CN1129002C - Application of vitrification technique in preparing enzyme-electrode test strip of electrochemical sensor for testing blood sugar - Google Patents
Application of vitrification technique in preparing enzyme-electrode test strip of electrochemical sensor for testing blood sugar Download PDFInfo
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Abstract
The present invention relates to a vitrification technique for preparing an enzyme electrode test strip of an electrochemical sensor for testing blood sugar. The production technique of the present invention comprises the steps that step one, vitrescent glucose oxidase is produced, and a solid state vitrification carrier is added into a glucose oxidase solution to be vitrified; step two, the vitality and the stability of the vitrescent glucose oxidase is tested; step three, the glucose oxidase electrode test strip is vitrified. The present invention for producing the enzyme electrode test strip uses an automatic wound vitrification technique and is implemented in a self-control room temperature vitrification machine. The finished product packaging of the present invention is finished in a local dehumidify cabinet in a decontamination chamber of 100, 000 grades, and relative moisture in the cabinet is automatically kept in 25%. The enzyme electrode test strip can be stored for a long time at room temperature with unchanged vitality by the room temperature vitrification technique.
Description
The present invention relates to biological chemistry, enzyme engineering and medical clinic applications technology, particularly a kind of production that room temperature vitreous technology is applied to the electrochemical sensor of detection by quantitative blood sugar with disposable vitrescence glucose oxidase electrode strip.
Since 1967 You Pudaike (Updike) immobilized glucose oxidase is covered in platinum electrode, make it quantitative measurement blood sugar, and proposed since " enzyme electrode " this notion various biology sensor (biosensor first; Be called for short BS) flourish like the mushrooms after rain.Although academia is always to the promise well of BS technology, the paper number of delivering not to descend 1,000,000 pieces, yet the kind of the genus that really comes into the market " enzyme electrode " class still has only several so far, and its quality is often reproached.Trace it to its cause, mainly be to form the instability of the key component (as immobilised enzymes) of recognition means.For example report recently, two enzyme membranes (forming) of the mensuration blood sugar made from conventional BS technology by immobilized glucose oxidase and peroxidase, after using once, be stored in 4 ℃ of refrigerators, detect after taking-up is installed on electrode surface after spending one month, enzyme activity reduces to 95.5%, take out to detect after spending two months and then reduce to 89.5% and (see Li Haihong, Yan Shaohua, lacquer moral precious jade etc. for details, the biology sensor of beta-cyclodextrin cross-linked immobilized enzyme and clinical practice: biological chemistry and biophysics progress, 1998,25 (2): 162-166).Obviously, this does not allow in regular clinical examination, and in conventional BS technology, and The above results also is considered to high stability, recurrent then is that the immobilised enzymes vigor descends at random along with the increase of access times, thereby is difficult to realize commercialization.
In addition, the operation of conventional BS technology also bothers.As the running program in the above-mentioned document be: earlier the fixed enzyme membrane of depositing in refrigerator is taken out, be installed on electrode surface carefully; Adjust the conversion of signals device; Add blood sample in reactant liquor, measure also and shut down behind the reading; Take off enzyme membrane more carefully, put into refrigerator after drying; Clean used electrode, to remove electrode surface because of the formed oxide layer of electrochemical reaction.Obviously, complicated operations must be carried out by the professional like this, is difficult to family and tests oneself.And dried the neglecting of neglecting of enzyme membrane wet, and the easier immobilised enzymes vigor that makes descends.How to correct these key weakness in the conventional BS technology, make it to become family easy and simple to handle, that enzyme activity the is constant instrument of testing oneself? nineteen ninety, people (Nankai S.Kawaguri M.Iijima T. such as the Japan South Sea, Biosensor and method for making thesame:US patent, G01N, 4897173,1990-01-30) proposed all kinds of diagnostic test technology in vogue are at that time incorporated the design of biosensor technology, make enzyme electrode become disposable test strips.The irregular key weakness of degradation down takes place with the access times increase in the immobilised enzymes vigor in the traditional B S technology because of overcoming in this design, is subjected to the attention that countries in the world comprise states colleagues such as moral, American and Britain immediately, and competitively development and improvement.So far, existing two classes are suitable for the disposable test strips formula biology sensor commodity appearance that family tests oneself.One class system is according to sensor (the Hoenes J.Schaoffler J. of electrochemical principle design, Methodand sensor electrode system for the electrochemical determination of an analyst oran oxidoreductase as well as the use of suitable compounds therefor:US patent, G01N, 5286362,1994-02-15), on its appended disposable enzyme strip electrode is arranged; Another kind of system is according to tintmeter (the Milpitas S.A.K.McGarraugh G.Yeung S. of reflectance spectrum principle design, control solution for a blood glucose monitor:US patent, G01N, 5605837,1997-02-25), electrodeless on its appended disposable enzyme strip, strip promptly