CN107941880B - Reaction reagent for improving storage stability of glucose sensor comprising betaine derivative, and glucose sensor - Google Patents
Reaction reagent for improving storage stability of glucose sensor comprising betaine derivative, and glucose sensor Download PDFInfo
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Abstract
The invention belongs to the technical field of medical equipment, and relates to a reaction reagent containing a betaine derivative for improving the storage stability of a glucose sensor and the glucose sensor. The invention provides a reaction reagent containing betaine derivatives for improving the storage stability of a glucose sensor, which comprises an enzyme, an electron mediator composition, at least one betaine derivative and a buffer solution, and also comprises at least one surfactant and at least one cellulose-based high polymer. The invention also provides a glucose sensor applying the reaction reagent, and the betaine derivative is added into the reaction reagent, so that the storage stability of the glucose sensor is obviously improved, and the accuracy in the validity period is ensured. The glucose sensor has the advantages of reasonable structure, simple process, easily obtained raw materials of reaction reagents and contribution to industrial production.
Description
Technical Field
The invention belongs to the technical field of medical equipment, and relates to a reaction reagent containing a betaine derivative for improving the storage stability of a glucose sensor and the glucose sensor.
Background
In recent years, disposable biosensors with electrode systems are often used in point of care testing (POCT). Taking the daily blood sugar test of a diabetic as an example, the patient can know the current blood sugar level only by using the disposable biosensor at home, thereby helping the patient to know the self physical condition in time and adjust the self diet structure, or leading a doctor to adjust the dosage of the medicine according to the daily monitored data. Therefore, the accuracy of such disposable biosensors is important in daily monitoring of diseases, and one of the important factors affecting the accuracy of disposable biosensors is their long-term storage stability.
In a disposable biosensor such as a glucose sensor, an electrode system including a working electrode and a counter electrode is formed on an insulating substrate, and a reagent constituting a reaction such as an enzyme and an electron mediator is disposed thereon. In the current mainstream technical route, dehydrogenases (GDH) which do not use molecular oxygen as an electron mediator are often used as the reaction reagent; a glucose sensor using FAD-GDH with Flavin Adenine Dinucleotide (FAD) as a coenzyme is the mainstream product in the market at present.
In general, factors affecting the long-term storage stability of glucose sensors mainly come from two aspects: on one hand, poor stability of the electron mediator causes the background current to change, thereby influencing the accuracy of the glucose sensor; another aspect is that the total enzyme activity present on the glucose sensor is significantly reduced due to denaturation of the enzyme by both heat treatment and oxidation processes, resulting in erroneous test results when using the glucose sensor.
Both the pretreatment and storage conditions of disposable biosensors may seriously affect the enzyme activity. For example, heat treatment during manufacture or storage may inactivate the enzymes of the disposable biosensor. As another example, many enzymes are sensitive to oxidation processes, however, many enzymes in disposable biosensors are often in a substantially solvent-free dry environment during storage, which may exacerbate such oxidation processes. Furthermore, the reactive agent layer may contain other components that exacerbate the oxidation process. However, glucose dehydrogenase, an important diagnostic tool, is quite sensitive to thermal treatment and oxidation reactions. Therefore, it is necessary to protect the enzymatic activity of FAD-GDH in a dried reaction reagent to improve the storage stability of the glucose sensor.
There are currently many reports on improving the long-term storage stability of biosensors:
patent CN 101896618A discloses the use of 3- (3',5' disulfonated monophenylimino) -3H-phenothiazine as an electron mediator for glucose sensors and found that the background current using these phenothiazines is much smaller than previously used mediators, while a smaller background current is advantageous for extending the shelf life of the sensor. In addition, it was shown that the saccharide surfactant heptanoyl-N-methylglucamide (MEGA-8) can improve the thermal stability of the glucose sensor.
Patent CN 104145023B discloses the use of tetrahydropyrimidine or derivatives thereof for reducing the decrease of the enzymatic activity of glucose dehydrogenase in a dry composition of diagnostic test elements.
Patent CN 106290329A discloses a compound R1-R2-R3The polymer is applied to the polymer with the general structural formula, and the stability of the polymer is improved through interaction with the enzyme and the color indicator, so that various in-vitro detection items can keep the stability of the enzyme and the color indicator under extreme environments (such as high temperature), the deviation of a detection result is small, and the accuracy is higher.
