WO2022016639A1 - Subcutaneously implanted glucose sensor with three electrodes and manufacturing method therefor - Google Patents

Abstract

A subcutaneously implanted glucose sensor with three electrodes and a manufacturing method therefor. The sensor adopts the form of three electrodes, i.e. a working electrode (1), a reference electrode (2) and an auxiliary electrode (3). A test micro-current passes through the working electrode (1) and the auxiliary electrode (3), and the reference electrode (2) substantially has no current passing therethrough, so that the service life of the reference electrode (2) is prolonged. By improving the manufacturing process of the sensor electrodes, the process becomes simple and the quality is controllable, and the stability of glucose oxidase of the working electrode (1) is maintained, thereby ensuring the accuracy of sensor testing data. In addition, carriers such as bovine serum albumin are removed, and the activity and stability of glucose oxidase are ensured by means of bifunctional coupling of glutaraldehyde and silane, thereby reducing potential safety risks to the human body and reducing rejection reactions of the human body. By means of the detachable snap-fitting of a transmitter and the sensor, the sensor and the transmitter can be separated conveniently, and the transmitter can also be reused after the sensor is replaced, reducing the costs of the users.

Description

A three-electrode subcutaneously implanted glucose sensor and method of making the same technical field

The invention relates to the field of needle-shaped sensors for real-time monitoring of blood sugar in diabetics, and more particularly to a three-electrode subcutaneously implanted glucose sensor and a manufacturing method thereof.

Background technique

A continuous blood glucose monitoring system (RGMS) is a new type of continuous blood glucose monitoring system that has been put into clinical use in recent years. The diameter of the probe head is small, and the patient has no obvious pain and discomfort when it is placed. The instrument receives an electrical signal reflecting blood sugar changes from the detector at certain intervals, and converts the average value of the electrical signals collected multiple times into a blood sugar value for storage. Hundreds of blood glucose values can be recorded every day. The dynamic blood glucose monitor can also store meal, exercise, medication and other times at the same time. This allows patients to no longer endure the pain of acupuncture every day, and it can provide daily blood sugar charts, multi-day blood sugar chart fluctuation trend analysis and daily blood sugar data summaries, which is a new breakthrough in blood sugar detection.

The detection needle of the continuous blood glucose monitor is the electrode. In order to improve the accuracy of blood glucose monitoring, the working electrode and the reference electrode are usually used together. The invention patent with publication number CN101530328B "Subcutaneously implanted glucose sensor and its manufacturing method" discloses a double-electrode subcutaneously implanted glucose sensor with a working electrode and a reference electrode, which improves and optimizes the performance of the actual glucose sensor , mutual compatibility and consistency. Although the performance and stability of the sensor have been guaranteed to a certain extent, the reference electrode will have current passing through, and the silver chloride contained in the reference electrode will react with the electrons AgCl+e ^- →Ag+Cl ^- , making silver\silver chloride The silver chloride in the layer is continuously consumed, which affects the service life of the reference electrode; in addition, the production of the sensor electrode uses many biochemical materials, and the process is too complicated, which is not conducive to the control of the quality of the electrode, and the scrap rate is high, which makes the cost of the sensor relatively high. High; In particular, the cross-linking of glucose oxidase requires the use of bovine serum albumin or human serum albumin as a carrier, which poses a certain biological safety hazard to the human body and increases the possibility of human rejection.

SUMMARY OF THE INVENTION

Aiming at the shortcomings of the prior art, the present invention provides a three-electrode subcutaneous implantable glucose sensor and a manufacturing method thereof. The three-electrode subcutaneous implantable glucose sensor of the present invention adopts a three-electrode form of a working electrode, a reference electrode and an auxiliary electrode. , the tested microcurrent passes through the working electrode and the auxiliary electrode, the reference electrode only measures the voltage, and basically no current passes through, thereby extending the service life of the reference electrode, that is, prolonging the service life of the sensor, which can only be implanted and worn. A few days are extended to implantation and wearing for half a month to a month; through the improvement of the sensor electrode fabrication process and the simplification of biochemical materials, the complex process becomes simple, the quality is controllable, the cost of the sensor is reduced, and through process improvement The stability of the working electrode glucose oxidase is maintained to ensure the accuracy of the sensor test data; in addition, the carrier of glucose oxidase such as bovine serum albumin or human serum albumin is cancelled, and the bifunctional coupling method of glutaraldehyde and silane is used to ensure The activity and stability of glucose oxidase reduce the potential biochemical safety hazards to the human body and the rejection reaction of the human body; After the sensor is replaced, the transmitter can be reused, which reduces the cost of use for the user.

The specific technical scheme of the present invention is as follows: a three-electrode subcutaneously implanted glucose sensor, comprising a needle-shaped working electrode, a reference electrode and an auxiliary electrode, wherein the working electrode, the reference electrode and the auxiliary electrode are fixed to the sensor seat;

The working electrode includes a first conductive substrate, a first metal transition layer, a first precious metal layer, an inner coupling layer, an immobilized enzyme layer, an outer coupling layer, and a first polymer film layer from the inside out;

The reference electrode includes a second conductive matrix, a second metal transition layer, a silver\silver chloride layer, and a second polymer film layer from the inside out;

The auxiliary electrode includes a third conductive matrix, a third metal transition layer, a second noble metal layer, a coupling layer, and a third polymer film layer from the inside out.

