CN107290417A - A kind of biology sensor for improving reagent crystal habit - Google Patents

Abstract

The invention discloses a biosensor for improving the crystal form of reagents, which comprises an insulating substrate, an electrode system, an intermediate layer and a covering layer from bottom to top; the electrode system includes at least a working electrode and a counter electrode; the electrode system is fixed on the Cover at least the detection reagent of the working electrode; the intermediate layer is hollowed out in the sample channel and the reaction chamber area; the covering layer and the intermediate layer and the insulating substrate are constructed into a reaction chamber and a sampling capillary channel; the reaction chamber has air holes ; The composition of the detection reagent contains chemical substances with amino groups. The present invention can effectively eliminate or reduce the "coffee ring" phenomenon after the detection reagent is cured after adding the chemical substance with amino group to the detection reagent component, making the shape of the detection reagent on the electrode system more uniform, meticulous and consistent, and significantly improving The precision performance of biosensor products is tested. In particular, the detection reagent using hexaamine ruthenium trichloride as the electron mediator has very remarkable effect.

Description

A Biosensor with Improved Reagent Crystalline Morphology

technical field

The invention relates to the field of biological detection instruments, in particular to a biosensor for improving the crystal form of reagents.

Background technique

A biosensor is an instrument that is sensitive to biological substances and converts its concentration into an electrical signal for detection. It is made of immobilized biosensitive materials as recognition elements (including enzymes, antibodies, antigens, microorganisms, cells, tissues, nucleic acids and other biologically active substances), appropriate physical and chemical transducers (such as oxygen electrodes, photosensitive tubes, field effect tubes, Piezoelectric crystals, etc.) and an analysis tool or system composed of a signal amplification device.

Electrochemical biosensors have been widely used in the field of in vitro diagnostics. These instrument systems can detect physiological substances in body fluids, which is convenient for doctors to analyze and diagnose patients' conditions. Some instrument systems are also used at home to facilitate self-monitoring of patients. These physiological Indicators include blood sugar, cholesterol, uric acid, triglycerides, lactic acid, ketone bodies, enzymes, etc. At present, biosensors have been widely used in clinical testing, and the most popular example is a portable blood glucose testing system.

The current biosensor test piece usually consists of the following parts: an insulating substrate, as a carrier of the product; an electrode system built on the insulating substrate, including at least a working electrode and a counter electrode; a detection reagent fixed on the electrode system; a septum The opening of the reaction chamber and the sampling channel covers the electrode system; the covering layer covers the intermediate layer and forms the reaction chamber together with the substrate and the intermediate layer.

The precision performance of clinically used biosensor products is very important, and whether the crystal form of the detection reagent on the working electrode is consistent directly affects the precision performance of the product. However, at present, the common problem in the detection reagent crystal morphology technology is the "coffee ring" phenomenon, especially the detection reagent using hexammonium ruthenium trichloride as the electron mediator, the specific performance is that the edge is high and the middle is low after the reagent is solidified, or the shape Mottled, coarse and uneven particles directly lead to uneven distribution of reagents on the electrode and inconsistent reactions, which is one of the main obstacles to further improving the precision performance of the product.

Contents of the invention

In order to overcome the above-mentioned defects in the prior art, the present invention aims to provide a biosensor with uniform crystal morphology of the detection reagent and good test precision, so as to solve the ubiquitous problem in the prior art - the "coffee ring" phenomenon.

The purpose of the present invention is achieved through the following technical solutions: a biosensor with improved reagent crystallization form can be used to detect the concentration or activity of specific substances in samples, and its composition includes: an insulating substrate; an electrode system built on the insulating substrate, the The electrode system includes at least a working electrode and a counter electrode; a detection reagent that is fixed on the electrode system and should at least cover the working electrode; hollowed out; the covering layer arranged above the intermediate layer, the covering layer, the intermediate layer and the insulating substrate are constructed into a reaction chamber and a sampling capillary channel, and the sample can be automatically injected by capillary siphon; the reaction chamber should There are air holes to form a siphon effect; wherein, the composition of the detection reagent contains chemical substances with amino groups.

