CN112226483A - High-stability reduced nicotinamide coenzyme determination reagent and preparation method thereof - Google Patents
High-stability reduced nicotinamide coenzyme determination reagent and preparation method thereof Download PDFInfo
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- CN112226483A CN112226483A CN202011219979.3A CN202011219979A CN112226483A CN 112226483 A CN112226483 A CN 112226483A CN 202011219979 A CN202011219979 A CN 202011219979A CN 112226483 A CN112226483 A CN 112226483A
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- reagent
- enzyme
- buffer solution
- nicotinamide coenzyme
- reduced
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 125
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 title claims abstract description 78
- 239000005515 coenzyme Substances 0.000 title claims abstract description 44
- 230000002829 reductive effect Effects 0.000 title claims abstract description 42
- 229960003966 nicotinamide Drugs 0.000 title claims abstract description 39
- 235000005152 nicotinamide Nutrition 0.000 title claims abstract description 39
- 239000011570 nicotinamide Substances 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 102000004190 Enzymes Human genes 0.000 claims abstract description 41
- 108090000790 Enzymes Proteins 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 16
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- 230000008569 process Effects 0.000 claims abstract description 8
- 239000007790 solid phase Substances 0.000 claims abstract description 8
- 238000003860 storage Methods 0.000 claims abstract description 6
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000002994 raw material Substances 0.000 claims abstract description 5
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- 239000001263 FEMA 3042 Substances 0.000 claims abstract description 4
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 claims abstract description 4
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 claims abstract description 4
- 229940033123 tannic acid Drugs 0.000 claims abstract description 4
- 235000015523 tannic acid Nutrition 0.000 claims abstract description 4
- 229920002258 tannic acid Polymers 0.000 claims abstract description 4
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 64
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 claims description 32
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 32
- 239000000243 solution Substances 0.000 claims description 22
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 6
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- 230000003647 oxidation Effects 0.000 claims description 4
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- RAXXELZNTBOGNW-UHFFFAOYSA-N 1H-imidazole Chemical compound C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 3
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- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
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- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 15
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
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- 108020005199 Dehydrogenases Proteins 0.000 description 2
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- 238000002791 soaking Methods 0.000 description 2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01049—Glucose-6-phosphate dehydrogenase (1.1.1.49)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
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Abstract
The invention discloses a reduced nicotinamide coenzyme determination reagent with good stability, which comprises a medium immobilized enzyme, a curing medium and a nicotinamide coenzyme stabilization reagent, wherein the medium immobilized enzyme comprises a matrix, an immobilized coating and enzyme molecules; the raw materials of the fixed coating comprise tannic acid and 3-aminopropyl triethoxysilane, the reduced nicotinamide coenzyme determination reagent with good stability and the preparation method thereof can fix glucose oxidase on a solid phase carrier, and the activity of the enzyme is more stable; the defect that the product performance is reduced due to degradation of a liquid reagent by using low-concentration enzyme is overcome; because the enzyme is fixed on the side wall of the reagent bottle, the liquid level gradually descends along with the gradual consumption of the reagent in the use process, and the enzyme capable of acting is also gradually reduced in proportion, so that the consistent stability of the reagent in storage, transportation and use is ensured; the influence of the reverse reaction in the liquid reagent on the detection is overcome.
Description
Technical Field
The invention relates to the technical field of biology, and relates to a reduced nicotinamide coenzyme determination reagent and a preparation method thereof, in particular to a reduced nicotinamide coenzyme determination reagent with good stability and a preparation method thereof.
