecular weight sodium carboxymethyl cellulose, wherein the content of high molecular weight sodium carboxymethyl cellulose is 0.1-1%.

In the creatine kinase biosensor, the coupling reaction combination reagent reacts with the analyte and can generate a current corresponding to the concentration of the total creatine kinase of the analyte;

The electron acceptor is in the group consisting of ferricyanate, methylene blue, ferrocene and its derivatives, p-benzoquinone, phenazine methyl sulfate, indophenol and its derivatives, and potassium β-naphthoquinone-4-sulfonate any of

The buffer is any one of the group consisting of phosphate buffer, TRIS buffer and MES buffer;

The enzyme activator is magnesium acetate or magnesium chloride;

The enzyme reducing agent is one of N-acetylcysteine, reduced glutathione or a sulfhydryl-containing substance;

The anticoagulant is any one of heparin, oxalate, citrate and EDTA;

The surfactant is any one of TritonX-100, diethanolamide, alkyl alcohol amide phosphoester salt, alcohol ether carboxylate, monoalkyl phosphate, sodium dodecylsulfonate or alkylphenol polyoxyethylene ether A sort of.

In the creatine kinase biosensor, the coupling reaction combination reagents include: creatine phosphate, glucose, adenosine monophosphate (AMP), adenosine diphosphate (ADP), coenzyme II (NADP) or coenzyme I ( NAD), hexokinase or glucokinase, glucose-6-phosphate dehydrogenase, diaphorase, all or part of these reagents.

A method for preparing a reagent used in a creatine kinase biosensor, comprising the following steps: a) first mixing creatine phosphate and glucose to prepare a solution R1; b) mixing adenosine monophosphate (AMP) and adenosine diphosphate (ADP) , coenzyme II (NADP) or coenzyme I (NAD), hexokinase or glucokinase, glucose-6-phosphate dehydrogenase and diaphorase, all or part of these reagents, buffers, enzyme activators, enzymes A reducing agent, an anticoagulant and a surfactant are mixed to form a solution R2; c) after the surface activation treatment of the electrode, the solutions R1, R2 and the electron acceptor solution are uniformly mixed, and then evenly added dropwise on the electrode nanomaterial layer; d) After drying the semi-finished product obtained in step c), cover it with an insulating material to form an encapsulation layer; e) use a strip cutter to cut the array of test strips into individual test strips, and then vacuum seal them for use.

In the preparation method of the reagent, the surface activation treatment of the electrode in the step c) is to rapidly activate the surface of the electrode by plasma method.

In the preparation method of the reagent, in the step d), the semi-finished product obtained in the step c) is dried in a drying oven at a temperature of 35-37° C. for 20-25 minutes, and then the encapsulation layer is covered with an insulating material.

The detection system of the creatine kinase biosensor adopts an amperometric method for detection.

Compared with current domestic and foreign products, the product of the present invention has many advantages. The test strip of the present invention and the method introduced are compatible with the commercialized series of portable current-type instruments, so they can be carried around, and the detection is simple and fast; because the production process of the disposable test strip adopts advanced micro-electromechanical systems (MEMS) The technology not only ensures the reliability and consistency of the detection, but also reduces the production cost of the test strip; adopts the simple and effective enzyme immobilization method, and adopts the principle of electrochemical current detection, which is more accurate than the principle of colorimetry, making the test strip The production of the strip is easier; the test strip adopts a capillary design, which makes the injection faster, requires less blood (3μl), and the system has a fast response and high sensitivity.

Description of drawings

Fig. 1 is a layered schematic diagram of a two-electrode system designed by the present invention;

Fig. 2 is a plan view of electrodes of the present invention.

Fig. 3 is a comparison chart of cyclic voltammetry of different concentrations of polymer carboxymethyl cellulose (CMC) in the embodiment of the present invention.

Fig. 4 is the real-time response graph of creatine kinase in the embodiment of the present invention.

