An electrochemical biosensor

Abstract

The present application belongs to the technical field of medical equipment, and relates to an electrochemical biosensor for metabolite detection equipment. The sensor has: (1) an electrode system comprising a working electrode and a counter electrode; (2) a reaction reagent arranged on the working electrode and the counter electrode. The reaction reagent comprises: (a) an enzyme; (b) an electron mediator composition; (c) a buffer solution; wherein the electron mediator composition comprises a ruthenium complex and an aniline derivative. The sensor of the present application adopts the same biosensing electrochemical technology route, and as long as the enzyme in the reaction reagent is replaced, electrochemical detection of corresponding metabolites such as blood lactic acid, blood ketone and blood glucose can be realized, which is beneficial to production quality control and improvement of product performance. The sensor of the present application has the advantages of simple manufacturing process, large detection range and rapid and accurate detection.

Description

Technical Field

[0001] This invention belongs to the field of medical device technology and relates to an electrochemical biosensor used in metabolite detection devices. Background Technology

[0002] Blood lactate monitoring can indicate tissue hypoxia or metabolic disorders, assess patient prognosis, and monitor the effectiveness of resuscitation treatment. For example, lactate levels are crucial for guiding the treatment of critically ill patients, especially those with myocardial infarction, heart failure, or tissue hypoxia caused by insufficient blood flow. Therefore, rapid response is essential, with diagnostically valuable results delivered within five minutes. Furthermore, measuring lactate helps identify fatigue levels, enabling the design of personalized training programs for athletes. Therefore, establishing a simple and rapid method for detecting blood lactate concentrations is of great significance.

[0003] In recent years, point-of-care testing (POC) products have been widely used in hospitals, clinics, and patients' homes, capable of detecting most routine clinical indicators. POC offers advantages such as rapid response, ease of operation, and patient-centered experience, enabling quick and appropriate diagnosis, nursing care, and pathological observation, thereby improving medical quality and patient satisfaction. Therefore, electrochemical biosensors, as powerful tools for achieving POC, must be accurate, easy to mass-produce, and inexpensive. Patent CN107735027B discloses a method for monitoring lactate concentration in the body. Patent CN102507915B discloses a liquid reagent kit for determining lactate. However, these technical approaches are insufficient to meet the needs of portable POC products. Patent CN103018291B discloses a dry lactate sensor, but the reagent layer preparation process is cumbersome.

[0004] To date, there are no products that utilize electron-intermediate compositions, especially ruthenium complexes and aniline derivatives, to achieve real-time detection of metabolites such as blood lactate, blood ketones, and blood glucose.

[0005] Using the same biosensor electrochemical technology route, the detection of corresponding metabolites can be achieved simply by replacing the enzymes in the reaction reagents. This is beneficial for production quality control, reducing product costs, and improving product performance. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the existing technology, the present invention provides an electrochemical biosensor containing a reaction reagent of aniline derivative.

[0007] The technical solution adopted by this invention to solve its technical problem is: An electrochemical biosensor having: (1) An electrode system comprising a working electrode and a counter electrode; (2) A reaction reagent disposed on the working electrode and the counter electrode, the reaction reagent comprising: (a) enzyme; (b) Electron mediator composition; (c) Buffer solution; The electron mediator composition includes a ruthenium complex and an aniline derivative.

[0008] The ruthenium complex is [Ru(NH3)5X]n+, where X is NH3, CN-, halide ions, etc., and n+ is the valence state of the entire chemical formula of the oxidized Ru3+ and other groups; preferably, the ruthenium complex is [Ru(NH3)6]3+.

[0009] The general formula (Ⅰ) of the aniline derivative is as follows:

[0010] Where R = O(CH2)nCH3, n = 0-8, and R can be in 1, 2, 3 or 4 positions; R'=(CH2)nCH3,n=0-8, R'=(CH2)nOH,n=1-8, R'=(CH2)nOCH3,n=1-8 or R'=(CH2)nOCOCH=CH2,n=1-8; R”=(CH2)nCH3,n=0-8, R”=(CH2)nOH,n=1-8, R”=(CH2)nOCH3,n=1-8 or R”=(CH2)nOCOCH=CH2,n=1-8.