shows the naked eyes visible color after adding blood sample.In fact strict, this class colorimetric strip and tintmeter can not be can be regarded as sensing technology.In addition, the haemoglobin of contained redness can make the absorption spectrum wavelength change in the blood sample, thereby produces certain error.These two classes commodity all entered Chinese market in 1998, the more type sale price of function is respectively up to 1900 yuan with 1100 yuan, than them 50 dollars and 40 dollars high nearly 4～5 times of the prices of its producing country.It should be noted that these two classes commodity all are only limited to mensuration blood sugar at present, and all be to utilize to realize based on the enzymatic reaction system of glucose oxidase.This is because this enzyme is more stable, easily the cause of making.Other oxidases does not then still have this kind disposable enzyme electrode strip formula product because of its instability and enters the market.
For this reason, have only the new disposable enzyme electrode strip of development could change traditional B S technology, this just should with the immunity-chromatography test strip technology with can be dissolved into these two technology of the vitrifacation technology of the long-term room temperature preservation of protein such as enzyme, antibody in the traditional B S technology, could obtain to have high stability, be used to detect disposable enzyme electrode strip blood sugar, that belong to galvanochemistry class sensor.
The purpose of this invention is to provide a kind of vitreous technology of using and produce the manufacture method of blood sugar with the enzyme electrode strip of electrochemical sensor; it makes enzyme electrode strip long-term storage and vigor is constant at room temperature by room temperature vitreous technology, solved this strip former under storage at room temperature the problem of poor stability.
Technical scheme of the present invention is as follows:
A kind of vitreous technology of using is produced the manufacture method of blood sugar with the enzyme electrode strip of electrochemical sensor, and its production technology comprises the following steps:
The first step, the production of vitrescence glucose oxidase:
Get glucose oxidase solution, add solid-state vitreous carrier, make its final concentration reach 10%, divide the bottle of packing into; Bottle was all put into the vitrification machine for greenhouse vitreous 12 hours; Vitreous is finished each bottle and is all sealed with rubber stopper in machine, takes out and seals up aluminium lid, and this is a vitrescence glucose oxidase finished product, stores to get final product under room temperature;
Second step, the vitreous of glucose oxidase enzyme electrode strip:
Disposable enzyme electrode strip of the present invention is added the vitreous carrier in traditional enzymatic reaction system, make the enzyme electrode strip be vitrescence and highly stable; Test strip electrode of the present invention adopts identical and the form that area does not wait of the two poles of the earth material, and electrode material is the metal spraying electrode, with anti-air oxidation, makes electric conductivity good; The vitreous operating process of enzymatic reaction system is as follows:
(1) preparation of enzymatic reaction system component:
Fixation support is the potpourri of hydroxyethyl cellulose and microcrystalline cellulose, and concentration is 4.5%; Cementing agent is a carboxymethyl cellulose, and concentration is 0.5%; The electron transport intermediate is the potassium ferricyanide, and concentration is 16%; Wetting agent is Triton X-100, and concentration is 0.08%; The dosage of vitrescence glucose oxidase is 1750 μ/ml; Buffer system is pH6.2,0.34mol/L kaliumphosphate buffer; During preparation, above-mentioned each reagent is mixed with the high concentration stock solution with pH6.2,0.34mol/L kaliumphosphate buffer respectively, combination in the desired amount again during use, and mistake leaching clear liquid, calculate in this liquid behind the amount volume and contained vitreous carrier amount, add solid-state vitreous carrier again and make its final concentration reach 10%, make enzyme reaction vitreous liquid;
(2) preparation of enzyme electrode strip:
Get the two poles of the earth area inequality metal spraying electrode strip of new production, at its enzyme-added pond and two electrode contact places, earlier with liquid detergent cleaning, distilled water rinse, being stained with absolute ethyl alcohol with disinfecting cotton swab again cleans, after treating that the ethanol volatilization is clean, each electrode strip application of sample box of packing into uniformly, be put on the full-automatic liquid filler operating platform that is located in 100,000 grades of decontamination chambers, spray adds enzyme reaction vitreous liquid in the enzyme-added pond of strip; In the reservoir of automatic liquid filler, pour into the enzyme reaction vitreous liquid that is prepared in above-mentioned the 1st operating process in advance; Required each parameter of application of sample also is provided with on its computer controller in advance and finishes;
(3) vitreous of enzyme electrode strip:
The strip that will add enzyme reaction vitreous liquid connects box and puts into the vitrification machine for greenhouse vitreous, and vitrification point is 45 ℃, and the vitreous time is 40 minutes; After vitreous is finished; in air chamber each box covered and enclosed; immigration is arranged in the local moisture eliminating case of 100,000 grades of decontamination chambers; wet in control is under the condition of relative humidity 25%; behind each enzyme electrode addition pool album leave membrane cover; pack into the strip branch in the aluminium foil bag and seal, this promptly detects the enzyme electrode strip finished product of blood sugar with electrochemical sensor.