The sensor used in the continuous blood glucose monitoring of diabetic patients is a consumable and needs to be replaced regularly. The most critical factor affecting the service life of the sensor is the loss of the reference electrode. When electrons pass through the reference electrode, the silver chloride contained in the reference electrode will AgCl+e ^- → Ag+Cl ^- reacts with electrons, so that the silver chloride in the silver\silver chloride layer is continuously consumed, which affects the service life of the reference electrode, and by increasing the auxiliary electrode, it can be Make most of the electrons pass through the auxiliary electrode, reduce the consumption of the silver/silver chloride layer, and prolong the service life of the sensor; the working electrode is the electrode that plays the main electrochemical reaction, and there is a generation of glucose in the blood of the human body. The reacted biological enzyme, that is, the immobilized enzyme layer, and the outer layer is the first polymer membrane layer that limits the amount of glucose reacting with the biological enzyme. In order to improve the stability of the biological enzyme and maintain the activity, the immobilized enzyme is usually The enzyme layer is immobilized on the carrier, and a coupling agent is used for coupling between the immobilized enzyme layer and the first polymer film layer, and the carrier usually uses bovine serum albumin or human serum albumin, which is not suitable for human body. There are certain biological safety hazards.

As a preferred option of the present invention, the sensor base includes a plastic base, three metal connection points arranged in a triangle in the middle of the plastic base, a mounting hole is opened in the middle of the metal connection points, the working electrode, the reference The electrode and the tail end of the auxiliary electrode needle pass through the mounting hole respectively and are connected to the metal connection point and conduct.

The working electrode, the reference electrode and the auxiliary electrode are arranged in a triangle on the plastic substrate, which can ensure that the distance between the three electrodes is short, and the monitoring value of blood sugar is more accurate.

As a preference of the present invention, the three metal connection points are arranged in an equilateral triangle, and the side length of the equilateral triangle is 3mm-6mm.

When the distance between the working electrode, the reference electrode and the auxiliary electrode is too close, the resistance of implantation into the human body is large. ～6mm can not only ensure smooth implantation into the human body, but also ensure the data accuracy of the sensor.

As a preferred option of the present invention, the working electrode, the reference electrode and the auxiliary electrode are all installed perpendicular to the plastic substrate.

The vertical implantation of the working electrode, the reference electrode and the auxiliary electrode into the human body is relatively smooth, especially when an auxiliary wearing tool is used, the vertical implantation becomes extremely easy, and the human body basically does not feel pain. , the plastic substrate and the implanted surface of the human body have a good degree of fit, which can ensure the stability of the sensor after implantation.

As a preferred option of the present invention, the sensor base further includes at least three metal contacts located on the plastic base, the metal contacts are respectively connected to the metal connection points, and the plastic base is provided with a The engaging mechanism of the connector.

The transmitter provides the sensor with the voltage required for operation, and receives the current generated by the sensor electrode, the metal contact is opposite to the contact position on the transmitter, and the transmitter and the sensor base are snap-connected When the metal contacts are connected to the contacts on the transmitter to realize the transmission of current data; the sensor is a consumable and needs to be replaced regularly, the transmitter can be used for a long time, and the engaging mechanism can It is convenient to separate the sensor and the transmitter, and realize the reuse of the transmitter.

As a preferred option of the present invention, an operational amplifier is installed in the transmitter, the reference electrode is electrically connected to the inverting input end of the operational amplifier through the metal connection point and the metal contact, and the auxiliary electrode is electrically connected through the The metal connection point and the metal contact are electrically connected to the output terminal of the operational amplifier.

the reference electrode is connected to the inverting input terminal of the operational amplifier, and the auxiliary electrode is connected to the output terminal of the operational amplifier, so that most of the current passes through the auxiliary electrode, and almost no current flows through the reference electrode, That is, no electrons pass through, which weakens the ^{occurrence of the reaction AgCl+e −} →Ag+Cl ⁻ and prolongs the service life of the reference electrode.

As a preference of the present invention, the first conductive base material is stainless steel, the first metal transition layer is gold, the first noble metal layer is platinum, and the inner coupling layer and the outer coupling layer are silane , the immobilized enzyme layer is glucose oxidase, and the first polymer film layer is polyurethane and/or polyethylene glycol.

The first conductive material is preferably 304 stainless steel or 316 stainless steel substrate, good toughness, low body rejection strong security; the immobilized enzyme is glucose oxidase layer can be effectively catalyze Glucose + O ₂ → gluconic acid + H ₂ O ₂ reaction occurs, the first polymer film is a polyurethane and / or polyethylene glycol can limit entry of glucose into the immobilized enzyme involved in the reaction layer to ensure sufficient oxygen participate in the reaction, the glucose concentration is such that the H _{The determining factor of the amount of 2} O ₂ generated; the first metal transition layer is gold, and the first noble metal layer is platinum, which can effectively catalyze _{the occurrence of H 2} O ₂ →O ₂ +2H ⁺ +2e ^- reaction, so that H ₂ The _{amount of O 2} generation is converted into the amount of electron generation, and the electrons generate a microcurrent through the working electrode, and gold and platinum can reduce the signal-to-noise ratio, so that there is a linear positive correlation between the microcurrent and the glucose concentration; the inner coupling layer and the The outer coupling layer is preferably silane, which can not only achieve a good coupling effect, improve the stability of the immobilized enzyme layer, but also simplify the process, so that the quality of the working electrode is controllable.

As a preference of the present invention, the second conductive base material is stainless steel, the second metal transition layer is silver, and the second polymer film layer is polyurethane and/or polyethylene glycol.

The material of the second conductive matrix is preferably 304 stainless steel or 316 stainless steel, which has good toughness, small rejection reaction of the human body, and strong safety; the second metal transition layer is silver, and the silver\chloride transitions outside the silver layer. The silver layer has high accuracy and stability.

As a preference of the present invention, the third conductive base material is stainless steel, the third metal transition layer is gold, the second noble metal layer is platinum, the coupling layer is silane, and the third polymer film The layers are polyurethane and/or polyethylene glycol.

The material of the third conductive substrate is preferably 304 stainless steel or 316 stainless steel, which has good toughness, small human body rejection reaction, and strong safety; the third metal transition layer is gold, and the second precious metal layer is platinum, which can reduce the current signal. noise ratio, to ensure the current stability after forming a loop with the working electrode.

