CN115976157A - High-throughput screening method of cyclodextrin transferase for catalytic synthesis of L-ascorbic acid-2-glucoside - Google Patents
High-throughput screening method of cyclodextrin transferase for catalytic synthesis of L-ascorbic acid-2-glucoside Download PDFInfo
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- CN115976157A CN115976157A CN202211723747.0A CN202211723747A CN115976157A CN 115976157 A CN115976157 A CN 115976157A CN 202211723747 A CN202211723747 A CN 202211723747A CN 115976157 A CN115976157 A CN 115976157A
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- ascorbic acid
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- MLSJBGYKDYSOAE-DCWMUDTNSA-N L-Ascorbic acid-2-glucoside Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)=C1O MLSJBGYKDYSOAE-DCWMUDTNSA-N 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 24
- 229920000858 Cyclodextrin Polymers 0.000 title claims abstract description 19
- 238000013537 high throughput screening Methods 0.000 title claims abstract description 12
- 108090000992 Transferases Proteins 0.000 title claims abstract description 10
- 102000004357 Transferases Human genes 0.000 title claims abstract description 10
- 238000007036 catalytic synthesis reaction Methods 0.000 title description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 26
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- 229960005070 ascorbic acid Drugs 0.000 claims abstract description 13
- 235000010323 ascorbic acid Nutrition 0.000 claims abstract description 12
- 239000011668 ascorbic acid Substances 0.000 claims abstract description 12
- 238000011161 development Methods 0.000 claims abstract description 9
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 9
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 8
- 239000007800 oxidant agent Substances 0.000 claims abstract description 8
- 239000001116 FEMA 4028 Substances 0.000 claims abstract description 7
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims abstract description 7
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims abstract description 7
- 229960004853 betadex Drugs 0.000 claims abstract description 7
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 6
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 4
- 230000001590 oxidative effect Effects 0.000 claims abstract description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical group OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 14
- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical group O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 claims description 14
- 238000002835 absorbance Methods 0.000 claims description 9
- 238000012216 screening Methods 0.000 claims description 6
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 239000012295 chemical reaction liquid Substances 0.000 abstract description 2
- 238000002372 labelling Methods 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 230000003647 oxidation Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 9
- 238000010438 heat treatment Methods 0.000 description 7
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 6
- 229960001031 glucose Drugs 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 238000004811 liquid chromatography Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000012452 mother liquor Substances 0.000 description 4
- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 108010025880 Cyclomaltodextrin glucanotransferase Proteins 0.000 description 2
- STZCRXQWRGQSJD-GEEYTBSJSA-M methyl orange Chemical compound [Na+].C1=CC(N(C)C)=CC=C1\N=N\C1=CC=C(S([O-])(=O)=O)C=C1 STZCRXQWRGQSJD-GEEYTBSJSA-M 0.000 description 2
- 229940012189 methyl orange Drugs 0.000 description 2
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 239000002211 L-ascorbic acid Substances 0.000 description 1
- 235000000069 L-ascorbic acid Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- DDXLVDQZPFLQMZ-UHFFFAOYSA-M dodecyl(trimethyl)azanium;chloride Chemical compound [Cl-].CCCCCCCCCCCC[N+](C)(C)C DDXLVDQZPFLQMZ-UHFFFAOYSA-M 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002761 liquid phase assay Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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Abstract
The invention relates to a high-throughput screening method of cyclodextrin transferase for catalyzing and synthesizing L-ascorbic acid-2-glucoside, which comprises the following steps: adding beta-cyclodextrin and ascorbic acid into a culture system of a strain to be screened, and adjusting the pH value to 5.0-6.0 to perform catalytic reaction; removing the strain after the catalytic reaction, and adding an oxidant to carry out an oxidation reaction; after the oxidation reaction, a color-developing agent of L-ascorbic acid-2-glucoside is added for color development. The method removes the unreacted ascorbic acid through a pre-oxidation strategy, adds a substance capable of developing color with the L-ascorbic acid-2-glucoside, reacts under a certain condition, and can conveniently detect the L-ascorbic acid-2-glucoside in an enzyme-labeling instrument after the reaction, thereby accurately quantifying the content of the L-ascorbic acid-2-glucoside in a reaction liquid system and bringing convenience for high-throughput screening of mutant strains.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a high-throughput screening method of cyclodextrin transferase for catalyzing and synthesizing L-ascorbic acid-2-glucoside.