The present invention with traditional, be known as repeatedly used pillar enzyme electrode (actual for once, enzyme activity just descends) and change disposable enzyme electrode strip into, this strip with after promptly abandon, enzyme activity is guaranteed by manufacturer.Secondly, the present invention has added the vitreous carrier in traditional enzymatic reaction system, makes the enzyme electrode strip be vitrescence and highly stable.The invention solves the problem of enzyme electrode strip poor stability under storage at room temperature, make enzyme electrode strip long-term storage and vigor is constant at room temperature by room temperature vitreous technology.
Production sensor of the present invention has following three characteristics with the enzyme electrode strip of ambient-temp-stable:
The one, with the homemade liquid glucose oxidase that buys (being called for short GOD) vitrifacation, make it to place refrigerator, at room temperature long-term storage and vigor is constant.
The 2nd, the production of enzyme electrode strip has been adopted and has been created vitreous technology certainly, and implements in self-control room temperature vitrification machine for greenhouse.
The 3rd, finished product packing is to finish in the local moisture eliminating case in being arranged at 100,000 grades of decontamination chambers, and the relative humidity automatic constant is 25% in the cabinet.
It is constant that gained vitrescence glucose oxidase of the present invention reaches 21 days vigor through 45 ℃ of breaking tests, and two kinds of glucose oxidase dried frozen aquatic productses from U.S. sigma company (Sigma) in contrast, destroying vigor descended respectively 26% (Sigma G-2133) and 60% (Sigma G-7016) with under the condition.Equally, the electrochemical sensor that gained of the present invention detects blood sugar is with disposable vitrescence glucose oxidase electrode strip, reach 21 days through 45 ℃ of breaking tests, enzyme activity is no change still, between the average bar of 50 strips difference still can remain on before the breaking test ± 0.5mmol/L in, test 6.25,12.5 and 25mmol/L standard glucose solution respectively with this strip, it is linear that the gained typical curve is.
Below in conjunction with accompanying drawing, subordinate list and embodiment the present invention is elaborated.
Fig. 1 is a kind of structural representation of using vitreous technology production testing blood sugar with the enzyme electrode strip of electrochemical sensor.
Fig. 2 is that vitrescence glucose oxidase enzyme product of the present invention detects its stable experimental result through the famous chemical reagent Industrial Co., Ltd of Asahi Chemical Industry of Japan in Japanese general headquarters.
Fig. 3 is the disposable enzyme electrode strip of vitrescence that the present invention produces, and inserts gained linear diagram when measuring a series of concentration standard solution of glucose in the minitype digital electrochemical gaging instrument.
Fig. 4 is the stability experiment result of vitrescence glucose oxidase electrode of the present invention.
Table 1 is an experimental result of surveying each concentration glucose standard solution with the vitrescence GOD enzyme electrode strip that the present invention produces.
Table 2 is experimental results of surveying each concentration glucose standard solution with German Bao Lingman electrochemical sensor and appended enzyme electrode strip thereof.
Referring to Fig. 1, enzyme electrode strip of the present invention is provided with the addition pool 1 of enzyme-added and vitreous liquid, in the lower end of strip electrode leads to client 2 is arranged.