The invention also discloses a manufacturing method of a three-electrode subcutaneously implanted glucose sensor:

The sensor includes a needle-shaped working electrode, a reference electrode and an auxiliary electrode, and the working electrode, the reference electrode and the auxiliary electrode are fixed on the sensor base;

The working electrode includes a first conductive substrate, a first metal transition layer, a first precious metal layer, an inner coupling layer, an immobilized enzyme layer, an outer coupling layer, and a first polymer film layer from the inside out. A conductive base material is stainless steel, the first metal transition layer is gold, the first precious metal layer is platinum, the inner coupling layer and the outer coupling layer are silane, and the immobilized enzyme layer is glucose Oxidase, the first polymer film layer is polyurethane and/or polyethylene glycol;

The reference electrode includes a second conductive substrate, a second metal transition layer, a silver/silver chloride layer, and a second polymer film layer from the inside out, the second conductive substrate is made of stainless steel, and the second metal transition layer is made of stainless steel. The layer is silver, and the second polymer film layer is polyurethane and/or polyethylene glycol;

The auxiliary electrode includes a third conductive substrate, a third metal transition layer, a second precious metal layer, a coupling layer, and a third polymer film layer from the inside to the outside, the third conductive substrate is made of stainless steel, and the third The metal transition layer is gold, the second precious metal layer is platinum, the coupling layer is silane, and the third polymer film layer is polyurethane and/or polyethylene glycol;

The first metal transition layer of the working electrode is covered on the outer layer of the first conductive base by a cationic etching method; the first precious metal layer is covered on the first conductive base by a cationic etching method or an electrochemical deposition method. The outer layer of the metal transition layer; the inner coupling layer is covered on the outer layer of the first precious metal layer by dipping or coating; the immobilized enzyme layer is attached to the inner coupling by dipping or coating from an enzyme solution the outer coupling layer; the outer coupling layer is covered on the outer layer of the immobilized enzyme layer by dipping or coating; the first polymer film layer is covered on the outer coupling layer by dipping or coating outer layer.

The working electrode is electroplated with a gold layer on the outer layer of the stainless steel needle, a platinum layer is deposited on the outer layer of the gold layer by cationic etching or electrochemical deposition, and the outer layer of the platinum layer is dipped or coated with silane, and then glucose oxidase is dipped or coated on the outer layer. The outer layer of the enzyme layer is impregnated or coated with silane, and finally impregnated or coated with polyurethane and/or polyethylene glycol. The whole preparation method is simple, low in cost, controllable in quality, and can ensure the stability of glucose oxidase.

As a preferred option of the present invention, the second metal transition layer of the reference electrode is covered on the outer layer of the second conductive substrate by a cationic etching method; the silver\silver chloride layer is formed by the second metal transition layer. Chlorination is formed; the second polymer film layer is covered on the outer layer of the silver/silver chloride layer by dipping or coating.

The reference electrode is electroplated with a silver layer on the outer layer of the stainless steel needle, and the silver layer is chlorinated to generate a silver\silver chloride layer, and then dipping or coating polyurethane and/or polyethylene glycol on the outside of the silver\silver chloride layer to make The method is simple, the quality is controllable, and the reference electrode can ensure the voltage stability.

As a preferred option of the present invention, the third metal transition layer of the auxiliary electrode is covered on the outer layer of the third conductive base by cationic etching; the second noble metal layer is covered by cationic etching or electrochemical deposition. Covered on the outer layer of the third metal transition layer; the coupling layer is covered on the outer layer of the second noble metal layer by dipping or coating; the third polymer film layer is covered by dipping or coating on the outer layer. the outer layer of the coupling layer.

The auxiliary electrode is equivalent to the working electrode with the glucose oxidase layer removed, and other structures and fabrication methods are the same as those of the working electrode to ensure the stability of the current after forming a circuit with the working electrode.

As a preference of the present invention, the solute of the enzyme solution is glucose oxidase, the solution is a phosphate buffer solution, and the concentration of glucose oxidase is 0.02 g/ml to 0.2 g/ml.

When the concentration of glucose oxidase in phosphate buffer solution is 0.02g/ml～0.2g/ml, the activity is better, and the effect on the catalytic reaction of human blood sugar is better.

As a preference of the present invention, the glucose oxidase is cured by glutaraldehyde cross-linking.

The solution of glutaraldehyde and glucose oxidase is mixed in a certain proportion, covered on the silane layer by dipping or coating, and then dipping or coating the silane layer on the outer layer; the free glucose oxidase is passed through the bifunctional coupling of glutaraldehyde and silane. Linked, cross-linked and solidified to form a sandwich-like sandwich layer, free enzymes are combined into aggregates, making the active sites of the enzyme more dense, high stability, and stable and lasting reaction ability in continuous glucose monitoring.

As a preference of the present invention, the cross-linking temperature of the glucose oxidase and the glutaraldehyde is 25° C. to 35° C., and the single cross-linking time is 20 min to 60 min.

Cross-linking the glucose oxidase and the glutaraldehyde for 20min-60min at a temperature of 25°C to 35°C can ensure stable enzyme activity and better aggregation, and ensure the effect of human blood glucose catalytic reaction.

As a preference of the present invention, the number of times of impregnation or coating of the glucose oxidase and the glutaraldehyde on the working electrode is not less than 3 times.

Through multiple dipping or coating, the distribution of the glucose oxidase on the working electrode is more uniform, the thickness of the immobilized enzyme layer is thicker, the continuous glucose monitoring of the human body is more stable and lasting, and the service life of the working electrode is prolonged. .