Patent CN 105136874a discloses a detection formula in an enzyme biosensor, which is composed of citric acid or citrate, malic acid or malate, polyacrylic acid or polyacrylate, potassium ferricyanide, enzyme and non-reactive components. The detection formula can inhibit potassium ferricyanide from being reduced, and improve the stability of a potassium ferricyanide mediator, thereby reducing the influence of background current on a detection result.
The above-mentioned methods for improving the long-term storage stability of biosensors mainly have the drawbacks of difficulty in obtaining raw materials, complicated manufacturing processes, and high cost. To date, there is no report on adding betaine derivatives to the reaction reagents of glucose sensors to significantly improve the storage stability of glucose sensors.
Disclosure of Invention
In order to solve the above problems, it is an object of the present invention to provide a reaction reagent for improving storage stability of a glucose sensor, comprising a betaine derivative, the reaction reagent comprising (a) an enzyme, (b) an electron mediator composition, (c) at least one betaine derivative, (d) a buffer solution; also comprises at least one surfactant and at least one cellulose high polymer.
The invention also aims to provide the glucose sensor which is optimized in reagent compatibility, reasonable in structural design and high in detection accuracy.
In order to achieve the purpose, the invention adopts the following technical scheme:
a reaction reagent comprising a betaine derivative for improving storage stability of a glucose sensor, the reaction reagent comprising: (a) an enzyme; (b) an electron mediator composition; (c) at least one betaine derivative; (d) and (4) buffer solution.
The betaine derivative refers to the following sulfobetaine derivatives:
wherein R is1And R3Is methyl, R2Is- (CH)2)nCH3And n is the polymerization degree, wherein the polymerization degree n is 12-18.
The betaine derivative is one or more of 3-sulfopropyl dodecyl dimethyl betaine (CAS number: 96702-03-3), 3-sulfopropyl tetradecyl dimethyl betaine (CAS number: 96702-03-3), 3-sulfopropyl hexadecyl dimethyl betaine (CAS number: 96702-03-3) and 3-sulfopropyl octadecyl dimethyl betaine (CAS number: 13177-41-8).
Preferably, the betaine derivative is 3-sulfopropyltetradecyldimethyl betaine.
Preferably, the betaine derivative is present at a concentration of about 0.25-1.25 wt.%. More preferably, the concentration of betaine derivative is about 0.85 wt.%.
The dehydrogenase (GDH) used in the present invention is glucose dehydrogenase, and is Flavin Adenine Dinucleotide (FAD) -GDH having FAD as a coenzyme.
The electron mediators employed in the context of the present invention are well known in the art and include, for example, potassium ferricyanide, ruthenium hexaammine chloride, osmium complexes or phenazines, among others. The electron mediator composition contains hexaammine ruthenium chloride and phenazine; the phenazine is one or the combination of 5-methylphenazinium methyl sulfate, 5-ethylphenazine ethyl sulfate or 1-methoxy-5-methylphenazinium methyl sulfate. Preferably, the electron mediator composition is hexaammine ruthenium chloride and 1-methoxy-5-methylphenazinium methyl sulfate. Details on how to apply the electron mediator composition can be found in patent CN206339517U, patent CN 103018292B.
The buffer solution is selected from biological buffer solution or zwitterion buffer solution, the pH of the buffer solution is 5.0-9.0, and the buffer solution comprises Good's buffer solution, organic acid buffer solution, phosphate buffer solution and the like. Preferably, MES buffer solution, TES buffer solution, ACES buffer solution, or a combination thereof is used. More preferably, ACES buffer solution is used, the concentration is 0.1-1M, and the pH is 6.5-7.5.
The reaction reagent also comprises at least one surfactant, which can be a nonionic surfactant, preferably, the surfactant is used
X-100。
The X-100 causes the liquid reaction reagent to be uniformly distributed and spread on the surface of the electrode after the liquid is spotted, and simultaneously improves the hydrophilicity of the dried reaction reagent, thereby improving the sample introduction rate of the glucose sensor when detecting the blood sample.
The reaction reagent also comprises at least one cellulose-based high polymer, preferably hydroxymethyl cellulose. The high polymer plays a role of a bracket in the reaction reagent, is beneficial to the dispersion and the stability of the enzyme, and simultaneously ensures that the dried reaction reagent has good film forming and strong adhesive force.