Fig. 5 is a linear relationship diagram of creatine kinase in the embodiment of the present invention.

Detailed ways:

The preparation method of the sensor test strip electrode of the present invention refers to the applied patents: 00410030214.X and 200410031948.X.

For the technology of rapidly activating the electrode surface by plasma method, please refer to the applied patent 02154524.3.

The invention provides a method for assembling a layer of high molecular nano particles in the reaction area on the electrode surface. By evenly dropping different concentrations of hydrophilic macromolecule sodium carboxymethyl cellulose on the surface of the electrode, and drying at a temperature of 37° C., electrode nano-layers with different pore sizes are obtained. The method further strengthens the hydrophilicity of the electrode surface and increases the effective surface area of the electrode. It is not only beneficial to the immobilization of enzyme reagents, but also increases the electrochemical activity of the electrode.

The present invention provides methods for immobilizing reaction reagents on nanoparticles.

In the present invention, the reaction reagent is fixed on the electrode to form a reagent layer, and the reaction reagent fixed on the polymer nano layer includes an electron acceptor, which reacts with the analyte and can generate a coupling reaction corresponding to the concentration of the analyte A combination of reagents that also includes buffers, enzyme activators, reducing agents, anticoagulants, and surfactants.

The reaction principle on which the reagent combination of the present invention is based is as follows:

Among them, Creatine phosphate creatine phosphate; Creatine creatine; CK creatine kinase; Glucose glucose; Glucose-6-phosphate 6-phosphate-glucose; Gluconolactone-6-phosphate6-phosphate gluconolactone; HK hexokinase; Dehydrogenase; NADPH NADP ⁺ (Coenzyme II) reduced/oxidized nicotinamide adenine dinucleotide phosphate; DIA diaphorase; Fe(CN) ₆ ^4- /Fe(CN) ₆ ^4- reduced/oxidized Potassium ferricyanide

First, mix appropriate concentrations of creatine phosphate and glucose to prepare a solution R1, and mix appropriate concentrations of adenosine monophosphate (AMP), adenosine diphosphate (ADP), coenzyme II (NADP), hexokinase, glucose-6- Phosphate dehydrogenase, diaphorase, magnesium acetate, reduced glutathione, EDTA, TritonX-100 were mixed to prepare solution R2, after the electrode surface was activated, the solutions R1, R2 and potassium ferricyanate solution Mix evenly, and then evenly drop it on the electrode polymer nano-layer with a sampler. After drying in a drying oven at 35-37° C. for 20-25 minutes, cover and package with an insulating material, and cut the array of test strips into individual test strips with a strip cutter. Then vacuum encapsulated. spare.

The applicable concentration range of creatine phosphate in the reagent is 10-1000mM, and the optimal reaction range is 50mM-100mM. The applicable concentration range of glucose is 10-1000mM, and the optimal reaction range is 50mM-100mM. The applicable concentration range of adenosine monophosphate is 1-100mM, and the optimal reaction range is 5mM-20mM. The applicable concentration range of adenosine diphosphate is 0.5-100mM, and the optimal reaction range is 1mM-20mM. The applicable concentration range of coenzyme II is 0.5-100mM, and its optimal reaction range is 1mM-20mM. The applicable concentration range of hexokinase, glucose-6-phosphate dehydrogenase and diaphorase is 1-2000U/ml, and the optimal reaction range is 1-40U/ml.

The electron acceptor that plays a redox role is composed of ferricyanate, methylene blue, ferrocene and its derivatives, p-benzoquinone, phenazine methyl sulfate, indophenol and its derivatives, and β-naphthoquinone-4-sulfonate One selected from the group consisting of potassium acid potassium; through the electron mediator, the electrons generated during the enzyme reaction are transferred from the enzyme reaction center to the electrode surface, so that the response speed and detection sensitivity of the amperometric enzyme sensor are improved, while reducing the reaction voltage. The electron acceptor is potassium ferricyanate, which reduces the working potential to 0.2V and reduces the interference of other active substances in the blood. The applicable concentration range of potassium ferricyanate: 0.1mM ~ 300mM, and its optimal reaction range: 10mM ~ 100mM.