To sum up, the present invention has the following beneficial effects:

1. The three-electrode form of working electrode, reference electrode and auxiliary electrode is adopted. The micro-current to be tested passes through the working electrode and the auxiliary electrode. The reference electrode only measures the voltage, and basically no current passes through, thereby prolonging the service life of the reference electrode, namely The service life of the sensor has been extended, from the original one that can only be implanted and worn for a few days to half a month to one month of implantation;

2. Through the improvement of the sensor electrode fabrication process and the simplification of biochemical materials, the complex process becomes simple, the quality is controllable, the cost of the sensor is reduced, and the stability of the working electrode glucose oxidase is maintained through the process improvement to ensure The sensor test data is accurate;

3. The carrier of glucose oxidase such as bovine serum albumin or human serum albumin is cancelled, and the bifunctional coupling method of glutaraldehyde and silane is adopted to ensure the activity and stability of glucose oxidase, which reduces the potential biochemical safety hazard to the human body , reduce the body's rejection response;

4. Through the detachable snap connection between the transmitter and the sensor, the sensor and the transmitter can be easily separated, and the transmitter can be reused after the sensor is replaced, which reduces the user's use cost.

Description of drawings

FIG. 1 is a perspective view of the present invention with the tip of the sensor electrode facing upward;

FIG. 2 is a perspective view of the sensor electrode needle tip side facing down according to the present invention;

3 is a schematic structural diagram of the working electrode of the sensor of the present invention;

4 is a schematic structural diagram of a sensor reference electrode of the present invention;

5 is a schematic structural diagram of an auxiliary electrode of a sensor of the present invention;

6 is a schematic diagram of the sensor reference electrode and the auxiliary electrode connected to an operational amplifier according to the present invention;

FIG. 7 is a broken line diagram of the linear correlation change of the three-electrode sensor and the two-electrode sensor of the present invention;

FIG. 8 is a test comparison diagram of the reference electrode of the three-electrode sensor and the reference electrode of the two-electrode sensor of the present invention;

In the figure, 1-working electrode, 11-first conductive substrate, 12-first metal transition layer, 13-first precious metal layer, 14-internal coupling layer, 15-immobilized enzyme layer, 16-external coupling layer , 17-first polymer film layer, 2-reference electrode, 21-second conductive matrix, 22-second metal transition layer, 23-silver\silver chloride layer, 24-second polymer film layer, 3- Auxiliary electrode, 31-third conductive base, 32-third metal transition layer, 33-second precious metal layer, 34-coupling layer, 35-third polymer film layer, 4-sensor base, 41-plastic base, 411 - snap mechanism, 42 - metal connection point, 43 - metal contact.

detailed description

The present invention will be further described below through specific embodiments in conjunction with the accompanying drawings.

Example 1: As shown in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, a three-electrode subcutaneously implanted glucose sensor, including a needle-shaped working electrode 1, a reference electrode 2 and an auxiliary electrode 3, the working electrode 1. The reference electrode 2 and the auxiliary electrode 3 are fixed on the sensor base 4;

The working electrode 1 includes a first conductive substrate 11, a first metal transition layer 12, a first noble metal layer 13, an inner coupling layer 14, an immobilized enzyme layer 15, an outer coupling layer 16, and a first polymer film from the inside out. layer 17;

The reference electrode 2 includes a second conductive base 21, a second metal transition layer 22, a silver/silver chloride layer 23, and a second polymer film layer 24 from the inside out;

The auxiliary electrode 3 includes a third conductive base 31 , a third metal transition layer 32 , a second noble metal layer 33 , a coupling layer 34 , and a third polymer film layer 35 from the inside out.

The sensor used in the continuous blood glucose monitoring of diabetic patients is a consumable item and needs to be replaced regularly. The most critical factor affecting the service life of the sensor is the loss of the reference electrode 2. When the reference electrode 2 has electrons passing through, the silver chloride contained in it will be lost. AgCl+e ^- → Ag+Cl ^- reacts with electrons, so that the silver chloride in the silver\silver chloride layer 23 is continuously consumed, which affects the service life of the reference electrode 2, and by adding the auxiliary electrode 3, most of the The electrons all pass through the auxiliary electrode 3, which reduces the consumption of the silver/silver chloride layer 23 and prolongs the service life of the sensor; the working electrode 1 is the electrode that plays the main electrochemical reaction, and there are biological enzymes that react with human blood glucose inside it, namely The immobilized enzyme layer 15, the outer layer is the first polymer film layer 17 that limits the amount of glucose reacting with the biological enzyme, in order to improve the stability of the biological enzyme and maintain the activity, the immobilized enzyme layer 15 is usually immobilized on the carrier, and is A coupling agent is used for coupling between the immobilized enzyme layer 15 and the first polymer film layer 17 , and the carrier usually uses bovine serum albumin or human serum albumin, which poses a certain biological safety hazard to the human body.

1 and 2, the sensor base 4 includes a plastic base 41, three metal connection points 42 arranged in a triangle in the middle of the plastic base 41, a mounting hole is opened in the middle of the metal connection point 42, the working electrode 1, the reference electrode 2 and the The tail ends of the pins of the auxiliary electrodes 3 pass through the mounting holes respectively and are connected to the metal connection point 42 and conduct.

The working electrode 1 , the reference electrode 2 and the auxiliary electrode 3 are arranged in a triangle on the plastic substrate 41 , which can ensure that the distance between the three electrodes is short, and the monitoring value of blood sugar is more accurate.

As shown in FIG. 1 and FIG. 2 , the three metal connection points 42 are arranged in an equilateral triangle, and the side length of the equilateral triangle is 3 mm˜6 mm.

When the distance between the working electrode 1, the reference electrode 2 and the auxiliary electrode 3 is too close, the resistance of implantation in the human body is large, and when the distance is long, the data deviation of blood glucose monitoring is large. It can not only ensure smooth implantation into the human body, but also ensure the accuracy of sensor data.