In addition, according to the reaction reagent of the present invention, a glucose sensor for measuring the glucose content in blood in vitro using the reaction reagent is further contemplated. The glucose sensor includes: (a) a working electrode and a counter electrode; (b) the reaction reagent; the reactive agent is disposed on the working electrode and the counter electrode, and the reactive agent comprises at least one betaine derivative.
The betaine derivative is one or more of 3-sulfopropyl dodecyl dimethyl betaine, 3-sulfopropyl tetradecyl dimethyl betaine, 3-sulfopropyl hexadecyl dimethyl betaine and 3-sulfopropyl octadecyl dimethyl betaine.
The glucose sensor is prepared by the following steps: after the liquid reaction reagent is prepared, fully stirring the liquid reaction reagent to dissolve and disperse the liquid reaction reagent to form a homogeneous solution; then, liquid reaction reagents are configured on a working electrode and a counter electrode of a glucose sensor reaction area in a liquid dropping mode; and removing the solvent in the liquid reaction reagent to obtain the glucose sensor with the dried reaction reagent.
Further, the reaction reagent of the glucose sensor of the present invention includes: concentration of FAD-GDH is 0.5-5wt.% (enzyme activity is 200u/mg), concentration of ruthenium hexaammine chloride is 0.5-5wt.%, concentration of 1-methoxy-5-methylphenazinium methyl sulfate is 0.005-0.05wt.%, concentration of 3-sulfopropyltetradecyldimethyl betaine is 0.25-1.25 wt.%, concentration of ACES buffer solution (ACES concentration in buffer solution is 0.1-1M, pH is 6.5-7.5) is 10-50wt.%,
the concentration of X-100 is 0.1-1wt.%, and the concentration of hydroxymethyl cellulose is 0.25-2.5 wt.%.
Specific structural details of the glucose sensor of the present invention can be found in patent CN 206459991U.
Preferably, the substrate layer is made of polyethylene terephthalate. The electrode layer is made of carbon, palladium and gold; more preferably, the material of the electrode layer is carbon. The insulating layer is made of polyacrylic resin. The material of the cushion high layer is modified polyacrylic acid double faced adhesive tape. The hydrophilic membrane subjected to single-side hydrophilic treatment is made of polyethylene terephthalate.
The specific method for testing the glucose sensor is as follows:
step 1, when a glucose sensor is inserted into a test instrument, a starting electrode and a counter electrode form a passage, and the test instrument is started;
step 2, after a blood sample is sucked, when a passage is formed between the counter electrode and the working electrode and the current reaches a set threshold value, and a passage is formed between the counter electrode and the detection electrode within a set time and the current reaches the set threshold value, applying a working voltage of 300mv between the counter electrode and the working electrode, and acquiring a glucose current value of the blood sample by adopting a timing current method at 5 seconds;
and 3, correspondingly converting the glucose current value of the blood sample to obtain the blood glucose value of the blood plasma.
The term "storage stability" as used herein refers to the stability of the electrochemical performance of the glucose sensor during its useful life. It will be appreciated by those skilled in the art that in order to determine the amount of analyte, a corresponding electrochemical signal needs to be obtained; more specifically, the chronoamperometric glucose sensor obtains a corresponding current signal of a blood sample with unknown blood glucose concentration, and then converts the current signal into a plasma blood glucose value of the blood sample through a calibration equation. How such calibration equations can be established and used is well known to those of ordinary skill in the art.
Further, the storage stability of the glucose sensor was evaluated, either with real storage stability or with accelerated aging. Patent CN100416266C relates to the evaluation of the stability of electron mediators, using experimental conditions of accelerated ageing at 56 ℃ for 2 weeks. The time cost can be greatly reduced by adopting the storage stability of accelerated aging; the theoretical basis is Vant Hoff rule and Arrhenius equation. It is deduced therefrom that 3 months of accelerated aging at 45 ℃ or 14 days of accelerated aging at 65 ℃ can be approximately regarded as 2 years under real storage conditions, as will be understood by those skilled in the art.