The buffer is one selected from the group consisting of phosphate buffer, TRIS buffer and MES buffer; the buffer is phosphate buffer. The buffer solution is used to provide a reaction environment with a stable pH value, the optimal pH value of the reaction is 6-8, and the concentration range is 10-100 mM.

In the creatine kinase reaction, magnesium ions are necessary, and the enzyme activator is magnesium acetate or magnesium chloride. Magnesium acetate is selected, its applicable concentration range: 1-100mM, and the optimum concentration range: 5-20mM.

Since most creatine kinase exists in an inactive form in serum, it must be activated in order to accurately quantify its activity. Commonly used enzyme reducing agents are N-acetylcysteine, reduced glutathione, etc. A kind of thiol substance; the enzyme reducing agent is reduced glutathione. Due to the poor stability of thiol compounds in solution, the reduced glutathione needs to be freshly prepared. Its applicable concentration range: 0.1 ~ 10mM, the optimal concentration range: 0.1 ~ 5mM.

Since the blood is easy to coagulate during the whole blood test, generally the blood begins to coagulate within 2 to 5 minutes. Therefore, in order for the reaction to proceed smoothly, it is necessary to add an anticoagulant to the reagent. The anticoagulant is one selected from heparin, oxalate, citrate and EDTA; the anticoagulant is EDTA. Its applicable concentration range: 0.1 ~ 10mM, the optimal concentration range: 0.5 ~ 5mM.

The surfactant is TritonX-100. Add 0.05-1% non-ionic surfactant. Preferably 0.1-0.5% concentration of surfactant.

The various purposes, methods, features and advantages of the present invention can be described in more detail below in conjunction with the accompanying drawings and embodiments.

Example 1

Fig. 1, Fig. 2 are two electrode figures that the present invention designs. First, the thin-film gold electrodes 2 and 3 are prepared on the insulating substrate 1 by micro-electro-mechanical system (MEMS) technology, and the second insulating layer 4 is adhered on the thin-film gold electrodes 2 and 3, and the second insulating layer 4 is provided with Capillary channel 7, which forms a reaction zone. Then, after the electrodes 2 and 3 are activated by the ion cleaning machine, a layer of macromolecule sodium carboxymethylcellulose (CMC) nanoparticle layer 8 is assembled in the reaction zone of the electrodes 2 and 3, that is, the capillary action channel 7, and electric heating at 37°C Dry it in a blast drying oven for 20 minutes, and take it out for later use.

Then add reagent, reagent is: creatine phosphate: 100mM; Glucose: 50mM; Magnesium acetate: 20mM; Adenosine monophosphate (AMP): 20mM; Adenosine diphosphate (ADP): 5mM; NADP): 5mM; Glucose 6-phosphate dehydrogenase: 4U/ml; Hexokinase: 4U/ml; Diaphorase: 2U/ml; Glutathione: 3mM; EDTA: 1mM; TritonX-100: 0.2% ; Potassium ferricyanate: 25mM; Phosphate buffer: 0.01M.

Add 5 μl of the above reagent solution dropwise in the capillary action channel 7, on the nanoparticle layer 8 covered with a polymer carboxymethylcellulose (CMC), and dry in a drying oven at 37°C for 20 minutes to form the reagent layer 9 . The test strip is taken out from the dry box, and covered with a transparent insulating packaging layer 5 to form a capillary channel 7 and capillary inlets 10 at both ends. The array of test strips is cut into individual test strips with a strip cutter. Then vacuum encapsulated.