As shown in FIG. 1 , the working electrode 1 , the reference electrode 2 and the auxiliary electrode 3 are all installed perpendicular to the plastic base 41 .

The working electrode 1, the reference electrode 2 and the auxiliary electrode 3 are vertically implanted into the human body relatively smoothly, especially when the auxiliary wearing tool is used, the vertical implantation becomes extremely easy, and the human body basically does not feel pain. After the vertical implantation, the plastic matrix 41 has a good degree of fit with the implanted surface of the human body, which can ensure the stability of the sensor after implantation.

As shown in FIG. 1 and FIG. 2 , the sensor base 4 also includes at least three metal contacts 43 located on the plastic base 41 . The metal contacts 43 are respectively connected to the metal connection points 42 . The engaging mechanism 411 .

The transmitter provides the sensor with the voltage required for operation, and receives the current generated by the sensor electrodes. The metal contact 43 is opposite to the contact position on the transmitter. When the transmitter and the sensor base 4 are snap-connected, the metal contact 43 is connected to the transmitter. The contacts on the transmitter are turned on to realize the transmission of current data; the sensor is a consumable item and needs to be replaced regularly, and the transmitter can be used for a long time.

Example 2: As shown in Figure 6, an operational amplifier is installed in the transmitter, the reference electrode 2 is electrically connected to the inverting input end of the operational amplifier through the metal connection point 42 and the metal contact 43, and the auxiliary electrode 3 is connected to the metal connection point 42 through the metal connection point 42. Contact 43 is electrically connected to the output of the operational amplifier.

The reference electrode 2 is connected to the inverting input terminal of the operational amplifier, and the auxiliary electrode 3 is connected to the output terminal of the operational amplifier, so that most of the current passes through the auxiliary electrode 3, and almost no current flows through the reference electrode 2, that is, no electrons pass through, which weakens the reaction. The occurrence of AgCl+e ⁻ →Ag+Cl ⁻ prolongs the service life of the reference electrode 2 .

Example 3: The material of the first conductive substrate 11 is stainless steel, the first metal transition layer 12 is gold, the first precious metal layer 13 is platinum, the inner coupling layer 14 and the outer coupling layer 16 are silane, and the immobilized enzyme layer 15 is Glucose oxidase, the first polymer membrane layer 17 is polyurethane and/or polyethylene glycol.

The material of the first conductive substrate 11 is preferably 304 stainless steel or 316 stainless steel, which has good toughness, small rejection reaction of the human body, and strong safety; the immobilized enzyme layer 15 is glucose oxidase, which can effectively catalyze glucose+O ₂ →gluconic acid+H ₂ O _2. The occurrence of the reaction, the first polymer membrane layer 17 is polyurethane and/or polyethylene glycol, which can restrict the entry of glucose into the immobilized enzyme layer 15 to participate in the reaction, and ensure that enough oxygen participates in the reaction, so that the glucose concentration is H ₂ O _{2 to} generate The first metal transition layer 12 is gold, and the first noble metal layer 13 is platinum, which can effectively catalyze _{the occurrence of H 2} O ₂ →O ₂ +2H ⁺ +2e ^- reaction, so that the _{amount of H 2} O ₂ can be converted into The amount of electrons generated, the electrons generate a microcurrent through the working electrode 1, and gold and platinum can reduce the signal-to-noise ratio, so that there is a linear positive correlation between the microcurrent and the glucose concentration; the inner coupling layer 14 and the outer coupling layer 16 are preferably silane, Not only can a good coupling effect be achieved, the stability of the immobilized enzyme layer 15 can be improved, but also the process can be simplified, so that the quality of the working electrode 1 can be controlled.

The second conductive base 21 is made of stainless steel, the second metal transition layer 22 is made of silver, and the second polymer film layer 24 is made of polyurethane and/or polyethylene glycol.

The material of the second conductive base 21 is preferably 304 stainless steel or 316 stainless steel, which has good toughness, small rejection reaction of the human body, and strong safety; the second metal transition layer 22 is silver, and the transition silver\silver chloride layer 23 has High accuracy and stability.

The material of the third conductive base 31 is stainless steel, the third metal transition layer 32 is gold, the second noble metal layer 33 is platinum, the coupling layer 34 is silane, and the third polymer film layer 35 is polyurethane and/or polyethylene glycol.

The material of the third conductive base 31 is preferably 304 stainless steel or 316 stainless steel, which has good toughness, small human body rejection reaction, and strong safety; the third metal transition layer 32 is gold, and the second precious metal layer 33 is platinum, which can reduce the current signal-to-noise ratio. Ensure the current stability after forming a loop with the working electrode 1.

According to the structure in Example 3, two sensors were fabricated, one of which was a three-electrode sensor, and the other was a two-electrode sensor with the auxiliary electrode removed. The two sensors were subjected to a current test in a glucose solution of phosphate buffered reagent in vitro. The test results as follows:

Table 1: In vitro test current and linear correlation of three-electrode sensor

Table 2: Two-electrode sensor in vitro test current and linear correlation

The linear correlation represents the correlation between the change of the sensor current value and the change of the concentration of the test solution. The closer to 100%, the better the performance of the sensor. It can be seen from the comparison between Table 1 and Table 2 that the three-electrode sensor is tested On the 18th day, the linear correlation was still above 99.9%, while the two-electrode sensor began to show a decline in the linear correlation on the 7th day of the test, and the test had already dropped significantly on the 9th day, so the test was no longer carried out.

Figure 7 is a comparison of the line graph of the linear correlation between the three-electrode sensor and the two-electrode sensor. It can be seen from the figure that the three-electrode sensor can maintain good accuracy when it works for 18 days, while the two-electrode sensor works for 6 days. After that, the stability dropped significantly.