Therefore, in the present inventionThe method for estimating the storage stability of the glucose sensor is as follows: respectively testing blood samples with certain blood glucose concentration by using a glucose sensor stored at room temperature of 23 ℃ as a control group and glucose sensors stored at high temperature (such as 45 ℃ and 65 ℃) of the same batch as a test group to obtain a current value I23℃，I45℃，I65℃(ii) a Further calculation yields the relative deviation, i.e. I45℃/I23℃100% and I65℃/I 23℃100%. The storage stability of the glucose sensor was evaluated quantitatively in terms of relative deviation.
The invention has the following beneficial effects:
1. according to the glucose sensor, the betaine derivative is added into the reaction reagent, and has a protection effect on the enzymatic activity of the FAD-GDH in the dry reaction reagent, so that the storage stability of the glucose sensor can be remarkably improved, and the accuracy in the effective period is guaranteed.
2. The betaine derivative only exists in a dry reaction reagent, and does not interfere an electrochemical detection system. In particular, the glucose sensor of the present invention has a low steady background current during the lifetime, which is beneficial for improving the accuracy of the glucose sensor during the low blood glucose concentration period.
3. The glucose sensor has the advantages of reasonable structure, simple process, easily obtained reaction reagent raw materials and low cost, and is beneficial to industrial production.
Drawings
FIG. 1 is a graph illustrating the effect of varying amounts of 3-sulfopropyltetradecyldimethyl betaine on the storage stability of a glucose sensor;
FIG. 2 is a graph of a linear evaluation of test values for a group C glucose sensor;
FIG. 3 is a graph of accuracy analysis of group C glucose sensors after accelerated aging;
FIG. 4 is an analytical graph comparing the effect of different betaine derivatives on the storage stability of glucose sensors;
FIG. 5 is an analytical graph comparing the effect of different enzyme protein protectants on storage stability of glucose sensors.
Detailed Description
The technical solutions of the present invention are further described in detail by the following specific examples, but it should be noted that the following examples are only used for describing the content of the present invention and should not be construed as limiting the scope of the present invention.
The validity of the glucose sensors currently on the market is usually as long as 1-2 years, however, for example, poor stability of the electron mediator may result in a high reading of the glucose sensor, and for example, a decrease in enzyme activity may result in a low reading of the glucose sensor. It is therefore necessary to improve the stability of the glucose sensor for long-term storage so as to ensure its accuracy.
The reaction reagent for improving storage stability of a glucose sensor, which comprises a betaine derivative, of the present embodiment comprises: (a) an enzyme; (b) an electron mediator composition; (c) at least one betaine derivative; (d) a buffer solution; also comprises at least one surfactant and at least one cellulose high polymer; also includes a solvent.
Depending on the solvent, the components of the reaction reagent should be dissolved under conditions which do not impair the function of the components of the reaction reagent, and in particular the enzymatic activity of glucose dehydrogenase. Suitable solvents for this embodiment include water, but may also be organic, such as alcohols. It will be appreciated that the solvent used may be a mixture of two or more of the above solvents. Preferably, the solvent of this embodiment is water.
In the manufacturing process of the glucose sensor of this embodiment, the initially liquid reaction reagent is dispensed onto the electrode system of the glucose sensor reaction region by means of a spot-on process, and then the solvent is removed by a suitable heat treatment process so that the remaining components are substantially free of the solvent, thereby obtaining the glucose sensor with the dried reaction reagent. Preferably, the heat treatment procedure: heat-treating in a drying tunnel at 30-70 deg.C for about 15-45 min. More preferably, the temperature of the drying tunnel is increased in steps by adopting a programmed heating mode to achieve the best drying effect, and the drying tunnel is sequentially subjected to heat treatment for 10 minutes in a drying tunnel section at the temperature of 30-40 ℃, a drying tunnel section at the temperature of 40-50 ℃ and a drying tunnel section at the temperature of 50-60 ℃.
It will be readily understood by those skilled in the art that the mass fraction of solvent or solvent mixture in the dried reaction reagent is at most 10%, at most 5%, at most 3%, at most 1%. In order to keep the dried reaction reagent after the heat treatment in a dry state, it is preferable to store the reaction reagent in the presence of a drying agent. Suitable drying agents are: preferably, it is a molecular sieve or silica gel. As will be readily appreciated by those of ordinary skill in the art, the glucose sensor is ultimately stored in a sealed aluminum foil pouch or sealed plastic cartridge with desiccant.
