CN114235790A - Preparation method of test paper for detecting content of lactic acid in body fluid - Google Patents
Preparation method of test paper for detecting content of lactic acid in body fluid Download PDFInfo
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- CN114235790A CN114235790A CN202111434825.0A CN202111434825A CN114235790A CN 114235790 A CN114235790 A CN 114235790A CN 202111434825 A CN202111434825 A CN 202111434825A CN 114235790 A CN114235790 A CN 114235790A
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- lactic acid
- test paper
- solution
- preparing
- body fluid
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 239000004310 lactic acid Substances 0.000 title claims abstract description 30
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 30
- 210000001124 body fluid Anatomy 0.000 title claims abstract description 11
- 239000010839 body fluid Substances 0.000 title claims abstract description 11
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 239000000243 solution Substances 0.000 claims abstract description 41
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 18
- 238000001035 drying Methods 0.000 claims abstract description 15
- 239000006173 Good's buffer Substances 0.000 claims abstract description 14
- 239000000758 substrate Substances 0.000 claims abstract description 14
- 108010073450 Lactate 2-monooxygenase Proteins 0.000 claims abstract description 12
- 239000004033 plastic Substances 0.000 claims abstract description 12
- 229920003023 plastic Polymers 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 10
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 9
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 9
- 238000002791 soaking Methods 0.000 claims abstract description 9
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 claims abstract description 8
- IRQRBVOQGUPTLG-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 IRQRBVOQGUPTLG-UHFFFAOYSA-M 0.000 claims abstract description 8
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229910052782 aluminium Inorganic materials 0.000 claims abstract description 6
- 238000005520 cutting process Methods 0.000 claims abstract description 6
- 239000011888 foil Substances 0.000 claims abstract description 6
- 239000008213 purified water Substances 0.000 claims abstract description 6
- 238000003756 stirring Methods 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000000203 mixture Substances 0.000 claims abstract description 5
- 239000002904 solvent Substances 0.000 claims abstract description 5
- 239000003381 stabilizer Substances 0.000 claims abstract description 5
- 229920000742 Cotton Polymers 0.000 claims abstract description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 7
- 239000004353 Polyethylene glycol 8000 Substances 0.000 claims description 7
- 229940098773 bovine serum albumin Drugs 0.000 claims description 7
- 229940085678 polyethylene glycol 8000 Drugs 0.000 claims description 7
- 235000019446 polyethylene glycol 8000 Nutrition 0.000 claims description 7
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims description 5
- 125000000979 2-amino-2-oxoethyl group Chemical group [H]C([*])([H])C(=O)N([H])[H] 0.000 claims description 5
- NUFBIAUZAMHTSP-UHFFFAOYSA-N 3-(n-morpholino)-2-hydroxypropanesulfonic acid Chemical compound OS(=O)(=O)CC(O)CN1CCOCC1 NUFBIAUZAMHTSP-UHFFFAOYSA-N 0.000 claims description 5
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 claims description 5
- CCIVGXIOQKPBKL-UHFFFAOYSA-N ethanesulfonic acid Chemical compound CCS(O)(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-N 0.000 claims description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 5
- 239000003223 protective agent Substances 0.000 abstract description 6
- 102000004190 Enzymes Human genes 0.000 abstract description 4
- 108090000790 Enzymes Proteins 0.000 abstract description 4
- 238000006243 chemical reaction Methods 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 230000009471 action Effects 0.000 abstract description 2
- 238000007598 dipping method Methods 0.000 abstract description 2
- 210000001215 vagina Anatomy 0.000 description 7
- 241000186660 Lactobacillus Species 0.000 description 6
- 206010046914 Vaginal infection Diseases 0.000 description 6
- 201000008100 Vaginitis Diseases 0.000 description 6
- 238000007705 chemical test Methods 0.000 description 4
- 239000002274 desiccant Substances 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 229930182830 galactose Natural products 0.000 description 3
- 229940039696 lactobacillus Drugs 0.000 description 3
- 239000002808 molecular sieve Substances 0.000 description 3
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 3
- 210000002700 urine Anatomy 0.000 description 3
- HFACYLZERDEVSX-UHFFFAOYSA-N benzidine Chemical group C1=CC(N)=CC=C1C1=CC=C(N)C=C1 HFACYLZERDEVSX-UHFFFAOYSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 208000036649 Dysbacteriosis Diseases 0.000 description 1
- 208000027244 Dysbiosis Diseases 0.000 description 1
- 241000194033 Enterococcus Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000207202 Gardnerella Species 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 208000007313 Reproductive Tract Infections Diseases 0.000 description 1
- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 206010000210 abortion Diseases 0.000 description 1