As shown in Figure 8, the changes of the sensor can also be clearly seen from the photos after three days of operation. The reference electrode of the three-electrode sensor only turns white on the tip of the needle, while the reference electrode of the two-electrode sensor has basically turned white.

Example 4: The manufacturing method of the three-electrode subcutaneously implanted glucose sensor in the above embodiment, the sensor includes a needle-shaped working electrode 1, a reference electrode 2 and an auxiliary electrode 3, a working electrode 1, a reference electrode 2 and an auxiliary electrode 3 fixed on the sensor base 4;

The working electrode 1 includes a first conductive substrate 11, a first metal transition layer 12, a first noble metal layer 13, an inner coupling layer 14, an immobilized enzyme layer 15, an outer coupling layer 16, and a first polymer film from the inside out. Layer 17, the material of the first conductive substrate 11 is stainless steel, the first metal transition layer 12 is gold, the first precious metal layer 13 is platinum, the inner coupling layer 14 and the outer coupling layer 16 are silane, and the immobilized enzyme layer 15 is glucose Oxidase, the first polymer film layer 17 is polyurethane and/or polyethylene glycol;

The reference electrode 2 includes a second conductive substrate 21, a second metal transition layer 22, a silver/silver chloride layer 23, and a second polymer film layer 24 from the inside out. The second conductive substrate 21 is made of stainless steel, and the second metal transition layer is made of stainless steel. The layer 22 is silver, and the second polymer film layer 24 is polyurethane and/or polyethylene glycol;

The auxiliary electrode 3 includes a third conductive base 31, a third metal transition layer 32, a second noble metal layer 33, a coupling layer 34, and a third polymer film layer 35 from the inside out. The three-metal transition layer 32 is gold, the second precious metal layer 33 is platinum, the coupling layer 34 is silane, and the third polymer film layer 35 is polyurethane and/or polyethylene glycol;

The first metal transition layer 12 of the working electrode 1 is covered on the outer layer of the first conductive base 11 by a cationic etching method; the first precious metal layer 13 is covered by a cationic etching method or an electrochemical deposition method on the outer layer of the first metal transition layer 12 The inner coupling layer 14 is covered on the outer layer of the first precious metal layer 13 by dipping or coating; the immobilized enzyme layer 15 is attached to the outer layer of the inner coupling layer 14 by dipping or coating with the enzyme solution; the outer coupling layer 16 covers the outer layer of the immobilized enzyme layer 15 by dipping or coating; the first polymer film layer 17 covers the outer layer of the outer coupling layer 16 by dipping or coating.

Working electrode 1: A gold layer is plated on the outer layer of the stainless steel needle, a platinum layer is deposited on the outer layer of the gold layer by cationic etching or electrochemical deposition, and the outer layer of the platinum layer is dipped or coated with silane, and then glucose oxidase is dipped or coated on the outer layer. , the outer layer of the enzyme layer is impregnated or coated with silane, and finally polyurethane and/or polyethylene glycol is impregnated or coated. The whole preparation method is simple, low in cost, controllable in quality, and can ensure the stability of glucose oxidase.

The second metal transition layer 22 of the reference electrode 2 is covered on the outer layer of the second conductive base 21 by cation etching; the silver\silver chloride layer 23 is formed by chlorination of the second metal transition layer 22; the second polymer film layer 24 The outer layer of the silver/silver chloride layer 23 is covered by dipping or coating.

The reference electrode 2 is electroplated with a silver layer on the outer layer of the stainless steel needle, and the silver layer is chlorinated to form a silver/silver chloride layer, and then immersed or coated with polyurethane and/or polyethylene glycol on the outside of the silver/silver chloride layer. Production method Simple, quality controllable, as a reference electrode can ensure voltage stability.

The third metal transition layer 32 of the auxiliary electrode 3 is covered on the outer layer of the third conductive base 31 by a cationic etching method; the second noble metal layer 33 is covered on the outer layer of the third metal transition layer 32 by a cationic etching method or an electrochemical deposition method ; The coupling layer 34 is covered on the outer layer of the second noble metal layer 33 by dipping or coating; the third polymer film layer 35 is covered on the outer layer of the coupling layer 34 by dipping or coating.

The auxiliary electrode 3 is equivalent to the working electrode 1 with the glucose oxidase layer removed, and other structures and manufacturing methods are the same as the working electrode, which ensures the stability of the current after forming a circuit with the working electrode 1 .

Example 5: The method of manufacturing the three electrodes is as in Example 4, wherein the specific cross-linking method of the immobilized enzyme layer 15 of the working electrode 1 is as follows.

The solute of the enzyme solution is glucose oxidase, the solution is a phosphate buffer solution, and the concentration of glucose oxidase is 0.02g/ml to 0.2g/ml.

When the concentration of glucose oxidase in phosphate buffer solution is 0.02g/ml to 0.2g/ml, the activity is better, and the effect on the catalytic reaction of human blood sugar is better.

Glucose oxidase is immobilized by glutaraldehyde cross-linking.

The solution of glutaraldehyde and glucose oxidase is mixed in a certain proportion, covered on the silane layer by dipping or coating, and then dipping or coating the silane layer on the outer layer; the free glucose oxidase is passed through the bifunctional coupling of glutaraldehyde and silane. Linked, cross-linked and solidified to form a sandwich-like sandwich layer, free enzymes are combined into aggregates, making the active sites of the enzyme more dense, high stability, and stable and lasting reaction ability in continuous glucose monitoring.

The cross-linking temperature of glucose oxidase and glutaraldehyde is 25℃～35℃, and the single crosslinking time is 20min～60min.

Glucose oxidase and glutaraldehyde are cross-linked at 25℃～35℃ for 20min～60min, which can ensure stable enzyme activity and better aggregation, and ensure the effect of human blood glucose catalytic reaction.