The betaine derivative of this example is a structurally related organic molecule that is present in the dried reaction reagent, significantly improving the storage stability of the glucose sensor. Preferably, the betaine derivative, if present in the dried reaction reagent according to the invention, will be able to improve the stability of the glucose sensor during manufacture and/or storage, in particular during storage above room temperature, e.g. 45 ℃ and even up to 65 ℃, to a statistically significant degree. Preferably, the betaine derivative, if present in the dried reaction reagent, should be capable of maintaining a relative deviation in the stability of the glucose sensor of at least 80%, at least 90% or even at least 95% compared to a control reaction reagent without said betaine derivative.
Preferably, the betaine derivative refers to 3-sulfopropyldodecyl dimethyl betaine (CAS number: 96702-03-3), 3-sulfopropyltetradecyl dimethyl betaine (CAS number: 96702-03-3), 3-sulfopropylhexadecyl dimethyl betaine (CAS number: 96702-03-3), and 3-sulfopropyloctadecyl dimethyl betaine (CAS number: 13177-41-8), or a combination thereof.
Preferably, the betaine derivative is present at a concentration of about 0.25-1.25 wt.%.
Preferably, the betaine derivative refers to 3-sulfopropyltetradecyldimethyl betaine (CAS number: 96702-03-3).
The studies conducted by the present invention have found that the storage stability of the existing glucose sensor, which generally has a dry reaction reagent, during storage is not good, and can be significantly improved by adding at least one betaine derivative as described in detail elsewhere herein, because the betaine derivative has a protective effect on the enzymatic activity of FAD-GDH in the dry reaction reagent.
The reaction reagent of the glucose sensor of the present invention comprises: concentration of FAD-GDH is 0.5-5wt.% (enzyme activity is 200u/mg), concentration of ruthenium hexaammine chloride is 0.5-5wt.%, concentration of 1-methoxy-5-methylphenazinium methyl sulfate is 0.005-0.05wt.%, concentration of 3-sulfopropyltetradecyldimethyl betaine is 0.25-1.25 wt.%, concentration of ACES buffer solution (ACES concentration in buffer solution is 0.1-1M, pH is 6.5-7.5) is 10-50wt.%,
the concentration of X-100 is 0.1-1wt.%, and the concentration of hydroxymethyl cellulose is 0.25-2.5 wt.%.
Example 1 evaluation of the Effect of the content of 3-sulfopropyltetradecyldimethyl betaine on the storage stability of a glucose sensor
Glucose sensors were fabricated according to the reaction reagent ratios in table 1, and were divided into group a, group B, group C, and group D in order. The specific manufacturing process is as follows: firstly, liquid reaction reagents are prepared, then the liquid reaction reagents are prepared to a reaction area of an electrode system in a liquid dropping mode, and the load capacity of each glucose sensor is 0.7-1.2 ul. Heat treatment is carried out in sequence in a drying tunnel section at 30-40 ℃, a drying tunnel section at 40-50 ℃ and a drying tunnel section at 50-60 ℃ for 10 minutes. Attaching a double-sided adhesive tape, a hydrophilic membrane and a shielding layer, cutting, and storing the glucose sensor in a closed plastic cylinder with a molecular sieve drying agent.
Experimental conditions for accelerated aging: the respective same batch of the barrel-packed glucose sensors are respectively stored in an oven at room temperature of 23 ℃ plus or minus 2 ℃, an oven at 45 ℃ plus or minus 1 ℃ and an oven at 65 ℃ plus or minus 1 ℃ for 14 days.
Experimental procedure for stability assessment: the cartridge glucose sensor in the oven was left at room temperature for more than 30 minutes. Adjusting the hematocrit ratio of the blood sample to 42% + -2%; the oxygen partial pressure is controlled to be 65mmHg +/-5 mmHg; venous blood samples for a total of 5 concentration segments: the blood glucose concentration of blood plasma is 0mg/dl, 50 + -5 mg/dl, 120 + -10 mg/dl, 350 + -20 mg/d and 550 + -20 mg/dl. The glucose sensors under the conditions of the oven with the room temperature of 23 +/-2 ℃, the oven with the temperature of 45 +/-1 ℃ and the oven with the temperature of 65 +/-1 ℃ respectively test 5 concentration section blood samples to obtain corresponding current values and converted blood glucose values of blood plasma. The YSI2300 is used as a reference measurement program in a laboratory to measure the blood glucose level in the blood sample.