Example 2

On the prepared electrodes 2 and 3, after washing with ionized water, 0.5% and 1% polymer carboxymethyl cellulose (CMC) was added dropwise, and dried in an electric blast drying oven at 37°C for 20 minutes. After taking it out, check its volt-ampere characteristic curve. In Fig. 3, it is the cyclic voltammogram of different concentrations of polymer carboxymethylcellulose (CMC) assembled on electrodes 2 and 3: (1) 0%; (2) 0.5%; (3) 1%. Scan rate: 100mV/s, solution base phase: 0.01M phosphate buffer (pH7.4), 0.03MK ₃ Fe(CN) ₆ . It can be seen from Figure 3 that as the concentration of polymer carboxymethyl cellulose (CMC) increases, its current increases, but as the concentration of polymer carboxymethyl cellulose (CMC) increases, the polymer nano-film thickens and electron transfer The rate decreases, affecting the reaction speed; the background reaction current increases. Therefore, an appropriate concentration needs to be selected. Generally choose 0.1 ~ 1%, according to the specific situation to determine. On the surfaces of the electrodes 2 and 3 where the polymer nanofilm is assembled, the surface area increases significantly. After adding the liquid reaction reagent, the nanomaterial layer 8 and the reagent form a sol layer, thereby generating a larger reaction current and increasing the reaction sensitivity.

Example 3

A voltage of +0.2V was applied between the working and reference electrodes of the test strip. On the electrodes 2 and 3 modified with the polymer material sodium carboxymethylcellulose (CMC) nano-layer 8, add the mixed reagent dropwise, and after the reaction current (background current) is stable, add creatine kinase buffers with different activities, It interacts with the reagent and the polymer nano-layer 8 to generate a current, which flows out through the electrode lead 6, and the current corresponding to the activity of the total creatine kinase can be quickly detected on the electrochemical biosensor detection system. The resulting current is directly proportional to creatine kinase activity. And it shows a good linear correlation in a wide range. Therefore, the sensor test strip has good application value. See Figure 4a, which is the real-time response curve of the biosensor to different concentrations of creatine kinase. Creatine kinase activity: (1) 800U/ml; (2) 400U/ml; (3) 80U/ml; (4) 8U/ml; (5) 800U/L; (6) 400U/L; (7) 80U/L; (8) 40U/L; (9) 8U/L. Fig. 4b is an enlarged view of the dotted line in Fig. 4a. Figure 5 is a graph showing the linear relationship of creatine kinase. Figure 5a shows the high activity range of creatine kinase, and the response time is 140 seconds; Figure 5b shows the low activity range of creatine kinase, and the reaction time is 280 seconds.

The present invention is described using preferred embodiments, which are for the purpose of illustration only, not limitations of the present invention. Many modifications and variations of the present invention are possible on the basis of the above description. Therefore, within the scope of the appended claims, the invention may be practiced in other ways than those described above. For example: other combinations of reagent formulations, cover the filter membrane on the reagent layer to eliminate the interference of red blood cells for the detection of creatine kinase in whole blood, optical detection methods can be used, electrode reactions modified by other nanoparticles and non-nano materials can be used region, three-electrode form, preparation form of thick-film electrode, etc.

Claims (9)