Background
At present, in the synthesis of L-ascorbic acid-2-glucoside (AA-2G), the world leads the Japanese enterprise "forest resource", and the enzyme used is CGTase, namely "cyclodextrin transferase", which is industrially used for the synthesis of beta-cyclodextrin on a large scale. In order to increase the efficiency of catalytic synthesis of AA-2G, it is necessary to screen cyclodextrin transferases with higher activity. For screening of the enzyme, there are generally used a "phenolphthalein color change circle method" and a "methyl orange method". The method is based on the principle that CGTase enzyme is generated by bacteria to convert starch into cyclodextrin, and the cyclodextrin is used for wrapping phenolphthalein or methyl orange to discolor the solution, so that the aim of evaluating the enzyme activity is fulfilled. It is clear that this approach does not directly evaluate the activity of AA-2G synthesis and does not truly reflect the level of AA-2G synthesis using this enzyme.
Therefore, how to obtain a method for directly evaluating the activity of catalyzing and synthesizing AA-2G by cyclodextrin transferase, and apply the method to high-throughput screening to quickly and accurately obtain beneficial mutant strains from a saturated mutant library is a technical problem to be solved by the technical personnel in the field.
Disclosure of Invention
The invention aims to provide a high-throughput screening method based on direct evaluation of the activity of synthesizing L-ascorbic acid-2-glucoside by catalysis of cyclodextrin transferase. In order to realize the purpose of the invention, the following technical scheme is adopted:
the invention relates to a high-throughput screening method of cyclodextrin transferase for catalyzing and synthesizing L-ascorbic acid-2-glucoside, which comprises the following steps:
adding beta-cyclodextrin and ascorbic acid into a culture system of a strain to be screened, and adjusting the pH value to 5.0-6.0 to perform catalytic reaction;
removing the strain after the catalytic reaction, and adding an oxidant to carry out an oxidation reaction;
after the oxidation reaction, a color-developing agent of L-ascorbic acid-2-glucoside is added for color development.
According to the invention, the unreacted ascorbic acid is oxidized through an oxidation reaction, the L-ascorbic acid-2-glucoside is not damaged in the process, and the content of the L-ascorbic acid-2-glucoside can be quantitatively detected through color development of the color developing agent of the L-ascorbic acid-2-glucoside.
In a preferred embodiment of the present invention, the beta-cyclodextrin and ascorbic acid are added in a weight ratio of 1:1-2.
In a preferred embodiment of the present invention, the oxidizing agent is hydrogen peroxide, ozone, or Fe 3+ A combination of salt and one or more of air or oxygen; preferably, the oxidizing agent is hydrogen peroxide, and hydrogen peroxide is used as the oxidizing agent, and the redox product of the hydrogen peroxide has less influence on the reaction system.
In a preferred embodiment of the present invention, the color developer is phosphomolybdic acid, and is heated to 70 to 90 ℃ to develop color. AA-2G is decomposed into ascorbic acid and glucose in the color development process, so that phosphomolybdic acid is blue-green in color, and the system does not have any ascorbic acid in a free state, so that the color development process is not influenced; moreover, the glucose per se does not interfere with the reaction system through verification.
In a preferred embodiment of the invention, after the color reaction, the absorbance is detected by a microplate reader, and the content of the L-ascorbic acid-2-glucoside is quantitatively determined by the absorbance data.
The invention removes the unreacted ascorbic acid by a preoxidation strategy, adds a substance capable of developing color with AA-2G, reacts under a certain condition, and can conveniently detect in an enzyme-linked immunosorbent assay after the reaction, thereby accurately quantifying the content of AA-2G in a reaction liquid system and bringing convenience for high-throughput screening of mutant strains.