Background
The coenzymes NAD +/NADH and NADP +/NADPH are two very important participants in the methodology of diagnosing enzyme changes in vivo. NAD + is chemically named Nicotinamide adenine dinucleotide, also known as coenzyme I (Co I). NAD + is a coenzyme for many dehydrogenases in the body, which can take up 2H (2H + +2 e) detached from a metabolite and then transfer it to another hydrogen transferring body, flavoprotein. The pyridine nitrogen in the nicotinamide is pentavalent nitrogen and can reversibly accept electrons to form trivalent nitrogen. The carbon para to nitrogen can also be reversibly hydrodehydrogenated. In this reaction, the nicotinamide can accept a hydrogen atom and two electrons, and a proton remains in the mediator. NADP + chemically named Nicotinamide adenine dinucleotide phosphate, also known as coenzyme II (Co II). NADP + is also a coenzyme for many dehydrogenases in the body, and compared to NAD +, NADP + is structurally mainly distinguished by the replacement of the hydrogen at the 2' -hydroxyl position of its ribose by a phosphate group. NADP + functions essentially the same as NAD +.
NADH/NADPH has absorption peaks at 260nm and 340nm, while oxidized NAD +/NADP + has absorption peaks only at 260nm, so that the change of the absorption peaks at 340nm can be measured to observe the change of the amounts of NADH/NADPH and NAD +/NADP + in the reaction system. It is a common method in biochemical reagents.
For example, the concentration of ammonia in plasma is measured, ammonia reacts with α -ketoglutaric acid to form glutamate under the action of glutamate dehydrogenase, and simultaneously NADPH is oxidized to NADP +, the absorbance value of the reaction system at 340nm is decreased, and the decrease in absorbance at 340nm is measured. The ammonia concentration in the plasma can be calculated.
For example, the determination of the concentration of alanine Aminotransferase (ALT) in a serum sample, the ALT in the sample catalyzes the conversion of alanine amino group to alpha-oxoglutarate to generate pyruvic acid and L-lactic acid, and NADH is oxidized to NAD, and the absorbance value of the reaction system at 340nm is reduced. By measuring the decrease in absorbance at 340nm, the ALT concentration in the sample can be calculated.
In early clinical use, the reagent is prepared by each laboratory, so that the requirement on the stability of NADH or NADPH is not high, and the reagent can be prepared and used recently. With the popularization and application of commercial kits, the unstable influence of NADH or NADPH in the enzyme reagent becomes more obvious and becomes one of the key factors for restricting the reagent.
The adoption of the freeze-dried powder reagent to keep the stability of NADH or NADPH is feasible, but the production process has high requirements on the process, consumes a large amount of energy and increases the cost. And is inconvenient to use and needs to be redissolved before use.
Increasing the amount of NADH or NADPH used in the liquid reagent, even if they are slowly oxidatively degraded, ensures that the absorbance at 340nm of the reagent is greater than 1.200 when used. The increase of the amount of NADH or NADPH leads the detection sensitivity and stability of the reagent at different times of shelf life to be in an uncertain state, thereby influencing the stability and reliability of clinical detection results.
The addition of stabilizer to assist the stabilization of NADH or NADPH is another solution, for example, patent CN102863495 by adding trehalose and glycerol, etc., and patent CN108845140 by adding EGTA and alkyl glycoside (APG) to stabilize NADH or NADPH.
Joseph De Giorgio et al (see U.S. Pat. Nos. 5804402 and 5705356) invented a stable enzyme system that adds NADH or NADPH to the assay reagent, such as glucose-6-phosphate dehydrogenase and its specific substrate glucose, to slowly reduce NAD + or NADP + formed by the oxidation of NADH or NADPH to NADH or NADPH during reagent storage, thereby maintaining the concentration of nicotinamide coenzyme available in the assay reagent. Although the method can ensure the stability of NADH or NADPH, because the existence of glucose-6-phosphate dehydrogenase and glucose which is a specific substrate thereof in a reaction system has reverse reaction during detection reaction, the influence of a detected object is not great when the concentration is high, but the detection sensitivity of a reagent is influenced when the concentration is low, particularly near the detection limit, and the low value is caused.