Data processing for stability assessment experiments: respectively testing blood samples with certain blood glucose concentration by using glucose sensors stored at room temperature of 23 ℃ as a control group and glucose sensors stored at high temperature, such as 45 ℃ and 65 ℃, of the same batch as a test group to respectively obtain current values I23℃，I45℃，I65℃. Further calculated to obtain the relative deviation I45℃/I23℃100% and relative deviation I65℃/I23℃100%. The storage stability of the glucose sensor was evaluated quantitatively in terms of relative deviation.
TABLE 1 composition and amount of liquid reactants per 100g before drying
TABLE 2 stability evaluation results of group A glucose sensors after 14 days of storage at 23 deg.C, 45 deg.C, 65 deg.C
TABLE 3 stability evaluation results of group B glucose sensors after 14 days of storage at 23 deg.C, 45 deg.C, 65 deg.C
TABLE 4 stability evaluation results of group C glucose sensors after 14 days of storage at 23 deg.C, 45 deg.C, 65 deg.C
TABLE 5 stability evaluation results of group D glucose sensors after 14 days of storage at 23 deg.C, 45 deg.C, 65 deg.C
As can be seen from tables 2-5, the storage stability of the glucose sensor can be significantly improved by adding a suitable amount of 3-sulfopropyltetradecyldimethyl betaine (e.g., group C, 0.85g/100 g).
FIG. 1 is a graph showing the analysis of the effect of different amounts of 3-sulfopropyltetradecyldimethyl betaine on the storage stability of glucose sensors, wherein glucose sensors of groups A, B, C and D stored at 23 ℃ are used as control groups, and glucose sensors of groups A, B, C and D stored at 45 ℃ and 65 ℃ for 14 days are used as test groups to test blood samples of 550mg/dl concentration range and obtain corresponding current values I23℃，I45℃，I65℃. Further calculation yields the relative deviation, i.e. I45℃/I23℃100% and65℃/I 23℃100%. The storage stability of the glucose sensor was evaluated quantitatively in terms of relative deviation. It can be seen that the 3-sulfopropyltetradecyldimethyl betaine has a significant effect on the storage stability of the glucose sensor.
Fig. 2 is a linear evaluation graph of the test values of the glucose sensor in group C, and it can be seen that the glucose sensor in group C (the concentration of 3-sulfopropyltetradecyldimethyl betaine is 0.85g/100g, i.e. 0.85 wt.%) has reasonable structural design, optimized reaction reagent compatibility, and good linear range of the plasma glucose test values.
FIG. 3 is a graph of accuracy analysis of group C glucose sensors after accelerated aging. Group C glucose sensors after 14 days of accelerated aging storage at 45 ℃ and 65 ℃ test the blood glucose values C of blood samples with blood glucose concentrations of 0mg/dl, 50 + -5 mg/dl, 120 + -10 mg/dl, 350 + -20 mg/dl and 550 + -20 mg/dl for 5 concentration segments in totalMeasuring. Plasma blood of blood sample is determined using YSI2300 as laboratory reference measurement procedureSugar value CY. Comparing the absolute deviation from a reference value, i.e. CMeasuring-CY. As can be seen from fig. 3, the 3-sulfopropyltetradecyldimethyl betaine (concentration of 0.85 wt.%) of group C significantly improved the storage stability of the glucose sensor, thereby ensuring the accuracy of the glucose sensor during the lifetime.
Example 2: evaluation of the Effect of different betaine derivatives on the storage stability of glucose Sensors
Referring to example 1, glucose sensors were prepared according to the reaction reagent ratios in table 6, and were classified into glucose sensors of group E, group F, group G, group H, group I, and group J in this order.