1. A creatine kinase biosensor, comprising the following structure: There are a pair of parallel electrodes and electrode leads on the upper surface of the insulating substrate; The two electrodes are respectively the working electrode and the counter electrode, and the top of the two electrodes is covered with an insulating layer; There is a groove in the lateral direction of the insulating layer; The top of the groove is covered with an encapsulation layer; The space surrounded by the insulating substrate, the groove and the encapsulation layer forms a capillary action channel, and the two ends of the capillary action channel are capillary inlets, and the capillary action channel is an electrode reaction area, which is characterized in that: There is a nanomaterial layer on the two electrode surfaces of the capillary action channel, and a reaction reagent layer is fixed on the upper surface of the nanomaterial layer; the nanomaterial layer is one of platinum black particles or polymer nanomaterials; to obtain a highly active electrode surface and nanomaterial layers with high specific surface area. 2. creatine kinase biosensor as claimed in claim 1, is characterized in that: described reaction reagent comprises electron acceptor, coupling reaction combination reagent, damping fluid, enzyme activator, enzyme reducing agent, anticoagulant and surface active agent. 3. The creatine kinase biosensor as claimed in claim 2, characterized in that: the coupling reaction combination reagent reacts with the analyte and can generate an electric current corresponding to the concentration of the total creatine kinase of the analyte ; The electron acceptor is any one in the group consisting of ferricyanate, methylene blue, ferrocene, p-benzoquinone, phenazine methyl sulfate, indophenol and β-naphthoquinone-4-sulfonate potassium; The buffer is any one of the group consisting of phosphate buffer, TRIS buffer and MES buffer; The enzyme activator is magnesium acetate or magnesium chloride; The enzyme reducing agent is one of N-acetylcysteine, reduced glutathione or a sulfhydryl-containing substance; The anticoagulant is any one of heparin, oxalate, citrate and EDTA; The surfactant is any one of TritonX-100, diethanolamide, alkyl alcohol amide phosphoester salt, alcohol ether carboxylate, monoalkyl phosphate, sodium dodecylsulfonate or alkylphenol polyoxyethylene ether A sort of. 4. creatine kinase biosensor as claimed in claim 3, is characterized in that: described coupling reaction combination reagent comprises: creatine phosphate, glucose, adenosine monophosphate AMP, adenosine diphosphate ADP, coenzyme II NADP or Coenzyme INAD, hexokinase or glucokinase, glucose-6-phosphate dehydrogenase, diaphorase, all or part of these reagents. 5. The creatine kinase biosensor according to claim 3, wherein the concentration range of the electron acceptor: 10mM～100mM, the concentration range of the buffer solution: 10～100mM, the concentration range of the enzyme activator: 0.1～10mM , Enzyme reducing agent concentration range: 5-20mM, anticoagulant concentration range: 0.5-5mM, surfactant concentration range: 0.1-0.5%. 6. The creatine kinase biosensor according to claim 4, characterized in that: in the coupling reaction combination reagent, the concentration range of creatine phosphate is 10-1000mM; the concentration range of glucose is 10-1000mM; The concentration of adenosine ranges from 1 to 100mM; the concentration of adenosine diphosphate ranges from 0.5 to 100mM; the concentration of coenzyme II ranges from 0.5 to 100mM; the concentration of hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase The range is 1-2000U/ml. 7. A preparation method for a creatine kinase biosensor, characterized in that: comprising the following steps: a) first mixing creatine phosphate and glucose to prepare a solution R1; b) mixing adenosine monophosphate AMP and adenosine diphosphate ADP , coenzyme II NADP or coenzyme I NAD, hexokinase or glucokinase, glucose-6-phosphate dehydrogenase and diaphorase, all or part of these reagents, buffer, enzyme activator, enzyme reducer, anti Coagulant and surfactant are mixed to prepare solution R2; c) After the surface activation treatment of the electrode, the solution R1, R2 and the electron acceptor solution are mixed evenly, and then evenly added dropwise on the electrode nanomaterial layer; d) For c ) After the semi-finished product obtained in step 1 is dried, it is covered with an insulating material to form an encapsulation layer; e) The test strip array is cut into individual test strips with a strip cutter, and then vacuum-packed for future use. 8. The preparation method of creatine kinase biosensor as claimed in claim 7, is characterized in that: in the described c) step, to electrode surface activation treatment, is to activate electrode surface rapidly with plasma method. 9. The preparation method of creatine kinase biosensor as claimed in claim 7, characterized in that: said step d) is to dry the semi-finished product obtained in step c) in a drying oven with a temperature of 35-37°C for 20-20°C. After 25 minutes, cover the encapsulation layer with insulating material.

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