Patent CN1548547 is to add oxidized coenzyme (NADP + or NAD +, etc.) and its slow reaction enzyme and substrate (such as glucose-6-phosphate dehydrogenase and its specific substrate glucose, etc.) during the production or preparation of reagent to form a system of slow reaction enzyme-substrate-NAD/NADP, etc. The content of the effective reduced nicotinamide coenzyme (NADH, thio-NAD, NADPH, thio-NADPH) or the like in the measurement reagent is generated by this system. The system continuously generates reduced nicotinamide coenzyme or analogues thereof required by determination, so that the stability of the reagent is improved. This system is similar to the above-mentioned system of Joseph De Giorgio et al, except that the reduced coenzyme for the initial reaction is changed to the oxidized coenzyme, which, in addition to the disadvantages of the above-mentioned system of Joseph De Giorgio et al, cannot be used immediately after the preparation of the reagent, and needs to be left for a certain period of time to allow the amount of reduced coenzyme to a certain extent to be used.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a reduced nicotinamide coenzyme determination reagent with good stability and a preparation method thereof. When the detection is carried out on the machine, the liquid reagent does not contain reverse reaction enzyme, so that the influence of the reverse reaction on the activity of low-value enzyme is eliminated. The method provided by the invention can effectively improve the stability and sensitivity of the reagent; the consistency among different batches of reagents and the consistency of different shelf life reagents are improved. The invention aims to overcome the defects of the prior art and provide a reduced nicotinamide coenzyme (NADPH or NADH) determination reagent with good stability and low cost and a preparation method thereof.
In order to achieve the purpose, the invention provides the following technical scheme: a reagent for determining reduced nicotinamide coenzyme with good stability comprises a medium immobilized enzyme, a solidified medium and a nicotinamide coenzyme stabilizing reagent, wherein the medium immobilized enzyme comprises a substrate, an immobilized coating and enzyme molecules; the raw materials of the fixed coating comprise tannic acid and 3-aminopropyl triethoxysilane.
Furthermore, the immobilization medium may be the inner wall of the reagent bottle, or a solid rod or ball added into the reagent bottle.
A preparation method of a reduced nicotinamide coenzyme determination reagent with good stability comprises the following steps: the nicotinamide coenzyme stabilizing reagent fixes an enzyme capable of producing reduced nicotinamide coenzyme, such as glucose-6-phosphate dehydrogenase, on a solid phase carrier, NADH/NADPH and a substrate of the enzyme are placed in a liquid reagent, and NAD + or NADP + generated by slow oxidation in the storage process of the reagent is continuously reduced to NADH or NADPH by the solid phase enzyme and the substrate thereof, thereby ensuring the stability of the reagent.
Furthermore, the reduced nicotinamide coenzyme determination reagent can be combined with a determination reagent in the preparation process, a buffer solution, a preservative, a heavy metal complexing agent, a surfactant and an enzyme stabilizer are added, the buffer solution comprises a Tris buffer solution, an imidazole buffer solution, a diethanol buffer solution, a phosphate buffer solution and a Good's buffer solution, the concentration of the buffer solution is generally selected within the range of 10-200mM, the adopted preservative is sodium azide, the heavy metal complexing agent comprises EDTA, EGTA and HEDTA, and the enzyme stabilizer adopts bovine serum albumin and mannitol.