The present invention uses the enzyme electrode strip of vitreous technology production testing blood sugar with electrochemical sensor, and its production technology comprises the following steps:
The first step, the production of vitrescence glucose oxidase:
Get the glucose oxidase solution available from Great Wall, Baoding clinical reagent company, totally 33 ten thousand units add the solid-state vitreous carrier of IV type available from the biological high-tech company of Shanghai Baokang, make its final concentration reach 10%.With every bottle 1000 unit packing, totally 330 bottles, (referring to the Chinese invention patent publication number: CN1216677A) vitreous is 12 hours all to put into self-produced vitrification machine for greenhouse.Vitreous is finished, and each bottle all seals with rubber stopper in machine, takes out and seals up aluminium lid, and this is a vitrescence glucose oxidase finished product, at room temperature stores to get final product.
The vigor that carries out the vitrescence glucose oxidase detects and stability experiment:
Get 5 bottles of the above-mentioned vitrescence glucose oxidase finished products (every bottle 1000 unit) of at room temperature storing 3 months, put into 45 ℃ of constant temperature oven insulations 21 days, meanwhile, the reference substance of putting into is glucose oxidase (G-6125) dried frozen aquatic products available from U.S. Sigma company.Take out each sample when being incubated 21 days, survey enzyme activity by classical colourimetry (P.169, publishing house of Southeast China University publishes for Department of Medical Administration of People's Republic of China (PRC) volume, national clinical examination working specification, 1997).Consequently this vitrescence glucose oxidase enzyme activity does not become at all, and the enzyme activity of Sigma G-6125 glucose oxidase dried frozen aquatic products has lost 32%.
In order to make this experimental result have more cogency, special with vitrescence glucose oxidase (product batch number: 200113) 4 bottles, every bottle 1000 unit, give (Asahi Chemical Industry Co. to the famous chemical reagent Industrial Co., Ltd of Asahi Chemical Industry of Japan, Ltd) visitor, take back the check of our department of Japanese firm with oneself by them, its testing result as shown in Figure 2.This shows, vitrescence glucose oxidase produced according to the invention, though through 45 ℃ of breaking tests 21 days, the true no change of enzyme activity, difference is almost nil between bottle.And two kinds of glucose oxidase dried frozen aquatic productses (G-2133 and G-7018) of producing by U.S. Sigma company, its enzyme activity descends gradually with the increase of 45 ℃ of insulation fates, and enzyme activity has lost 32% and 60% respectively in the time of the 22nd day.Testing result shown in Fig. 2 confirms that effectively vitrescence glucose oxidase of the present invention has highest stabilizing.
Second step, the vitreous of glucose oxidase enzyme electrode strip:
Disposable enzyme electrode strip of the present invention is added the vitreous carrier in traditional enzymatic reaction system, make the enzyme electrode strip be vitrescence and highly stable; Test strip electrode is adopted identical and the form that area does not wait of the two poles of the earth material, and electrode material is the metal spraying electrode, with anti-air oxidation, makes electric conductivity good; The vitreous operating process of enzymatic reaction system is as follows:
1, the preparation of enzymatic reaction system component:
Fixation support is the potpourri of hydroxyethyl cellulose and microcrystalline cellulose, and optimum concentration is 4.5%; Cementing agent is a carboxymethyl cellulose, and optimum concentration is 0.5%; The electron transport intermediate is the potassium ferricyanide, and optimum concentration is 16%; Wetting agent is Triton X-100, and optimum concentration is 0.08%; The suitableeest dosage of vitrescence glucose oxidase is 1750 μ/ml; The suitableeest buffer system is pH value 6.2,0.34mol/L kaliumphosphate buffer.During preparation; above-mentioned each reagent is mixed with the high concentration stock solution with pH value 6.2,0.34mol/L kaliumphosphate buffer respectively; combination in the desired amount again during use; and mistake leaching clear liquid; calculate in this liquid behind the amount volume and contained vitreous carrier amount; add solid-state vitreous carrier again and make its final concentration reach 10%, make enzyme reaction vitreous liquid.