- 231100000176 abortion Toxicity 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical group SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 230000007140 dysbiosis Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
Abstract
The invention relates to a preparation method of test paper for detecting the content of lactic acid in body fluid, which comprises the following steps: preparing GOODS buffer solution: adding the solvent into purified water, and adjusting the pH value to 6.0-7.0 by using a sodium hydroxide solution; preparing a lactic acid test paper treatment solution: measuring GOODS buffer solution, sequentially adding a stabilizer, CHAPS, TOOS, lactate oxidase and peroxidase, and stirring until the mixture is completely dissolved to obtain a lactic acid test paper treatment solution; using filter paper or cotton fiber as a reagent carrier, soaking the lactic acid test paper treatment solution, drying, cutting into small blocks with certain size, adhering the small blocks on a plastic substrate, and placing the small blocks into a sealed plastic bottle or an aluminum foil bag to obtain the reagent. The invention utilizes lactate oxidase and peroxidase, tests the content of lactic acid through a series of reaction coloration, uses a novel protective agent in the formula, uses substrates CHAPS and TOOS under the action of the protective agent, and the two substrates stably exist in solution, thereby realizing the preparation and manufacturing one-step method without preparing enzyme solution and substrate solution twice and dipping and drying.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnosis test paper, and particularly relates to a preparation method of test paper for detecting the content of lactic acid in body fluid.
Background
Lower genital tract infection belongs to a common gynecological disease, is easy to cause abortion, infertility, cervical erosion and even cervical cancer, and is an important public health problem faced by developing China.
Vaginitis is mainly influenced by vaginal dysbacteriosis caused by various reasons and vaginal microecological balance destruction. Clinical cases in recent years have shown that common vaginitis in women is mostly caused by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, Salmonella typhi and enterococcus. The lactobacillus is a normal strain and a dominant flora in the vagina of a healthy woman, is a micro-aerobic flora, forms micro-ecological balance with the vaginal flora and restricts the growth of other bacteria. The metabolite (lactic acid) of the lactobacillus can reduce the pH value of the vagina and maintain the ecological balance of the vagina, and the lactobacillus vaginal cleanser has no toxicity or side effect and plays an important role in keeping the vagina clean and the like. Researches show that the number of the lactobacilli of patients with various vaginitis is obviously reduced, the protective effects of substitution, rejection, competition and the like of the lactobacilli are reduced or eliminated, mycoplasma, fungi, gardnerella or other external pathogenic microorganisms parasitizing in the vagina multiply in the vagina to cause the attack of various vaginitis, and the reduction of the number of the lactobacilli in the vagina plays a very key role in the pathogenesis process of various vaginitis.
The invention patent with the patent number of CN201811207828.9 provides a dry chemical test paper for lactic acid detection and a preparation method thereof, belonging to the technical field of in vitro diagnosis test paper. Solves the problem of how to provide a simple, quick, safe and effective lactic acid detection dry chemical test paper with high sensitivity and specificity for diagnosing vaginitis and a preparation method thereof. The dry chemical test paper for detecting lactic acid is filter paper dispersed with a substrate, an enzyme, a chelating agent, a protective agent and a buffer; wherein the substrate is benzidine or benzidine derivative; the enzyme is lactate oxidase and peroxidase. The dry chemical test paper can be used for measuring the content of the lactic acid in one step, and is simple, quick, safe, effective, high in sensitivity and good in specificity.
However, the product in the patent is prepared by using A, B liquid twice, the process is complicated, the labor cost is increased, and the probability of error of the product is increased by one more manufacturing process.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a preparation method of test paper for detecting the content of lactic acid in body fluid, which comprises the following steps in sequence:
(1) preparing GOODS buffer solution: adding the solvent into purified water, and adjusting the pH value to 6.0-7.0 by using a sodium hydroxide solution;
(2) preparing a lactic acid test paper treatment solution: measuring GOODS buffer solution, sequentially adding a stabilizer, CHAPS, TOOS, lactate oxidase and peroxidase, and stirring until the mixture is completely dissolved to obtain a lactic acid test paper treatment solution;
(3) using filter paper or cotton fiber as a reagent carrier, soaking the lactic acid test paper treatment solution, drying, cutting into small blocks with certain size, adhering the small blocks on a plastic substrate, and placing the small blocks into a sealed plastic bottle or an aluminum foil bag to obtain the reagent.