Compared with the prior art, the invention has the beneficial effects that: the reduced nicotinamide coenzyme determination reagent with good stability and the preparation method thereof have the following advantages:
1. after the glucose oxidase is fixed on the solid phase carrier, the activity of the enzyme is more stable. The defect that the product performance is reduced due to degradation of a liquid reagent by using low-concentration enzyme is overcome;
2. because the enzyme is fixed on the side wall of the reagent bottle, the liquid level gradually descends along with the gradual consumption of the reagent in the use process, and the enzyme capable of acting is also gradually reduced in proportion, so that the consistent stability of the reagent in storage, transportation and use is ensured;
3. the influence of the reverse reaction in the liquid reagent on the detection is overcome. The enzyme is directly added into the reagent, although the quantity is small, the influence on most detection is not great, but when the content of the target enzyme in the detection sample is low, the influence cannot be ignored;
4. overcomes the defect that the reagent using oxidized nicotinamide coenzyme (NADPH or NADH) can not be used immediately. The reagent of the invention can be used immediately after being prepared because of using reduced nicotinamide coenzyme (NADPH or NADH);
5. the dosage (1/5-1/10 of a common reagent) of reduced nicotinamide coenzyme (NADPH or NADH) in the liquid double reagent/single reagent is effectively overcome, and the reagent cost is effectively reduced;
6. because the initial dosage of reduced nicotinamide coenzyme (NADPH or NADH) is reduced, the reactivity and the sensitivity of the reagent are greatly improved, and the linear range is enlarged.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to embodiments 1 to 3 of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: a reagent for determining reduced nicotinamide coenzyme with good stability comprises a medium immobilized enzyme, a solidified medium and a nicotinamide coenzyme stabilizing reagent, wherein the medium immobilized enzyme comprises a substrate, an immobilized coating and enzyme molecules; the raw materials of the fixed coating comprise tannic acid and 3-aminopropyltriethoxysilane, and the fixed medium can be the inner wall of a reagent bottle, and can also be a solid rod or a solid ball added into the reagent bottle.
A preparation method of a reduced nicotinamide coenzyme determination reagent with good stability comprises the following steps: the nicotinamide coenzyme stabilizing reagent fixes an enzyme capable of producing reduced nicotinamide coenzyme, such as glucose-6-phosphate dehydrogenase, on a solid phase carrier, NADH/NADPH and a substrate of the enzyme are placed in a liquid reagent, and NAD + or NADP + generated by slow oxidation in the storage process of the reagent is continuously reduced to NADH or NADPH by the solid phase enzyme and the substrate thereof, thereby ensuring the stability of the reagent; the reduced nicotinamide coenzyme determination reagent can be combined with a determination reagent in the preparation process, a buffer solution, a preservative, a heavy metal complexing agent, a surfactant and an enzyme stabilizer are added, the buffer solution comprises a Tris buffer solution, an imidazole buffer solution, a diethanol buffer solution, a phosphate buffer solution and a Good's buffer solution, the concentration of the buffer solution is generally selected within the range of 10-200mM, the adopted preservative is sodium azide, the heavy metal complexing agent comprises EDTA, EGTA and HEDTA, and the enzyme stabilizer adopts bovine serum albumin and mannitol.
Example 1:
preparing an alanine aminotransferase reagent, which consists of the following two parts: s11 reagent I treatment reagent bottle and S12 reagent II treatment reagent bottle.
S11: reagent I treatment reagent bottle: dissolving 3-aminopropyltriethoxysilane in absolute ethanol to obtain a 10 g/L3-aminopropyltriethoxysilane solution; adding the mixture into a reagent bottle, carrying out ultrasonic reaction for 1 hour, washing for 2 times by using absolute ethyl alcohol, drying, adding a buffer solution containing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride with the concentration of 0.05mol/L, N-hydroxysuccinimide with the concentration of 0.1mol/L and 2- (N-morpholine) ethanesulfonic acid with the concentration of 0.05mol/L and the pH of 5.0, mixing for 30 minutes, discarding the liquid, washing once by using a buffer solution containing 2- (N-morpholine) ethanesulfonic acid with the concentration of 0.05mol/L and the pH of 5.0, adding 1ml of 2- (N-morpholine) ethanesulfonic acid with the concentration of 0.05mol/L and the pH of 5.0, adding 5mg of glucose-6-phosphate dehydrogenase, ultrasonically mixing for 1 hour, and discarding the liquid; washing with PBS twice; the reagent bottle is filled with a liquid reagent I, and the liquid reagent I comprises the following components in parts by weight:
100mmol/L Tris buffer pH 7.20
500mmol/L L alanine
0.18mmol/L NADH
1700U/L LDH
0.1mmol/L pyridoxal phosphate
6.0mmol/L EDTANa2
5.0mmol/L dipotassium hydrogen phosphate
80mmol/L D-glucose
1.5mmol/L sodium azide
The solvent is purified water.