Glucose oxidase and glutaraldehyde are impregnated or coated on the working electrode 1 for no less than three times.

Through multiple dipping or coating, the distribution of glucose oxidase on the working electrode 1 is more uniform, the thickness of the immobilized enzyme layer 15 is thicker, the continuous glucose monitoring of the human body is more stable and lasting, and the service life of the working electrode 1 is prolonged.

Under the condition of temperature of 25°C, different concentrations of enzyme solutions were cross-linked for different times (single cross-linking time was 30 min), and then the sensitivity in vitro test was carried out using three-electrode sensors made of different working electrodes 1, and the following test data were obtained. :

Concentrationg/ml	0.01	0.02	0.04	0.06	0.08	0.1	0.12	0.14	0.16	0.18	0.2	0.22
Apply once	4.28	7.28	13.58	18.55	23.67	29.54	35.04	40.43	45.82	51.24	56.24	60.54
2 coats	5.46	9.13	16.74	22.46	28.15	34.52	40.21	45.59	50.75	55.77	60.13	63.59
Coating 3 times	6.39	10.56	19.13	25.38	31.45	38.13	43.92	49.23	54.19	58.87	62.76	65.62
4 coats	6.52	10.77	19.52	25.89	32.09	38.90	44.81	50.23	55.29	60.06	64.03	66.95
5 coats	6.94	11.42	20.60	27.21	33.58	40.54	46.49	51.90	56.89	61.54	65.33	68.02
Coating 6 times	7.44	12.18	21.86	28.72	35.25	42.33	48.28	53.60	58.43	62.87	66.37	68.73

Table 3: Sensor sensitivity (nA/mmol) obtained after different concentrations of enzyme solutions were coated for different times

When the sensitivity of the sensor is above 10nA/mmol, it can ensure more accurate monitoring of blood sugar level. It can be seen from the test data that when the concentration of glucose oxidase in the enzyme solution exceeds 0.02g/ml, the glucose oxidase is mixed with glutaraldehyde and then dipped or coated. When the number of times is not less than 3 times, the sensitivity of the three-electrode sensor can meet the requirements, and when the concentration of glucose oxidase in the enzyme solution exceeds 0.2g/ml, the sensitivity of the sensor even reaches more than 60nA/mmol, and when the sensitivity reaches 60nA/mmol In the above, the sensitivity is no longer the main factor affecting the performance of the sensor; in addition, from the current data obtained by the user wearing the sensor, when the enzyme solution is only cross-linked for a single time, the sensitivity of the sensor fluctuates greatly with the wearing time; For problems such as efficiency and sensor stability, when the concentration of glucose oxidase in phosphate buffer solution is generally controlled to be 0.02g/ml to 0.08g/ml, the mixed solution of glucose oxidase and glutaraldehyde is dipped or coated on the working electrode 1 for 5 times; when the concentration of glucose oxidase in the phosphate buffer solution is 0.08g/ml ~ 0.14g/ml, the mixed solution of glucose oxidase and glutaraldehyde is dipped or coated on the working electrode 1 for 4 times; When the concentration in the solution is 0.14 g/ml to 0.2 g/ml, the mixed solution of glucose oxidase and glutaraldehyde is dipped or coated on the working electrode 1 three times.

The above-mentioned embodiments are only to describe the preferred embodiments of the present invention, and do not limit the concept and scope of the present invention. On the premise of not departing from the design concept of the present invention, various modifications and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope of the present invention, and the technical content claimed in the present invention has been fully recorded in the claims.

Claims (16)

1. A three-electrode subcutaneous implantable glucose sensor, characterized in that it comprises a needle-shaped working electrode (1), a reference electrode (2) and an auxiliary electrode (3), the working electrode (1), the reference electrode (2) and the auxiliary electrode (3) are fixed on the sensor base (4);

   The working electrode (1) includes a first conductive substrate (11), a first metal transition layer (12), a first precious metal layer (13), an inner coupling layer (14), and an immobilized enzyme layer (14) from the inside out. 15), an outer coupling layer (16), a first polymer film layer (17);

   The reference electrode (2) includes a second conductive matrix (21), a second metal transition layer (22), a silver/silver chloride layer (23), and a second polymer film layer (24) from the inside out;