Preferably, the solvent in step (1) is one of 2- (N-morpholino) ethanesulfonic acid, 2[ (2-amino-2-oxoethyl) amino ] ethanesulfonic acid, piperacha-N, N-bis (2-ethanesulfonic acid) or 3- (N-morpholino) -2-hydroxypropanesulfonic acid.
In any of the above embodiments, preferably, the stabilizer is: bovine serum albumin and polyethylene glycol 8000.
In any of the above embodiments, the concentration of GOODS buffer is preferably between 0.1M and 1M, and the pH is preferably in the range of 6.0 to 7.0.
In any of the above embodiments, preferably, the concentration of bovine serum albumin is 0.1% to 1%, and the concentration of polyethylene glycol 8000 is 0.05% to 0.5%.
In any of the above schemes, preferably, in step (3), the drying conditions are: drying the mixture for 10 to 20 minutes in an oven at the temperature of between 60 and 80 ℃.
In any of the above embodiments, it is preferable that the protecting agent is dithiothreitol (DDT), and the terminal sulfur atom of lactate oxidase tends to form a dimer in the solution, and the dimer largely reduces the efficiency of the coupling reaction, and the addition of DDT in the solution of lactate oxidase can effectively reduce dimerization, thereby improving the reaction efficiency of lactate oxidase.
The invention utilizes lactate oxidase and peroxidase, tests the content of lactic acid through a series of reaction coloration, uses a novel protective agent in the formula, uses substrates CHAPS and TOOS under the action of the protective agent, and the two substrates stably exist in solution, thereby realizing the preparation and manufacturing one-step method without preparing enzyme solution and substrate solution twice and dipping and drying.
Detailed Description
In order that the invention may be further understood, the invention will now be described in detail with reference to specific examples.
Example one
1. Preparing GOODS buffer solution; adding one reagent of 2- (N-morpholino) ethanesulfonic acid, 2[ (2-amino-2 oxoethyl) amino ] ethanesulfonic acid, piperacha-N, N-di (2-ethanesulfonic acid), 3- (N-morpholino) -2-hydroxypropanesulfonic acid and the like serving as a solute into purified water, and adjusting the pH to 6.0-7.0 by using a sodium hydroxide solution with a corresponding concentration;
2. preparing a lactic acid test paper treatment solution: accurately measuring GOODS buffer solution, sequentially adding bovine serum albumin, polyethylene glycol 8000, CHAPS, TOOS, lactate oxidase, peroxidase and the like, and stirring for complete dissolution;
3. soaking and drying: soaking 3mm filter paper of GE company in the solution for 10-30 seconds, drying the filter paper for 10-20 minutes at the temperature of 60-80 ℃, and taking out the dried filter paper;
4. product assembly: cutting the dried test paper into small blocks with certain size, and sticking the small blocks on a plastic substrate. Putting into sealed plastic bottle or aluminum foil bag, using 13X type molecular sieve as desiccant, and storing at room temperature for 18 months.
Example two
1. Preparing GOODS buffer solution; adding one reagent of 2- (N-morpholino) ethanesulfonic acid, 2[ (2-amino-2 oxoethyl) amino ] ethanesulfonic acid, piperacha-N, N-di (2-ethanesulfonic acid), 3- (N-morpholino) -2-hydroxypropanesulfonic acid and the like serving as a solute into purified water, and adjusting the pH to 6.0-7.0 by using a sodium hydroxide solution with a corresponding concentration;
2. preparing a lactic acid test paper treatment solution: accurately measuring GOODS buffer solution, sequentially adding bovine serum albumin, polyethylene glycol 8000, CHAPS, TOOS, lactate oxidase, peroxidase and the like, and stirring for complete dissolution;
3. soaking and drying: soaking 3mm filter paper of GE company in the solution for 10-30 seconds, drying the filter paper for 10-20 minutes at the temperature of 60-80 ℃, and taking out the dried filter paper;
4. product assembly: cutting the dried test paper into small blocks with certain size, and sticking the small blocks on a plastic substrate. Putting into sealed plastic bottle or aluminum foil bag, using 13X type molecular sieve as desiccant, and storing at room temperature for 18 months.