[0011] From a manufacturability perspective, better water solubility helps reduce the difficulty of preparing liquid reaction reagents and improves the homogeneity of the solution. Therefore, this invention preferably uses aniline derivatives containing shorter carbon chains or more hydroxyl groups.

[0012] From the perspective of improving the stability of reaction reagents, electron mediators that possess good storage stability while meeting electrochemical performance requirements are more desirable. Therefore, this invention preferably uses aniline derivatives containing fewer active functional groups.

[0013] The preferred aniline derivative of this invention is (II), (III), or (IV), and its structural formula is as follows:

[0014] More preferably, the aniline derivative is m-methoxy-N,N-di(hydroxyethyl)-p-nitrosoaniline, with the following structural formula (Ⅳ):

[0015] From the perspectives of detection sensitivity and product considerations, the concentration of hexaammineruthenium chloride is 0.25-5 wt.%, and the concentration of m-methoxy-N,N-di(hydroxyethyl)-p-nitrosoaniline (CAS#:733686-00-5) is 0.005-0.1 wt.%.

[0016] The enzymes mentioned are glucose oxidase, glucose dehydrogenase, lactate oxidase, lactate dehydrogenase, β-hydroxybutyrate dehydrogenase, uricase oxidase, cholesterol oxidase, creatine hydrolase, ascorbic acid oxidase, etc. The amount of enzyme used in a single electrochemical biosensor is preferably 0.1-10 U, which will be readily understood by those skilled in the art.

[0017] The buffer solution is a Good's buffer solution, an organic acid buffer solution, or a phosphate buffer solution; preferably, the buffer solution is a Good's buffer solution, which may be one or more of ACES, TES, and MES, with a concentration of 0.1-1M and a pH of 6.5-7.5.

[0018] The reaction reagents configured on the working electrode and the counter electrode in this invention refer to the liquid reaction reagents that are precisely dispensed onto the surface area of ​​the electrode system using a dispensing device.

[0019] The electrode system can be made of metallic materials, such as gold, platinum, palladium, or their alloys, or other inert conductive materials such as carbon. Preferably, metallic materials are used.

[0020] As is well known to those skilled in the art, the reaction reagent also includes a protein stabilizer, which may be selected from sugars, such as sucrose; polyols, such as sorbitol; amino acids, such as monosodium glutamate; proteins, such as silk fibroin, etc. Preferably, sucrose is used at a concentration of 0.1-1 wt.%. Adding an excessive amount of protein stabilizer increases the solid content of the liquid reaction reagent, leading to a decrease in the response current of the electrochemical biosensor, which is readily understood by those skilled in the art.

[0021] The reaction reagent also includes at least one surfactant, which can be an ionic or nonionic surfactant. The addition of the surfactant allows the liquid reaction reagent to spread rapidly and uniformly on the electrode surface. Preferably, [the surfactant is used]. The concentration is 0.05-1 wt.%. It is easy for those skilled in the art to understand that adding excessive surfactant will cause the liquid reaction reagent to spread out and the film thickness to be uneven.

[0022] The reaction reagent also includes a polymer. The polymer used in this invention is well-known in the art. An appropriate amount of polymer results in good film formation and strong adhesion of the dried reaction reagent, but excessive polymer will increase film thickness, lengthen response time, and decrease sensitivity. Preferably, for example, methylcellulose, at a concentration of 0.25-2.5 wt.%.

[0023] The detection sample of the electrochemical biosensor can be a blood sample such as whole blood, plasma, or serum, or other types of samples such as urine, sweat, tears, or interstitial fluid.

[0024] The specific fabrication process of the electrochemical biosensor of the present invention is as follows: A metal layer attached to a polymer material is used to achieve corresponding electrode patterns through laser etching or mask sputtering; the electrode material can be gold, platinum, palladium, or their alloys. For example, the electrode pattern is laser-etched on a gold film with polyethylene terephthalate (PET) as the substrate. A liquid reaction reagent is prepared according to the above-mentioned reaction reagent ratio, and it is thoroughly stirred to dissolve and disperse, forming a uniform solution; then, the liquid reaction reagent is applied to the surface of the electrode system by dotting; finally, the solvent water in the liquid reaction reagent is removed by drying, thereby obtaining a dry reagent layer of uniform thickness, thus obtaining an electrochemical biosensor.