   The auxiliary electrode (3) includes a third conductive substrate (31), a third metal transition layer (32), a second precious metal layer (33), a coupling layer (34), and a third polymer film layer from the inside to the outside (35).
2. A three-electrode subcutaneously implanted glucose sensor according to claim 1, characterized in that: the sensor seat (4) comprises a plastic base (41), and three triangles located in the middle of the plastic base (41) are in the shape of a triangle. Arranged metal connection points (42), a mounting hole is formed in the middle of the metal connection point (42), and the pin tails of the working electrode (1), the reference electrode (2) and the auxiliary electrode (3) are respectively After passing through the installation hole, it is connected to the metal connection point (42) and conducted.
3. The three-electrode subcutaneous implantable glucose sensor according to claim 2, wherein the three metal connection points (42) are arranged in an equilateral triangle, and the side length of the equilateral triangle is 3mm-6mm.
4. A three-electrode subcutaneous implantable glucose sensor according to claim 3, characterized in that: the working electrode (1), the reference electrode (2) and the auxiliary electrode (3) are all perpendicular to the The plastic base (41) is installed.
5. A three-electrode subcutaneously implanted glucose sensor according to claim 2, characterized in that: the sensor seat (4) further comprises at least three metal contacts (43) located on the plastic substrate (41) , the metal contacts (43) are respectively connected to the metal connection points (42), and the plastic base (41) is provided with a snap mechanism (411) connected to the transmitter.
6. A three-electrode subcutaneously implanted glucose sensor according to claim 5, characterized in that: an operational amplifier is installed in the transmitter, and the reference electrode (2) passes through the metal connection point (42) and the The metal contact (43) is electrically connected to the reverse input end of the operational amplifier, and the auxiliary electrode (3) is connected to the operational amplifier through the metal connection point (42) and the metal contact (43) The output terminal is electrically connected.
7. A three-electrode subcutaneous implantable glucose sensor according to claim 1, characterized in that: the material of the first conductive substrate (11) is stainless steel, the first metal transition layer (12) is gold, and the material of the first conductive substrate (11) is gold. The first precious metal layer (13) is platinum, the inner coupling layer (14) and the outer coupling layer (16) are silane, the immobilized enzyme layer (15) is glucose oxidase, and the first The polymer film layer (17) is polyurethane and/or polyethylene glycol.
8. A three-electrode subcutaneous implantable glucose sensor according to claim 1, characterized in that: the material of the second conductive substrate (21) is stainless steel, the second metal transition layer (22) is silver, and the material of the second conductive base (21) is stainless steel. The second polymer film layer (24) is polyurethane and/or polyethylene glycol.
9. A three-electrode subcutaneous implantable glucose sensor according to claim 1, characterized in that: the material of the third conductive substrate (31) is stainless steel, the third metal transition layer (32) is gold, and the material of the third conductive substrate (31) is gold. The second noble metal layer (33) is platinum, the coupling layer (34) is silane, and the third polymer film layer (35) is polyurethane and/or polyethylene glycol.
10. A method for manufacturing a three-electrode subcutaneously implanted glucose sensor, characterized in that the sensor comprises a needle-shaped working electrode (1), a reference electrode (2) and an auxiliary electrode (3), and the working electrode (1) ), the reference electrode (2) and the auxiliary electrode (3) are fixed on the sensor base (4);

    The working electrode (1) includes a first conductive substrate (11), a first metal transition layer (12), a first precious metal layer (13), an inner coupling layer (14), and an immobilized enzyme layer (14) from the inside out. 15), an outer coupling layer (16), a first polymer film layer (17), the material of the first conductive substrate (11) is stainless steel, the first metal transition layer (12) is gold, and the first metal transition layer (12) is made of gold. A precious metal layer (13) is platinum, the inner coupling layer (14) and the outer coupling layer (16) are silane, the immobilized enzyme layer (15) is glucose oxidase, the first high The molecular film layer (17) is polyurethane and/or polyethylene glycol;

    The reference electrode (2) includes a second conductive substrate (21), a second metal transition layer (22), a silver/silver chloride layer (23), and a second polymer film layer (24) from the inside out, so The second conductive substrate (21) is made of stainless steel, the second metal transition layer (22) is made of silver, and the second polymer film layer (24) is made of polyurethane and/or polyethylene glycol;

    The auxiliary electrode (3) includes a third conductive substrate (31), a third metal transition layer (32), a second precious metal layer (33), a coupling layer (34), and a third polymer film layer from the inside to the outside (35), the material of the third conductive substrate (31) is stainless steel, the third metal transition layer (32) is gold, the second noble metal layer (33) is platinum, and the coupling layer (34) is silane, and the third polymer film layer (35) is polyurethane and/or polyethylene glycol;

    The first metal transition layer (12) of the working electrode (1) is covered on the outer layer of the first conductive base (11) by an electrochemical deposition method; the first noble metal layer (13) is etched by cation method or electrochemical deposition method to cover the outer layer of the first metal transition layer (12); the inner coupling layer (14) is covered on the outer layer of the first noble metal layer (13) by dipping or coating; The immobilized enzyme layer (15) is attached to the outer layer of the inner coupling layer (14) by dipping or coating from the enzyme solution; the outer coupling layer (16) is covered on the outer coupling layer (16) by dipping or coating. the outer layer of the immobilized enzyme layer (15); the first polymer film layer (17) is covered on the outer layer of the outer coupling layer (16) by dipping or coating.
11. The method for manufacturing a three-electrode subcutaneously implanted glucose sensor according to claim 10, characterized in that: the second metal transition layer (22) of the reference electrode (2) is covered by an electrochemical deposition method on the The outer layer of the second conductive substrate (21); the silver/silver chloride layer (23) is formed by chlorination of the second metal transition layer (22); the second polymer film layer (24) is made of The outer layer of the silver/silver chloride layer (23) is covered by dipping or coating.
12. The method for manufacturing a three-electrode subcutaneously implanted glucose sensor according to claim 10, characterized in that: the third metal transition layer (32) of the auxiliary electrode (3) is covered by an electrochemical deposition method on the the outer layer of the third conductive substrate (31); the second noble metal layer (33) is covered on the outer layer of the third metal transition layer (32) by a cationic etching method or an electrochemical deposition method; the coupling The layer (34) is covered on the outer layer of the second noble metal layer (33) by dipping or coating; the third polymer film layer (35) is covered on the coupling layer (34) by dipping or coating ) outer layer.
13. The method for making a three-electrode subcutaneously implanted glucose sensor according to claim 10, wherein the solute of the enzyme solution is glucose oxidase, the solution is a phosphate buffer solution, and the concentration of glucose oxidase is 0.02 g /ml～0.2g/ml.
14. The method for manufacturing a three-electrode subcutaneously implanted glucose sensor according to claim 13, wherein the glucose oxidase is cured by cross-linking with glutaraldehyde.
15. The method for manufacturing a three-electrode subcutaneously implanted glucose sensor according to claim 14, wherein the cross-linking temperature of the glucose oxidase and the glutaraldehyde is 25°C to 35°C, and a single cross-linking The time is 20min～60min.
16. The method for manufacturing a three-electrode subcutaneously implanted glucose sensor according to claim 15, wherein the glucose oxidase and the glutaraldehyde are impregnated or coated on the working electrode (1). The number of times is not less than 3 times.

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