The insulating substrate is the carrier of the biosensor. In the present invention, the insulating substrate is preferably a flexible plastic sheet with a certain stiffness, including but not limited to PET, PP, PVC, acrylic and other materials. There are currently a variety of commercial products to choose from, such as Dupont's ST 504, ST 339, etc.

The electrode system is built on an insulating substrate, including at least a counter electrode and a working electrode. The electrode material includes but is not limited to one of graphite, silver, gold, platinum, palladium, ITO, and IZO, which can be constructed on an insulating substrate by screen printing, vacuum sputtering, and laser processing.

The intermediate layer is built above the electrode system, and the reaction chamber area is hollowed out. Combined with the cover layer, the reaction chamber, that is, the sampling channel, is formed. The thickness of the intermediate layer is 0.025 mm to 0.5 mm, preferably, the thickness of the intermediate layer is 0.075 mm to 0.125 mm. The material of the intermediate layer can be a tape with or without a base material, which is bonded after processing; it can also be glue or polymer paste, which is printed on by screen printing; if there is no other insulating material on the electrode, The material of the intermediate layer must be a good insulating material.

The cover layer, above the intermediate layer, together with the intermediate layer and the insulating substrate are constructed into a reaction chamber and a sample injection channel, so as to automatically inject samples through siphon action. PET material can be used for the cover layer, preferably transparent hydrophilic treated material, transparent can help users easily confirm the sample injection status in the reaction area, hydrophilic can make the sample injection smoother, non-reaction chamber area can be opaque, and the sample injection logo can be printed and Logo, available commercial materials are Adhesives Research, Inc's 92804, Kemafoil HHNW of Coveme Company, 9971 of 3M Company, etc.

Vent holes, the reaction chamber should have vent holes to form a siphon effect, and the vent holes can be realized by punching holes in the tail end area of the sampling channel on the cover layer or insulating substrate, or a vent can be left in the tail end area of the sampling channel through the middle compartment accomplish.

Detection reagent is one of the core technologies of biosensors. The detection reagent is fixed on the electrode system and should at least cover the working electrode. The detection reagent can be processed and fixed on the electrode by spotting, screen printing, inkjet printing, slot extrusion and other technologies. Detection reagents include enzymes, electron mediators, buffers, surfactants, polymers and other components. The role of the enzyme is to specifically react with the analyte; the electron mediator obtains electrons from the reaction between the enzyme and the analyte, and diffuses to the electrode to release electrons and form a signal, which can be captured by the tester; buffer Ensure a suitable reaction environment; surfactants can optimize reaction conditions and promote sample diffusion; polymers can form a network structure and immobilize enzymes to a certain extent.

Main feature of the present invention is to comprise the chemical substance that has amino group in the detection reagent, and the chemical general formula that this substance has is as follows:

The R1, R2, R3 variable groups of the above chemical general formula include not limited to H, hydrocarbon groups, acyl groups and derivatives of hydrocarbon groups, acyl derivatives, and two or all three groups of R1, R2, R3 groups can be Form a ring. Typical substances in this chemical formula include amino acids (such as serine, threonine, proline, alanine, lysine, histidine, lysine, etc.), imidazole and imidazole derivatives.

The inventors have found that adding chemical substances with amino groups to the detection reagent can effectively eliminate or reduce the "coffee ring" phenomenon after the detection reagent is solidified, making the shape of the detection reagent on the electrode system more uniform, detailed and consistent, and significantly improving Test precision performance of biosensor products. In particular, the detection reagent using hexaamine ruthenium trichloride as the electron mediator has very remarkable effect.

In the present invention, the concentration of the chemical substance having an amino group in the detection reagent preparation solution may be 1 mM-500 mM, preferably 10 mM-100 mM.