2, the preparation of enzyme electrode strip:
Get the two poles of the earth area inequality metal spraying electrode strip of new production; at its enzyme-added pond and two electrode contact places; earlier with liquid detergent cleaning, distilled water rinse; being stained with absolute ethyl alcohol with disinfecting cotton swab again cleans; after treating that the ethanol volatilization is clean; each electrode strip application of sample box of packing into uniformly, be put on the operating platform that is located at 100,000 grades of full-automatic liquid fillers of XYZ3000 type (U.S. Bio-Dot company product) in the decontamination chamber, spray adds enzyme reaction vitreous liquid in the enzyme-added pond of strip.In the reservoir of automatic liquid filler, pour into the enzyme reaction vitreous liquid that is prepared in above-mentioned the 1st operating process in advance.Required each parameter of application of sample also is provided with on its computer controller in advance and finishes, that is: spacing is 9.0mm, every pond adds enzyme reaction solution, and to drip number be 6～12, every drop volume is 0.5 μ l, and every pond cumulative volume is 3～6 μ l, and the application of sample frequency is 100.00, time is 0.50ms, multiplicity is 10, and the X-axis side-play amount is 9.0, and the Y-axis side-play amount is 0.
3, the vitreous of enzyme electrode strip:
The strip that will add enzyme reaction vitreous liquid connects box and puts into the vitrification machine for greenhouse vitreous, and vitrification point is 45 ℃, and the vitreous time is 40 minutes.During to the vitreous of enzyme electrode strip, each parameter of vitreous is set in vitrification machine for greenhouse: baseplate temp is 45 ℃, and the air chamber temperature is 35 ℃, and the air chamber air quantity is maximum.Air chamber relative humidity is 30%.After vitreous is finished; in air chamber each box covered and enclosed; immigration is arranged in the local moisture eliminating case of 100,000 grades of decontamination chambers; wet in control is under the condition of relative humidity 25%; behind the addition pool album leave membrane cover of each enzyme electrode; pack into the strip branch in the aluminium foil bag and seal, this promptly detects the enzyme electrode strip finished product of blood sugar with electrochemical sensor.
Carry out the Quality Control experiment of vitrescence GOD enzyme electrode strip:
According to the standard glucose solution preparation method of " national clinical examination working specification " the 169th page of defined of Department of Medical Administration of People's Republic of China (PRC) establishment, prepared the glucose standard solution of a series of concentration.Get each 15 of each lot number enzyme electrode strips at random.During mensuration, each strip inserts in the electrode plug receptacle of minitype digital electrochemical gaging instrument successively, and drips 10 μ, 1 each concentration glucose standard solution successively respectively and carry out glucose quantitation measure in the addition pool of this disposable enzyme electrode strip.Every kind of concentration is surveyed 5 altogether.Measurement result counts the Quality Control table.By table 1 as seen, difference is between bar: 4.8%; Difference between batch is: 5.9%.Equally, at random series concentration glucose standard solution is measured and mapped, can get linear standard curve as shown in Figure 3.For relatively, the present invention uses commercially available by (the Boehringer Mannheim of Bao Ling Man again; BM) (trade name: Advantage TM), repeat above-mentioned experiment, the result is as shown in table 2 for the electrochemical sensor of the detection blood sugar of Chu Pining.This shows that the quality of this product is suitable with the BM product.
Carry out the stability experiment of vitrescence GOD enzyme electrode strip:
Get 50 of above-mentioned vitrescence GOD enzyme electrode strip finished products at room temperature storing 3 months, put into 45 ℃ of constant temperature ovens insulations 21 days.Took out 10 every 7 days, survey 12.50mmol/L glucose titer with the self-control electrochemical sensor, ask this mean value of 10, the result as shown in Figure 4.This shows that vitrescence GOD enzyme electrode strip has high stability.