TABLE 6 composition of the reagents in liquid state and their contents per 100g before drying
TABLE 7 stability evaluation results of group E glucose sensors after 14 days of storage at 23 deg.C, 45 deg.C, 65 deg.C
TABLE 8 stability evaluation results of glucose sensors in group F after 14 days of storage at 23 deg.C, 45 deg.C, 65 deg.C
TABLE 9 stability evaluation results of group G glucose sensors after 14 days of storage at 23 deg.C, 45 deg.C, 65 deg.C
TABLE 10 stability evaluation results of group H glucose sensors after 14 days of storage at 23 ℃, 45 ℃, 65 ℃
TABLE 11 stability evaluation results of group I glucose sensors after 14 days of storage at 23 ℃, 45 ℃, 65 ℃
TABLE 12 stability evaluation results of group J glucose sensors after 14 days of storage at 23 deg.C, 45 deg.C, 65 deg.C
FIG. 4 is a graph showing the analysis of the effect of different betaine derivatives on the storage stability of glucose sensors, in which glucose sensors of groups E, F, G, H, I and J stored at 23 ℃ were used as control groups, and glucose sensors of groups E, F, G, H, I and J stored at 45 ℃ and 65 ℃ for 14 days in the same lot and accelerated aging were used as test groups, and blood samples at a concentration of 550mg/dl were measured to obtain current values I23℃，I45℃，I65℃. Further calculation yields the relative deviation, i.e. I45℃/I23℃100% and I65℃/I 23℃100%. The storage stability of the glucose sensor was evaluated quantitatively in terms of relative deviation. It can be seen that betaine derivatives, specifically, 3-sulfopropyldodecyl dimethyl betaine, 3-sulfopropyltetradecyl dimethyl betaine, 3-sulfopropylhexadecyl dimethyl betaine, and 3-sulfopropyloctadecyl dimethyl betaine, have an obvious effect on the storage stability of the glucose sensor.
As can be seen from the evaluation results of example 2 and fig. 4, the storage stability effects of the H group and the I group were the best. It is considered that although the background current (i.e., the test current of the glucose sensor in the 0mg/dl concentration range) of each group of glucose sensors is lower, the background current increases as the alkyl carbon chain of the betaine derivative is lengthened, and the smaller background current is more favorable for improving the accuracy of the glucose sensor in the low concentration range. Therefore, the group H to which 3-sulfopropyltetradecyldimethyl betaine has been added is preferable.
Example 3: evaluation of the Effect of other different enzyme protein stabilizers on the storage stability of glucose Sensors
Enzyme protein stabilizers which are readily known to those of ordinary skill in the art are: sugars, such as trehalose; polyols, such as sorbitol; amino acids such as sodium glutamate; proteins, such as silk protein; and tetrahydropyrimidine (CAS:96702-03-3) disclosed in, for example, MEGA-10(CAS:85261-20-7), patent CN 104145023B, and the like.
Referring to example 1, glucose sensors were prepared according to the reaction reagent ratios in table 13, and were classified into K-group, L-group, M-group, N-group, O-group, and P-group glucose sensors in this order.
TABLE 13 composition of reactants per 100g liquid before drying and their contents
Respectively testing blood samples in a 550mg/dl concentration section by taking glucose sensors in groups K, L, M, N, O and P stored at 23 ℃ at room temperature as a control group and glucose sensors in groups K, L, M, N, O and P stored for 14 days at 45 ℃ and 65 ℃ in the same batch as a test group respectively to obtain corresponding current values I23℃，I45℃，I65℃. Further calculation yields the relative deviation, i.e. I45℃/I23℃100% and I65℃/I 23℃100%. . The storage stability of the glucose sensor was evaluated quantitatively in terms of relative deviation.
FIG. 5 is a graph comparing the effect of different enzyme protein protective agents on the storage stability of glucose sensors, and it can be seen that, in the control studies of the present invention, no effect of improving the storage stability of glucose sensors, which is significantly comparable to betaine derivatives, was found in the dried reaction reagent. While the use of a protein stabilizer solution (supplied by the Gvent Group, Q2030317P49stabiliser solution) has the effect of improving the storage stability of the glucose sensor, it significantly increases the product cost.
Claims (4)
1. A reaction reagent for improving storage stability of a glucose sensor, comprising a betaine derivative, wherein the reaction reagent comprises:
(a) an enzyme;
(b) an electron mediator composition;
(c) a betaine derivative;
(d) a buffer solution;
the structural formula of the betaine derivative is as follows:
wherein R is1And R3Is methyl, R2Is- (CH)2)nCH3N is the degree of polymerization, and n = 12-18;
the betaine derivative is 3-sulfopropyl tetradecyl dimethyl betaine; the concentration of betaine derivative was 0.85 wt.%.