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

The present invention can effectively eliminate or reduce the "coffee ring" phenomenon after the detection reagent is cured after adding the chemical substance with amino group to the detection reagent component, making the shape of the detection reagent on the electrode system more uniform, meticulous and consistent, and significantly improving Test precision performance of biosensor products. In particular, the detection reagent using hexaamine ruthenium trichloride as the electron mediator has very remarkable effect.

The experimental test results show that the same concentration response curve of the control example has large dispersion and the precision is not very ideal; while the same concentration response curve of the embodiment of the present invention is very consistent, and the precision of the biosensor has been significantly improved.

Description of drawings

FIG. 1A and FIG. 1B are respectively a schematic diagram of an exploded structure and a schematic diagram of a complete structure of a biosensor with improved reagent crystal form according to the present invention.

Fig. 2 is a schematic structural view of the electrode system of a biosensor with improved reagent crystal form according to the present invention.

FIG. 3 is a crystallographic diagram of the detection reagent of Comparative Example 1.

Fig. 4 is a crystallographic diagram of the detection reagent of Example 1 of the present invention.

Fig. 5 is a crystallographic diagram of the detection reagent of Example 2 of the present invention.

Fig. 6 is a crystallographic diagram of the detection reagent of Example 3 of the present invention.

Fig. 7 is a crystallographic diagram of the detection reagent of Example 4 of the present invention.

Fig. 8 is a crystallographic diagram of the detection reagent of Example 5 of the present invention.

Fig. 9 is a crystallographic diagram of the detection reagent of Example 6 of the present invention.

Figure 10 is a response curve diagram of Comparative Example 1.

Fig. 11 is a reaction curve diagram of Example 1 of the present invention.

Fig. 12 is the reaction curve diagram of Example 2 of the present invention.

Fig. 13 is a reaction curve diagram of Example 3 of the present invention.

Fig. 14 is the reaction curve diagram of Example 4 of the present invention.

Fig. 15 is a reaction curve diagram of Example 5 of the present invention.

Fig. 16 is a reaction curve diagram of Example 6 of the present invention.

detailed description

The content of the present invention will be described in more detail below in conjunction with the embodiments and accompanying drawings. It should be understood that the implementation of the present invention is not limited to the following examples, and any modifications and/or changes made to the present invention will fall within the protection scope of the present invention.

In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or commonly used in this industry. The methods in the following examples, unless otherwise specified, are conventional methods in the art.

Description of the composition of the biosensor test piece in the embodiment:

Insulating substrate, the material includes but not limited to one of PET, PP, PVC, and acrylic.

Electrode system, built on an insulating substrate. The electrode system includes a first pair of electrodes, a working electrode, and a second pair of electrodes, and the first pair of electrodes, the working electrode, and the second pair of electrodes are arranged sequentially from the sample inlet to the far end of the reaction channel. The electrode material includes but is not limited to one of graphite, silver, gold, platinum, palladium, ITO, and IZO, which can be constructed on an insulating substrate by screen printing, vacuum sputtering, and laser processing.

The detection reagent, fixed on the electrode system, covers at least the working electrode. The detection reagent is first prepared into a solution or slurry, and then fixed on the electrode system by spraying, screen printing, slot extrusion, inkjet printing and other processes. The present invention takes the glucose detection reagent as an example.

The intermediate layer is built above the electrode system, and the reaction chamber area is hollowed out, and combined with the cover layer, the reaction chamber, that is, the sampling channel, is formed. The thickness of the intermediate layer is 0.025 mm to 0.5 mm, preferably, the thickness of the intermediate layer is 0.075 mm to 0.125 mm. The material of the middle layer can be a tape with or without a base material, which is bonded after processing; it can also be glue or polymer paste, which is printed by screen printing; if there is no other insulating material on the electrode , The material of the intermediate layer must be a good insulating material.

The cover layer, above the intermediate layer, and the intermediate layer and the insulating substrate are constructed to form a reaction chamber, that is, a sampling channel, so as to automatically inject samples through siphon action. PET material can be used for the cover layer, preferably transparent hydrophilic treated material, transparent can help users easily confirm the sample injection status in the reaction area, hydrophilic can make the sample injection smoother, non-reaction chamber area can be opaque, and the sample injection logo can be printed and Logo, available commercial materials are Adhesives Research, Inc's 92804, Kemafoil HHNW of Coveme Company, 9971 hydrophilic membrane of 3M Company, etc.