S12: reagent II handles reagent bottle, and reagent II's composition and corresponding content are:
15mmol/L alpha-ketoglutaric acid
According to the method for preparing the reagent solution of S11 and S12, after the alanine aminotransferase is prepared, when the blank absorbance of the reagent is greater than 1.20, 340nm and the optical path of 10cm is used for measuring the sample, a speed method is adopted, 100 microliters of the sample is added, 1000 microliters of the reagent I is added, the temperature is 37 ℃ for warm bath for 300 seconds, then 100 microliters of the reagent II is added, the mixture is uniformly mixed, the ALT catalytic reaction is started, the delay time is 30 seconds at the wavelength of 340nm, the absorbance decrease rate is continuously measured for 180 seconds, and the reading frequency is greater than 6 points; ALT viability was calculated from- Δ A/Min in the linear phase.
Example 2:
preparing an ammonia reagent: comprises the steps of S21 solid enzyme carrier-plastic tube treatment, S22 enzyme fixation and S23 ammonia reagent preparation;
s21: three plastic tubes with the inner diameter of 0.5cm and the length of 7cm are immersed into 10g/L of 3-aminopropyltriethoxysilane ethanol solution, ultrasonic mixing is carried out for 30 minutes at room temperature, the plastic tubes are immersed into the solution completely, air bubbles are removed, then the plastic tubes are taken out, washed twice by absolute ethyl alcohol and dried.
S22: mixing 0.05 mol/L1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 0.05 mol/L2- (N-morpholine) ethanesulfonic acid buffer solution with pH of 5.0 and 500ml of each buffer solution, wetting the plastic tube treated in the step S21, taking out the plastic tube, adding 50mg of glucose-6-phosphate dehydrogenase into the mixed solution, quickly mixing uniformly, adding the mixture into the plastic tube again, and performing ultrasonic bonding for 30 minutes; then the plastic tube is taken out and washed twice by 2- (N-morpholine) ethanesulfonic acid buffer solution with the concentration of 0.05mol/L and the pH value of 5.0, and then the plastic tube is dried for standby.
Preparation of S23 Ammonia reagent: taking 100mM Tris buffer solution, taking 15mM alpha-ketoglutaric acid with the pH of 8.0, taking 10 mM bovine serum albumin solution with the concentration of 5g/L, taking 3mM adenosine diphosphate sylvite, taking 5000U/L glutamate dehydrogenase, 0.20mM NADPH, 50mM D-glucose, KH2PO 45 mM, EDTANa 25 mM and 4.0mM sodium azide for mixing; and putting one plastic tube labeled with glucose-6-phosphate dehydrogenase into the mixed reagent in a reagent bottle, and storing at 2-8 ℃ for one year without degrading NADPH.
Example 3:
preparing a CO2 reagent, comprising the following two steps: s31 solid enzyme carrier- -treatment of plastic ball, S32 enzyme immobilization and preparation of S33 CO2 reagent;
s31: solid enzyme carrier-treatment of plastic balls: soaking four plastic balls with the diameter of 1.0cm into 10 g/L3-aminopropyl triethoxysilane ethanol solution, ultrasonically mixing at room temperature for 30 minutes, soaking all the plastic balls into the solution, continuously turning over in the combination process, taking out the plastic balls, cleaning twice with absolute ethyl alcohol, and drying.
S32: enzyme immobilization, namely mixing 500ml of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride with the concentration of 0.05mol/L and 500ml of 2- (N-morpholine) ethanesulfonic acid buffer solution with the concentration of 0.05mol/L and the pH value of 5.0, wetting the plastic balls in the step S41 with the mixed solution, taking out the plastic balls, adding 50mg of glucose-6-phosphate dehydrogenase into the mixed solution, quickly mixing the mixture uniformly, adding the mixture into the plastic balls again, ultrasonically bonding the mixture for 30 minutes, enabling the plastic balls to completely invade the solution, and continuously overturning the plastic balls in the bonding process; the plastic balls are taken out and washed twice by 2- (N-morpholine) ethanesulfonic acid buffer solution with the concentration of 0.05mol/L and the pH value of 5.0, and then are dried for standby.