[0025] The testing method for an electrochemical biosensor according to the present invention is as follows: After the blood sample is drawn by capillary action, an excitation voltage of 100mV-600mV, preferably 150mV-350mV, is applied between the working electrode and the counter electrode. The current value of the blood sample is obtained by chronoamperometry at 3-30 seconds, and then converted into the concentration value of the analyte by calibration curve.

[0026] The beneficial effects of this invention are as follows: 1. The present invention provides an electrochemical biosensor that adopts the same biosensing electrochemical technology route. By simply replacing the enzyme in the reaction reagent, it is possible to achieve electrochemical detection of corresponding metabolites such as blood lactate, blood ketones, and blood glucose. This is beneficial for production quality control, reducing product costs, and improving product performance.

[0027] 2. The electrochemical biosensor of this invention features a simple process, allowing for the preparation of reaction reagents on the electrode in a single dispensing operation. The raw materials for the reaction reagents are readily available and low in cost, facilitating industrial production and demonstrating strong practicality.

[0028] 3. The electrochemical biosensor of the present invention has a short detection time, acquiring data in 3 to 30 seconds, which can achieve the purpose of real-time detection. Attached Figure Description

[0029] Figure 1 This is the response current diagram of a blood lactate electrochemical biosensor involving three groups of aniline derivatives; Figure 2 This is a calibration curve for a blood lactate electrochemical biosensor according to the present invention; Figure 3 This is a calibration curve for a blood ketone electrochemical biosensor according to the present invention; Figure 4 This is a calibration curve for a blood glucose electrochemical biosensor according to the present invention. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below through specific embodiments. However, it should be noted that the following embodiments are only used to describe the content of the invention and do not constitute a limitation on the scope of protection of the present invention.

[0031] Example 1: Preparation and measurement method of the electrochemical biosensor of the present invention.

[0032] The preparation method of the blood lactate electrochemical biosensor in this embodiment is as follows: First, the liquid reaction reagents were prepared according to Table 1. Lactate oxidase (model: T-47, 42 U / mg) was purchased from Asahi Kasei Corporation, Japan; three aniline derivatives (see Table 2) were all purchased from PharmaBlock Sciences (Nanjing), Inc. A 30 nm gold layer was vacuum sputtered onto poly(ethylene terephthalate) (PET) sheets, and electrode patterns were laser-etched. The liquid reaction reagents were applied to the working and counter electrodes via spotting, with each blood lactate electrochemical biosensor having a loading of 1.0 mg. The sensors were then heat-dried sequentially in a 45°C drying tunnel for 20 minutes and a 70°C drying tunnel for 10 minutes. After bonding, pressing, and cutting, the finished lactate electrochemical biosensors were stored in a sealed plastic cylinder containing a molecular sieve desiccant.

[0033] Table 1: Composition of liquid reaction reagents

[0034] Table 2: Three aniline derivatives

[0035] Those skilled in the art will understand that, in order to determine the amount of an analyte, a corresponding electrochemical signal is required. More specifically, a chronoamperometric blood lactate electrochemical biosensor acquires the corresponding current signal from a blood sample with an unknown lactate concentration, and then converts it into a lactate concentration value of the blood sample using a calibration equation. How such a calibration equation can be established and used is well known to those skilled in the art.

[0036] Experimental Procedure 1: Three sets of tubular blood lactate electrochemical biosensors were placed at room temperature (23℃±2℃) for at least 30 minutes. The blood sample requirements were as follows: hematocrit adjusted to 42%±2%; oxygen partial pressure controlled at 65mmHg±5mmHg; three whole blood lactate samples were prepared within a concentration range of 0-35mM. The venous whole blood samples were assigned values ​​using a Radiometer Medical Aps (ABL90 FLEX Analyzer / Sensor Cassette Pack). Two blood lactate electrochemical sensors were tested for each sample. In this embodiment, an excitation voltage of 300mV was used, and the current value was read after 10 seconds to obtain the current data. A graph was plotted with the average test current for each concentration range (n=2) as the ordinate and the Radiometer Medical Aps test value as the whole blood lactate value as the abscissa to evaluate the current response and sensitivity (uA / mM). The results are shown in [Figure number missing]. Figure 1 .