The reaction chamber should have air holes so as to form a siphon effect, and the air holes in the embodiment of the present invention are realized by punching holes in the tail end area of the sampling channel in the covering layer.

Comparative example 1

The structure of the biosensor is shown in Figure 1A, Figure 1B and Figure 2. An electrode system 200 is built on the insulating substrate 100; the electrode system 200 includes three electrodes: a first pair of electrodes 201, a working electrode 202, and a second pair of electrodes 203. The carbon slurry is solidified and formed; the detection reagent 300 is fixed above the electrode system, the detection reagent covers at least the working electrode, and the detection reagent solution is distributed on the electrode by a glue dispenser to dry; The interlayer is hollowed out by laser engraving or mold cutting to make the sample injection channel and the opening of the reaction chamber, and the middle interlayer is attached to the electrode system; the covering layer 500 is made of transparent hydrophilic sheet, which is placed in the corresponding reaction chamber by laser engraving or mold cutting The air vent 501 is mainly made at the end, and the cover layer 500 is attached to the middle layer. The cover layer, the middle layer and the insulating substrate are constructed into a reaction chamber and a sample injection channel 401, which can automatically inject samples through a siphon; then use the machine Press to make a complete biosensor test piece 600, one end of the opening of the middle compartment 400 is a sample inlet 601, and each electrode lead wire of the electrode system 200 extends to the other end of the biosensor test piece to form a connecting contact pin 602. The pin end is inserted into the port of the detection instrument, and the connecting contact pin of each electrode will be connected with the corresponding contact in the port.

Wherein detection reagent 300 is formulated into aqueous solution first, and its composition is as follows: 100mM sodium phosphate buffer solution (pH is 7.0), 1.2% (w/v) methylcellulose (Sigma M6385), 0.5% (w/v) polyethylene glycol Alcohol 4000 (Sigma 95904), 0.5% (w/v) Triton X-100 (Aladdin T109026), 1.86% (w/v) hexaamine ruthenium trichloride (CMI 8015), 2000U/mL glucose oxidase (BBI Solutions GO3A). The detection reagent aqueous solution was dispensed and fixed on the electrode system of the biosensor test piece using a dispenser, and each biosensor test piece was dispensed with 1.0uL detection reagent aqueous solution, and then placed in an oven at 60 degrees Celsius for 10 minutes to dry.

Example 1

The structure of the biosensor of Example 1 is the same as that of Comparative Example 1, except that the detection reagent components are different.

The composition of the detection reagent aqueous solution is as follows: 100mM sodium phosphate buffer (pH 7.0), 1.2% (w/v) methylcellulose (Sigma M6385), 0.5% (w/v) polyethylene glycol 4000 (Sigma 95904), 0.5% (w/v) Triton X-100 (Aladdin T109026), 50mM imidazole (Aladdin reagent I108707), 1.86% (w/v) hexaamine ruthenium trichloride (CMI8015), 2000U/mL glucose oxidase ( BBI Solutions GO3A). The detection reagent aqueous solution was dispensed and fixed on the electrode system of the biosensor test piece using a dispenser, and each biosensor test piece was dispensed with 1.0uL detection reagent aqueous solution, and then placed in an oven at 60 degrees Celsius for 10 minutes to dry.

Example 2

The structure of the biosensor of Example 2 is the same as that of Comparative Example 1, except that the detection reagent components are different.