| Measure the date | Sample (glucose titer) | Measurement result (mmol/L) | Poor between bar (%) | Strip lot number (date of manufacture) | |||||
| No.1 | ?No.2 | No.3 | ?No.4 | No.5 | On average | ||||
| 1999/12/23 | ?6.25mmol/L | 5.9 | ?6.0 | 6.6 | ?5.7 | 6.2 | 6.1 | 5.6 | 990813 |
| ?12.50mmol/L | 12.9 | ?13.1 | 12.8 | ?12.1 | 12.4 | 12.7 | 3.1 | ||
| ?25.00mmol/L | 25.6 | ?26.1 | 25.3 | ?25.8 | 24.9 | 25.5 | 1.8 | ||
| 2000/3/27 | ?6.25mmol/L | 5.4 | ?6.3 | 5.7 | ?5.7 | 6.2 | 5.9 | 6.4 | 990907 |
| ?12.50mmol/L | 12.5 | ?11.8 | 12.1 | ?12.6 | 12.1 | 12.2 | 2.7 | ||
| ?25.00mmol/L | 24.9 | ?25.7 | 25.1 | ?24.8 | 25.0 | 25.1 | 1.4 | ||
| 2000/6/15 | ?6.25mmol/L | 6.2 | ?5.9 | 5.4 | ?6.0 | 5.7 | 5.8 | 5.2 | 991024 |
| ?12.50mmol/L | 12.1 | ?13.0 | 11.9 | ?12.7 | 12.4 | 12.4 | 3.5 | ||
| ?25.00mmol/L | 26.0 | ?24.9 | 25.4 | ?24.9 | 25.3 | 25.3 | 1.8 | ||
| 2000/6/15 | ?6.25mmol/L | 7.0 | ?6.8 | 6.2 | ?6.6 | 6.9 | 6.7 | 4.8 | 991105 |
| ?12.50mmol/L | 13.1 | ?12.9 | 13.0 | ?13.1 | 12.6 | 12.9 | 1.6 | ||
| ?25.00mmol/L | 25.8 | ?25.8 | 25.4 | ?25.1 | 25.4 | 25.5 | 1.0 | ||
| 2000/6/15 | ?6.25mmol/L | 5.8 | ?5.9 | 5.8 | ?6.1 | 5.9 | 5.9 | 2.0 | 000302 |
| ?12.50mmol/L | 12.1 | ?12.5 | 12.3 | ?11.9 | 12.4 | 12.2 | 1.9 | ||
| ?25.00mmol/L | 24.7 | ?24.6 | 24.7 | ?25.1 | 24.9 | 24.8 | 0.8 | ||
| Difference between batch | ?6.25mmol/L | 5.9％ | |||||||
| ?12.50mmol/L | 2.5％ | ||||||||
| ?25.00mmol/L | 1.2％ | ||||||||
Table 1
| Measure the date | Sample (glucose titer) | Measurement result (mmol/L) | The explanation of enzyme electrode strip | |||||
| ?No.1 | No.2 | ?No.3 | No.4 | ?No.5 | On average | |||
| 1999/9/14 | ?25.00mmol/L | ?26.1 | 27.5 | ?26.2 | ?26.6 | Code number: 431; Packing: 100/tin | ||
| ?12.50mmol/L | ?12.7 | 12.6 | ?12.3 | ?12.5 | ||||
| 1999/10/22 | ?25.00mmol/L | ?27.3 | 28.3 | ?27.5 | 28.0 | ?error | ||
| 1999/10/25 | ?12.50mmol/L | ?16.5 | 9.2 | ?16.4 | ||||
| ?6.25mmol/L | ?6.9 | 7.4 | ||||||
| 1999/11/1 | ?25.00mmol/L | ?26.4 | 26.1 | ?26.6 | ||||
| ?12.50mmol/L | ?7.4 | 12.6 | ?12.3 | 12.2 | ?12.0 | |||
| ?6.25mmol/L | ?5.2 | 5.5 | ||||||
| ?3.125mmol/L | ?1.9 | 2.0 | ||||||
| 2000/3/22 | ?25.00mmol/L | ?25.0 | 25.0 | |||||
| ?12.50mmol/L | ?11.3 | 11.4 | ||||||
| ?6.25mmol/L | ?5.3 | 5.2 | ||||||
| 1999/11/5 | ?25.00mmol/L | ?25.0 | 25.6 | ?25.3 | Code number: 459 packings: 50/tin | |||
| ?12.50mmol/L | ?9.4 | 4.4 | ?12.0 | 11.9 | ?6.1 | |||
| ?6.25mmol/L | ?5.0 | 5.2 | ?4.8 | 5.4 | ?3.9 | |||
Table 2
Claims (2)
1. a manufacture method of using vitreous technology production blood sugar with the enzyme electrode strip of electrochemical sensor is characterized in that its production technology comprises the following steps:
The first step, the production of vitrescence glucose oxidase:
Get glucose oxidase solution, add solid-state vitreous carrier, make its final concentration reach 10%, divide the bottle of packing into; Bottle was all put into the vitrification machine for greenhouse vitreous 12 hours; Vitreous is finished each bottle and is all sealed with rubber stopper in machine, takes out and seals up aluminium lid, and this is a vitrescence glucose oxidase finished product, stores to get final product under room temperature;
Second step, the vitreous of glucose oxidase enzyme electrode strip:
Disposable enzyme electrode strip of the present invention is added the vitreous carrier in traditional enzymatic reaction system, make the enzyme electrode strip be vitrescence and highly stable; Test strip electrode of the present invention adopts identical and the form that area does not wait of the two poles of the earth material, and electrode material is the metal spraying electrode, with anti-air oxidation, makes electric conductivity good; The vitreous operating process of enzymatic reaction system is as follows:
(1) preparation of enzymatic reaction system component:
Fixation support is the potpourri of hydroxyethyl cellulose and microcrystalline cellulose, and concentration is 4.5%; Cementing agent is a carboxymethyl cellulose, and concentration is 0.5%; The electron transport intermediate is the potassium ferricyanide, and concentration is 16%; Wetting agent is Triton X-100, and concentration is 0.08%; The dosage of vitrescence glucose oxidase is 1750 μ/ml; Buffer system is pH6.2,0.34mol/L kaliumphosphate buffer; During preparation, above-mentioned each reagent is mixed with the high concentration stock solution with pH6.2,0.34mol/L kaliumphosphate buffer respectively, combination in the desired amount again during use, and mistake leaching clear liquid, calculate in this liquid behind the amount volume and contained vitreous carrier amount, add solid-state vitreous carrier again and make its final concentration reach 10%, make enzyme reaction vitreous liquid;
(2) preparation of enzyme electrode strip:
Get the two poles of the earth area inequality metal spraying electrode strip of new production, at its enzyme-added pond and two electrode contact places, earlier with liquid detergent cleaning, distilled water rinse, being stained with absolute ethyl alcohol with disinfecting cotton swab again cleans, after treating that the ethanol volatilization is clean, each electrode strip application of sample box of packing into uniformly, be put on the full-automatic liquid filler operating platform that is located in 100,000 grades of decontamination chambers, spray adds enzyme reaction vitreous liquid in the enzyme-added pond of strip; In the reservoir of automatic liquid filler, pour into the enzyme reaction vitreous liquid that is prepared in above-mentioned the 1st operating process in advance; Required each parameter of application of sample also is provided with on its computer controller in advance and finishes;
(3) vitreous of enzyme electrode strip:
The strip that will add enzyme reaction vitreous liquid connects box and puts into the vitrification machine for greenhouse vitreous, and vitrification point is 45 ℃, and the vitreous time is 40 minutes; After vitreous is finished; in air chamber each box covered and enclosed; immigration is arranged in the local moisture eliminating case of 100,000 grades of decontamination chambers; wet in control is under the condition of relative humidity 25%; behind each enzyme electrode addition pool album leave membrane cover; pack into the strip branch in the aluminium foil bag and seal, this promptly detects the enzyme electrode strip finished product of blood sugar with electrochemical sensor.
2. application vitreous technology according to claim 1 is produced the manufacture method of blood sugar with the enzyme electrode strip of electrochemical sensor; it is characterized in that; during to the vitreous of enzyme electrode strip; each parameter of vitreous is set in vitrification machine for greenhouse: baseplate temp is 45 ℃; the air chamber temperature is 35 ℃; the air chamber air quantity is maximum, and air chamber relative humidity is 30%.
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| JP5032769B2 (en) | 2003-03-25 | 2012-09-26 | アークレイ株式会社 | Sensor storage container |
| TWI385379B (en) * | 2004-10-12 | 2013-02-11 | Bayer Healthcare Llc | Concentration determination in a diffusion barrier layer |
| US20150160151A1 (en) * | 2013-12-06 | 2015-06-11 | Google Inc. | Formulation and Storage Method to Enhance the Enzyme and Sensor Stabilities |
| AU2015373937A1 (en) * | 2014-12-31 | 2017-07-27 | Trividia Health, Inc. | Glucose test strip with interference correction |
| EP3589746B1 (en) * | 2017-03-03 | 2021-05-19 | Siemens Healthcare Diagnostics Inc. | Nanobead containing biosensors and methods of production and use thereof |
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| CN1340704A (en) | 2002-03-20 |
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