EXAMPLE III
1. Preparing GOODS buffer solution; adding one reagent of 2- (N-morpholino) ethanesulfonic acid, 2[ (2-amino-2 oxoethyl) amino ] ethanesulfonic acid, piperacha-N, N-di (2-ethanesulfonic acid), 3- (N-morpholino) -2-hydroxypropanesulfonic acid and the like serving as a solute into purified water, and adjusting the pH to 6.0-7.0 by using a sodium hydroxide solution with a corresponding concentration;
2. preparing a lactic acid test paper treatment solution: accurately measuring GOODS buffer solution, sequentially adding bovine serum albumin, polyethylene glycol 8000, CHAPS, TOOS, lactate oxidase, peroxidase and the like, and stirring for complete dissolution;
3. soaking and drying: soaking 3mm filter paper of GE company in the solution for 10-30 seconds, drying the filter paper for 10-20 minutes at the temperature of 60-80 ℃, and taking out the dried filter paper;
4. product assembly: cutting the dried test paper into small blocks with certain size, and sticking the small blocks on a plastic substrate. Putting into sealed plastic bottle or aluminum foil bag, using 13X type molecular sieve as desiccant, and storing at room temperature for 18 months.
1. Critical value: urine galactose concentration is 1mmol/L, urine galactose concentration is positive when being less than 1mmol/L, and urine galactose concentration is negative when being more than or equal to 1 mmol/L.
2. Establishing a positive judgment value: the positive judgment value of the product is verified by 180 normal people.
It will be understood by those skilled in the art that the method for preparing the test strip for measuring the lactic acid content in a body fluid according to the present invention includes any combination of the above-mentioned summary of the present invention and the portions shown in the detailed description, which is not described in detail and is not intended to simplify the description. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A preparation method of test paper for detecting the content of lactic acid in body fluid comprises the following steps in sequence:
(1) preparing GOODS buffer solution: adding the solvent into purified water, and adjusting the pH value to 6.0-7.0 by using a sodium hydroxide solution;
(2) preparing a lactic acid test paper treatment solution: measuring GOODS buffer solution, sequentially adding a stabilizer, CHAPS, TOOS, lactate oxidase and peroxidase, and stirring until the mixture is completely dissolved to obtain a lactic acid test paper treatment solution;
(3) using filter paper or cotton fiber as a reagent carrier, soaking the lactic acid test paper treatment solution, drying, cutting into small blocks with certain size, adhering the small blocks on a plastic substrate, and placing the small blocks into a sealed plastic bottle or an aluminum foil bag to obtain the reagent.
2. The method of claim 1, wherein the solvent used in step (1) is one of 2- (N-morpholino) ethanesulfonic acid, 2[ (2-amino-2 oxoethyl) amino ] ethanesulfonic acid, piperacha-N, N-bis (2-ethanesulfonic acid), and 3- (N-morpholino) -2-hydroxypropanesulfonic acid.
3. The method for preparing a test paper for detecting the content of lactic acid in body fluid according to claim 1, wherein the stabilizer is: bovine serum albumin and polyethylene glycol 8000.
4. The method for preparing test paper for detecting the content of lactic acid in body fluid as claimed in claim 1, wherein the concentration of GOODS buffer is between 0.1M and 1M, and the pH range is 6.0-7.0.
5. The method for preparing test paper for detecting the content of lactic acid in body fluid according to claim 3, wherein the concentration of bovine serum albumin is 0.1% -1%, and the concentration of polyethylene glycol 8000 is 0.05% -0.5%.
6. The method for preparing a test paper for detecting the content of lactic acid in body fluid according to claim 1, wherein in the step (3), the drying conditions are as follows: drying the mixture for 10 to 20 minutes in an oven at the temperature of between 60 and 80 ℃.
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| CN202111434825.0A CN114235790A (en) | 2021-11-29 | 2021-11-29 | Preparation method of test paper for detecting content of lactic acid in body fluid |
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| CN202111434825.0A CN114235790A (en) | 2021-11-29 | 2021-11-29 | Preparation method of test paper for detecting content of lactic acid in body fluid |
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| CN202111434825.0A Pending CN114235790A (en) | 2021-11-29 | 2021-11-29 | Preparation method of test paper for detecting content of lactic acid in body fluid |
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