[0037] Depend on Figure 1 It can be seen that all three groups of aniline derivatives involved in the blood lactate electrochemical biosensors have linear gradients, among which the sensitivity (uA / mM) of group C, which involves m-methoxy-N,N-di(hydroxyethyl)-nitrosoaniline, is the highest.

[0038] Experimental Procedure 2: The C-group tubular electrochemical biosensor for blood lactate was placed at room temperature (23℃±2℃) for at least 30 minutes. The blood sample requirements were as follows: hematocrit adjusted to 42%±2%; oxygen partial pressure controlled at 65mmHg±5mmHg; nine whole blood lactate samples were prepared within a concentration range of 0-35mM, and values ​​were assigned to the venous whole blood samples using a Radiometer Medical Aps (ABL90 FLEX Analyzer / Sensor Cassette Pack). Eight blood lactate electrochemical sensors were tested for each sample. In this embodiment, an excitation voltage of 300mV was used, and the current value was read after 10 seconds, resulting in the current data shown in Table 3. A linear fit was performed with the average test current for each concentration range as the x-axis and the Radiometer Medical Aps test value as the whole blood lactate value as the y-axis, establishing the calibration curve as shown below. Figure 2 As shown.

[0039] Table 3: Sample test current data of the blood lactate electrochemical biosensor in Group C

[0040] from Figure 2 As can be seen, the calibration curve established by the Reidumite blood gas analyzer using whole blood lactate value and average test current shows a correlation coefficient squared (R²) greater than 0.99, indicating good fit. Compared with Examples 1-3 in patent CN103018291B, the blood lactate electrochemical biosensor of this invention also exhibits good linear current response to high concentrations (e.g., 30 mM) samples, and can provide detection results in a shorter time (e.g., 10 seconds).

[0041] Example 2: Preparation and measurement method of the electrochemical biosensor of the present invention.

[0042] The preparation method of the blood ketone electrochemical biosensor in this embodiment is as follows: First, the liquid reaction reagents were prepared according to Table 4. β-hydroxybutyrate dehydrogenase (model: HBD-301, 167 U / mg) was purchased from Toyobo, Japan; myofibril enzyme (approximately 30 U / mg, derived from Clostridium difficile) was purchased from Aladdin; and coenzyme nicotinamide adenine dinucleotide (β-NAD+) was purchased from Oriental Yeast. A 30 nm gold layer was vacuum sputtered onto polyethylene terephthalate (PET) sheets, and electrode patterns were laser-etched. The liquid reaction reagents were applied to the working and counter electrodes via spotting, with each blood ketone electrochemical biosensor having a loading of 1.0 mg. The sensors were then heat-dried sequentially in a 45°C drying tunnel for 20 minutes and a 70°C drying tunnel for 10 minutes. After bonding, pressing, and cutting, the finished blood ketone electrochemical biosensors were stored in a sealed plastic cylinder containing a molecular sieve desiccant.

[0043] Table 4: Composition of Liquid Reagents

[0044] Experimental Procedure: The tubular blood ketone electrochemical biosensor was placed at room temperature (23℃±2℃) for at least 30 minutes. The blood sample requirements were as follows: hematocrit adjusted to 42%±2%; oxygen partial pressure controlled at 65mmHg±5mmHg; eight whole blood ketone samples were prepared within a concentration range of 0-8mM, and values ​​were assigned to the venous whole blood samples using Abbott's Freestyle Optium Neo / Freestyle Optium Blood β-Ketone Test Strips (LOT75001H61). Eight blood ketone electrochemical biosensors were tested for each sample. In this embodiment, an excitation voltage of 300mV was used, and the current value was read after 10 seconds, resulting in the current data shown in Table 5. A linear fit was performed with the average test current for each concentration range as the x-axis and the test value from Abbott's Freestyle Optium Neo / Freestyle Optium Blood β-Ketone Test Strips (LOT75001H61) as the whole blood ketone value. The established calibration curve is shown below. Figure 3 As shown.