S33: preparation of CO2 reagent: mixing 50mM Tris buffer solution, 2.0mM phosphoenolpyruvate with the pH of 8.0, 600U/L phosphoenolpyruvate carboxylase, 1500U/L malate dehydrogenase, 2g/L bovine serum albumin, 48 mM MgSO, 2.5mM sodium oxamate, 0.18mM NADH, 50mM D-glucose, KH2PO 45 mM and 4mM sodium azide together; 6 plastic balls marked with glucose-6-phosphate dehydrogenase are put into the reagent bottle after the reagent is prepared, and the reagent bottle is stored for one year at the temperature of 2-8 ℃, and NADH is not degraded.
Because CO2 widely exists in the environment, the CO2 reagent is prepared by boiling and cooling fresh deionized water, and the prepared reagent is completely sealed and stored.
The invention aims to overcome the defects of the prior art and provide a reduced nicotinamide coenzyme (NADPH or NADH) determination reagent with good stability and low cost and a preparation method thereof, and the invention also discloses a preparation and production method of the saved and improved determination reagent, because the amount of the NADPH or the NADH used in the preparation of the reagent is greatly reduced, the raw material cost of the reagent is greatly reduced, and the invention also discloses an improved determination reagent which comprises ALT, AST, CO2 reagents and the like.
After the reagents of the above examples 1-3 are prepared, the application method is the same as the application method of the common reagents in the related field, and the details are not repeated herein.
The above formulation of the reagents of examples 1-3 are merely illustrative of the principles and applications of the present invention and are not intended to be limiting. The scope of the above examples; in addition, although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. A good-stability reduced nicotinamide coenzyme determination reagent is characterized in that: comprises a medium immobilized enzyme, a immobilized medium and a nicotinamide coenzyme stabilizing reagent, wherein the medium immobilized enzyme comprises a substrate, an immobilized coating and enzyme molecules; the raw materials of the fixed coating comprise tannic acid and 3-aminopropyl triethoxysilane.
2. The reagent of claim 1, wherein the reagent is characterized by comprising: the immobilized medium can be the inner wall of the reagent bottle, and can also be a solid rod or ball added into the reagent bottle.
3. A preparation method of a reduced nicotinamide coenzyme determination reagent with good stability is characterized by comprising the following steps: the method comprises the following steps: the nicotinamide coenzyme stabilizing reagent fixes an enzyme capable of producing reduced nicotinamide coenzyme, such as glucose-6-phosphate dehydrogenase, on a solid phase carrier, NADH/NADPH and a substrate of the enzyme are placed in a liquid reagent, and NAD + or NADP + generated by slow oxidation in the storage process of the reagent is continuously reduced to NADH or NADPH by the solid phase enzyme and the substrate thereof, thereby ensuring the stability of the reagent.
4. The reagent for assaying reduced nicotinamide coenzyme according to claim 3, which has good stability, and the preparation method thereof are characterized in that: the reduced nicotinamide coenzyme determination reagent can be combined with a determination reagent in the preparation process, a buffer solution, a preservative, a heavy metal complexing agent, a surfactant and an enzyme stabilizer are added, the buffer solution comprises a Tris buffer solution, an imidazole buffer solution, a diethanol buffer solution, a phosphate buffer solution and a Good's buffer solution, the concentration of the buffer solution is generally selected within the range of 10-200mM, the adopted preservative is sodium azide, the heavy metal complexing agent comprises EDTA, EGTA and HEDTA, and the enzyme stabilizer adopts bovine serum albumin and mannitol.
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