2. The reagent for improving storage stability of a glucose sensor comprising a betaine derivative according to claim 1, wherein the enzyme is FAD-GDH having flavin adenine dinucleotide FAD as a coenzyme; the electron mediator composition comprises hexamine ruthenium chloride and phenazine, wherein the phenazine is one or more of 5-methylphenazinyl methyl sulfate, 5-ethylphenazine ethyl sulfate and 1-methoxy-5-methylphenazinyl methyl sulfate; the buffer solution is one or more of ACES, TES and MES.
3. The glucose sensor using the reaction reagent comprising a betaine derivative according to claim 1 for improving storage stability of the glucose sensor, wherein the composition of the glucose sensor comprises (a) a working electrode and a counter electrode, and (b) the reaction reagent; the reactive agent is disposed on the working electrode and the counter electrode and becomes a dried reactive agent by heat treatment, and the reactive agent contains a betaine derivative.
4. The glucose sensor of claim 3, wherein the reactive agent comprises: concentration of FAD-GDH is 0.5-5wt.%, and enzyme activity is 200 u/mg; the concentration of ruthenium hexammine chloride is 0.5-5wt.%, the concentration of 1-methoxy-5-methylphenazinium methyl sulfate is 0.005-0.05wt.%, and the concentration of 3-sulfopropyltetradecyldimethyl betaine is 0.85 wt.%; the ACES buffer solution has a concentration of 10-50wt.%, an ACES concentration of 0.1-1M, and a pH of 6.5-7.5; TRITON (Triton on-insulator)®The concentration of X-100 is 0.1-1wt.%, and the concentration of hydroxymethyl cellulose is 0.25-2.5 wt.%.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103094564A (en) * | 2013-01-16 | 2013-05-08 | 天津大学 | Method for preparing composite nano material filled with lithium iron phosphate between graphene layers |
| CN104749122A (en) * | 2015-04-02 | 2015-07-01 | 山东博科生物产业有限公司 | Serum monoamine oxidase detection kit |
| CN106124595A (en) * | 2016-06-17 | 2016-11-16 | 浙江亿联健医疗器械有限公司 | A kind of biosensor and preparation method thereof and reaction reagent |
| CN106602039A (en) * | 2017-01-23 | 2017-04-26 | 陕西煤业化工技术研究院有限责任公司 | Functionalized graphene doped nickel cobalt lithium manganate composite cathode material and preparation method thereof |
| CN107064261A (en) * | 2017-05-01 | 2017-08-18 | 台州亿联健医疗科技有限公司 | A kind of biology sensor and detection method based on glucose dehydrogenase |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007123179A1 (en) * | 2006-04-19 | 2007-11-01 | Panasonic Corporation | Biosensor |
| CN101896618B (en) * | 2007-12-10 | 2015-04-22 | 拜尔健康护理有限责任公司 | Reagents and methods for detecting analytes |
| US8323467B2 (en) * | 2009-10-27 | 2012-12-04 | Lifescan Scotland Limited | Dual chamber, multi-analyte test strip with opposing electrodes |
| US20120199497A1 (en) * | 2011-02-07 | 2012-08-09 | Lifescan Scotland Limited | Electrochemical-based analytical test strip with diffusion-controlling layer and method for determining an analyte using such an test strip |
| JP5584740B2 (en) * | 2011-09-26 | 2014-09-03 | アークレイ株式会社 | Glucose sensor |
-
2017
- 2017-11-16 CN CN201711139385.XA patent/CN107941880B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103094564A (en) * | 2013-01-16 | 2013-05-08 | 天津大学 | Method for preparing composite nano material filled with lithium iron phosphate between graphene layers |
| CN104749122A (en) * | 2015-04-02 | 2015-07-01 | 山东博科生物产业有限公司 | Serum monoamine oxidase detection kit |
| CN106124595A (en) * | 2016-06-17 | 2016-11-16 | 浙江亿联健医疗器械有限公司 | A kind of biosensor and preparation method thereof and reaction reagent |
| CN106602039A (en) * | 2017-01-23 | 2017-04-26 | 陕西煤业化工技术研究院有限责任公司 | Functionalized graphene doped nickel cobalt lithium manganate composite cathode material and preparation method thereof |
| CN107064261A (en) * | 2017-05-01 | 2017-08-18 | 台州亿联健医疗科技有限公司 | A kind of biology sensor and detection method based on glucose dehydrogenase |
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