[0045] Table 5: Sample test current data of the blood ketone electrochemical biosensor

[0046] from Figure 3 It can be seen that the calibration curve established by Abbott's Synaptic Glucose / Ketoscale Meter and the average test current of whole blood ketone values ​​shows a correlation coefficient squared (R2) greater than 0.99, indicating a good fit.

[0047] Example 3: Preparation and Measurement Method of the Electrochemical Biosensor of the Present Invention

[0048] The preparation method of the blood glucose electrochemical biosensor in this embodiment is as follows: First, the liquid reaction reagents were prepared according to Table 6, including glucose dehydrogenase (model: GLD-351, 596 U / mg), purchased from Toyobo, Japan. A 30 nm gold layer was vacuum sputtered onto poly(ethylene terephthalate) (PET) sheets, and electrode patterns were laser-etched. The liquid reaction reagents were applied to the working and counter electrodes via spotting, with a loading of 1.0 mg per blood glucose electrochemical biosensor. The sensors were then heat-dried sequentially in a 45°C drying tunnel for 20 minutes and a 70°C drying tunnel for 10 minutes. After bonding, pressing, and cutting, the finished blood glucose electrochemical biosensors were stored in a sealed plastic container with a molecular sieve desiccant.

[0049] Table 6: Composition of Liquid Reagents

[0050] Experimental Procedure: The cylindrical glucose electrochemical biosensor was placed at room temperature (23℃±2℃) for at least 30 minutes. The blood sample requirements were as follows: hematocrit ratio adjusted to 42%±2%; oxygen partial pressure controlled at 65mmHg±5mmHg; 12 whole blood glucose samples were prepared within a concentration range of 0-35mM, and values ​​were assigned to the venous whole blood samples using a YSI2500 glucose / lactate analyzer from Jinquan Company. Eight glucose electrochemical biosensors were tested for each sample. In this embodiment, an excitation voltage of 300mV was used, and the current value was read after 5 seconds, resulting in the current data shown in Table 7. A linear fit was performed with the average test current for each concentration range as the x-axis and the YSI2500 glucose / lactate analyzer test value as the whole blood glucose value as the y-axis, establishing the calibration curve as shown below. Figure 4 As shown.

[0051] Table 7: Sample test current data of blood glucose electrochemical biosensor

[0052] from Figure 4 It can be seen that the calibration curve established by the blood glucose test value and the average test current of the YSI2500 glucose / lactic acid analyzer has a correlation coefficient squared (R2) greater than 0.99, indicating a good fit.

[0053] The above description is merely a preferred embodiment of the present invention and does not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. An electrochemical biosensor, comprising: (1) An electrode system comprising a working electrode and a counter electrode; (2) A reaction reagent disposed on the working electrode and the counter electrode, the reaction reagent comprising: (a) an enzyme; the enzyme is glucose oxidase, lactate oxidase, uricate oxidase, cholesterol oxidase, creatine hydrolase, or ascorbic acid oxidase. (b) Electron mediator composition; (c) Buffer solution; in, The electron mediator composition includes ruthenium complexes and aniline derivatives; The ruthenium complex is [Ru(NH3)6]. 3+ ; The aniline derivative is m-methoxy-N,N-di(hydroxyethyl)-p-nitrosoaniline, and its structural formula (Ⅳ) is as follows: 。 2. The sensor according to claim 1, characterized in that, The ruthenium complex is [Ru(NH3)5X] n+ In the formula, X represents NH3 and CN. - Halogen ions, etc., n+ is the oxidized state of Ru 3+ The valence state of the entire chemical formula when combined with other groups.

3. The sensor according to any one of claims 1-2, characterized in that, The buffer solution used is Good's buffer solution, organic acid buffer solution, or phosphate buffer solution.

4. The sensor according to any one of claims 1 to 2, characterized in that, The buffer solution is a Good's buffer solution, which is selected from one or more of ACES, TES, and MES.

Images