The composition of the detection reagent aqueous solution is as follows: 100mM sodium phosphate buffer (pH 7.0), 1.2% (w/v) methylcellulose (Sigma M6385), 0.5% (w/v) polyethylene glycol 4000 (Sigma 95904), 0.5% (w/v) Triton X-100 (Aladdin T109026), 50mM L-threonine (Aladdin reagent T108221), 1.86% (w/v) hexaamine ruthenium trichloride (CMI 8015), 2000U/ mL glucose oxidase (BBI Solutions GO3A). The detection reagent aqueous solution was dispensed and fixed on the electrode system of the biosensor test piece using a dispenser, and each biosensor test piece was dispensed with 1.0uL detection reagent aqueous solution, and then placed in an oven at 60 degrees Celsius for 10 minutes to dry.

Example 3

The structure of the biosensor of Example 3 is the same as that of Comparative Example 1, except that the detection reagent components are different.

The composition of the detection reagent aqueous solution is as follows: 100mM sodium phosphate buffer (pH 7.0), 1.2% (w/v) methylcellulose (Sigma M6385), 0.5% (w/v) polyethylene glycol 4000 (Sigma 95904), 0.5% (w/v) Triton X-100 (Aladdin T109026), 50mM L-proline (Aladdin reagent P108709), 1.86% (w/v) hexaamine ruthenium trichloride (CMI 8015), 2000U/ mL glucose oxidase (BBI Solutions GO3A). The detection reagent aqueous solution was dispensed and fixed on the electrode system of the biosensor test piece using a dispenser, and each biosensor test piece was dispensed with 1.0uL detection reagent aqueous solution, and then placed in an oven at 60 degrees Celsius for 10 minutes to dry.

Example 4

The structure of the biosensor of Example 4 is the same as that of Comparative Example 1, except that the detection reagent components are different.

The composition of the detection reagent aqueous solution is as follows: 100mM sodium phosphate buffer (pH 7.0), 1.2% (w/v) methylcellulose (Sigma M6385), 0.5% (w/v) polyethylene glycol 4000 (Sigma 95904), 0.5% (w/v) Triton X-100 (Aladdin T109026), 50mM L-serine (Aladdin reagent S103483), 1.86% (w/v) hexaamine ruthenium trichloride (CMI 8015), 2000U/mL glucose Oxidase (BBI Solutions GO3A). The detection reagent aqueous solution was dispensed and fixed on the electrode system of the biosensor test piece using a dispenser, and each biosensor test piece was dispensed with 1.0uL detection reagent aqueous solution, and then placed in an oven at 60 degrees Celsius for 10 minutes to dry.

Example 5

The biosensor structure of Example 5 is the same as that of Comparative Example 1, except that the detection reagent components are different.

The composition of the detection reagent aqueous solution is as follows: 100mM sodium phosphate buffer (pH 7.0), 1.2% (w/v) methylcellulose (Sigma M6385), 0.5% (w/v) polyethylene glycol 4000 (Sigma 95904), 0.5% (w/v) Triton X-100 (Aladdin T109026), 10mM L-serine (Aladdin reagent S103483), 1.86% (w/v) hexaamine ruthenium trichloride (CMI 8015), 2000U/mL glucose Oxidase (BBI Solutions GO3A). The detection reagent aqueous solution was dispensed and fixed on the electrode system of the biosensor test piece using a dispenser, and each biosensor test piece was dispensed with 1.0uL detection reagent aqueous solution, and then placed in an oven at 60 degrees Celsius for 10 minutes to dry.

Example 6

The biosensor structure of Example 6 is the same as that of Comparative Example 1, except that the detection reagent components are different.

The composition of the detection reagent aqueous solution is as follows: 100mM sodium phosphate buffer (pH 7.0), 1.2% (w/v) methylcellulose (Sigma M6385), 0.5% (w/v) polyethylene glycol 4000 (Sigma 95904), 0.5% (w/v) Triton X-100 (Aladdin T109026), 100mM L-serine (Aladdin reagent S103483), 1.86% (w/v) hexaamine ruthenium trichloride (CMI 8015), 2000U/mL glucose Oxidase (BBI Solutions GO3A). The detection reagent aqueous solution was dispensed and fixed on the electrode system of the biosensor test piece using a dispenser, and each biosensor test piece was dispensed with 1.0uL detection reagent aqueous solution, and then placed in an oven at 60 degrees Celsius for 10 minutes to dry.

test case

Use venous whole blood anticoagulated with heparin to prepare samples of 7 glucose concentration levels that run through the range of 0-700 mg/dL. Low-concentration samples can be obtained by glycolysis of blood samples, and high-concentration samples can be obtained by adding high-concentration glucose solution Preparation, the actual preparation concentration is: 0mg/dL, 58.4mg/dL, 115.7mg/dL, 174.2mg/dL, 291.5mg/dL, 432.6mg/dL, 645.6mg/dL. The 7 kinds of biosensor test pieces of the above-mentioned comparative example 1 and embodiment 1-6 are tested and compared on the test instrument, the potential difference between the working electrode and the counter electrode of the instrument is set to 0.30V, and each concentration sample of each test piece is tested 5, the test result uses a 5-second current.

The test results of Examples 1-6 and Comparative Example 1 are shown in Figures 3 to 16 and the following Tables 1-7.

Table 1: Test results of control example 1

Table 2: Example 1 test results

Table 3: Example 2 test results

Table 4: Example 3 test results

Table 5: Example 4 test results

Table 6: Example 5 test results

Table 7: Example 6 test results

As can be seen from Table 1 to Table 7, except for the 0 concentration, the average value of the coefficient of variation of Examples 1-6 is significantly lower than that of the control example 1 (the value of the 0 concentration factor is too low, and the relative variation of the coefficient of variation is too large, so it is not included in the comparison) , the average coefficient of variation of Comparative Example 1 is 3.1%, and the average coefficient of variation of Examples is 1.0% to 2.0%. It can be seen that the present invention can effectively improve the test precision of the biosensor test piece.

3 to 9 are the crystal morphology diagrams of the detection reagents of Comparative Example 1 and Examples 1-6. It can be seen that the "coffee ring" phenomenon in Comparative Example 1 is significant, and the morphology of the detection reagent is very heterogeneous; while the detection reagents in the six embodiments have good crystallization morphology and are uniform, without significant "coffee ring" phenomenon.

Figures 10 to 16 are response curves of seven concentration tests of Comparative Example 1 and Examples 1-6. It can be seen that the same concentration-response curve of the comparative example is more discrete, indicating that the precision of the biosensor of the comparative example is not very ideal; and the same concentration-response curves of the six examples are very consistent, indicating that the precision of the biosensor of the present invention has been significantly improved. improve.

Claims (6)

1. A biosensor for improving the crystalline form of reagents, comprising from bottom to top: an insulating substrate, an electrode system, an intermediate layer, and a cover layer; the electrode system includes at least a working electrode and a counter electrode; fixed on the electrode system is at least Covering the detection reagent of the working electrode; the intermediate layer is hollowed out in the sample channel and the reaction chamber area; the covering layer, the intermediate layer and the insulating substrate are constructed into a reaction chamber and a sampling capillary channel; the reaction chamber has air holes; It is characterized in that the composition of the detection reagent includes chemical substances with amino groups. 2. A kind of biosensor of improving reagent crystal form according to claim 1, is characterized in that, the chemical general formula of described chemical substance with amino group is as follows: The variable groups of R1, R2 and R3 in the above general chemical formula include H, hydrocarbyl, acyl and derivatives of hydrocarbyl and acyl. 3. A biosensor for improving the crystal form of reagents according to claim 2, characterized in that two or all three of the variable groups of R1, R2 and R3 form a ring. 4 . The biosensor for improving the crystal form of reagents according to claim 2 , wherein the chemical substances with amino groups include amino acids, imidazoles and imidazole derivatives. 5 . The biosensor for improving the crystal form of reagents according to claim 1 , wherein the concentration of the chemical substances with amino groups in the detection reagent preparation solution is 1 mM-500 mM. 6. A biosensor for improving the crystal form of reagents according to claim 1 or 5, characterized in that the concentration of the chemical substance with amino groups in the detection reagent preparation solution is 10 mM-100 mM.