Drawings
FIG. 1 is a 0 hour sample spectrum;
FIG. 2 is a sample spectrum after 2.0 hours of reaction;
FIG. 3 is a spectrum of a sample after 7.0 hours of reaction;
FIG. 4 is a spectrum of a sample after 24.0 hours of reaction;
FIG. 5 is a photograph of the solution before heating;
FIG. 6 is a photograph of the solution after heating;
FIG. 7 shows the relationship between the AA-2G consumption and the absorbance;
FIG. 8 is a photograph of a reaction mixture before heating;
FIG. 9 is a photograph after the reaction was heated at 80 ℃.
Detailed Description
The present invention is further illustrated by the following examples which are intended to be purely exemplary of the invention and are not intended to be limiting, and therefore any changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Example 1
(1) And (3) catalytic reaction: after culturing escherichia coli in a 96-well plate to OD =7, 50 μ L of β -cyclodextrin (50 g/L) and 50 μ L of ascorbic acid (50 g/L) were added thereto, and pH =5.0 to 5.5 was adjusted with 0.1M sodium hydroxide, incubated at 37 ℃, and reacted with shaking at 200rpm for 24 hours.
(2) Pre-oxidation reaction: after the thalli are removed by centrifugation, 10 microliters of 30% hydrogen peroxide is added for preoxidation reaction, the temperature is raised to 50 ℃ for continuous reaction for 24 hours, and the experimental result is shown in figures 1-4, wherein the L-ascorbic acid-2-glucoside in the system is almost kept unchanged along with the prolonging of time, the ascorbic acid is gradually reduced, and the L-ascorbic acid hardly disappears in the sample after 24 hours.
(3) And (3) color development reaction: 80 microliters of 47.62mg/g phosphomolybdic acid is added, and the temperature is kept at 80 ℃ for reaction for 1 hour. Transferring to a microplate reader, and detecting the absorbance at 660 nm. The greater the absorbance at this wavelength, the more AA-2G the system generates. The sample after the color reaction is also subjected to liquid chromatography detection, the experimental result is shown in table 1, and the experimental result shows that the method can realize the detection result similar to the liquid chromatography, but the method is simpler and lower in cost compared with the liquid chromatography, and brings convenience for high-throughput screening of mutant strains.
Detection conditions of liquid chromatography: a chromatographic column: c18 150 x 4.6mm,5um; mobile phase: a:10mMol/L dodecyl trimethyl ammonium chloride aqueous solution, pH3.0; b: methanol (volume ratio of A: B85; column temperature: 35 ℃, wavelength: 260nm; flow rate: 1ml/min.
Table 1: comparison of results of microplate reader and liquid chromatography in color development experiment
| Liquid phase assay results (mg/g) | Color development results (mg/g) Using phosphomolybdic acid |
| 0.924117333 | 0.959276 |
| 1.108176554 | 1.101726 |
Verification experiment 1: phosphomolybdic acid heat color reaction experiment
(1) 5G of phosphomolybdic acid solution of 0.1mg/G, 0.25mg/G, 0.5mg/G, 0.75mg/G and 1mg/gAA-2G, and 4.762mg/G were added to the test tube, and the photograph of the solution before heating is shown in FIG. 5.
(2) After the reaction at 80 ℃ for 1h, the photograph of the solution after the reaction is shown in FIG. 6, and it can be seen that AA-2G can react with phosphomolybdic acid by adding phosphomolybdic acid and heating, thereby causing a difference in color, which can be recognized even by naked eyes.
(3) The sample subjected to the heating reaction is transferred to an enzyme labeling instrument, the absorbance at 660nm is detected, the experimental results are shown in table 2 and figure 7, and the experimental results show that the phosphomolybdic acid spectrophotometer method has good linearity and accuracy and can be used for high-throughput screening of strains.
TABLE 2 Absorbance assay results
Verification experiment 2: interference experiment of glucose on reaction system:
phosphomolybdic acid mother liquor (47.62 mg/g): 1g of phosphomolybdic acid is taken and dissolved in 20g of water;
vc mother liquor (2 mg/g): 0.1g VC was dissolved in 50mL of water.
1. Diluting the phosphomolybdic acid mother liquor by 10 times, wherein the solution is light yellow;
2. the diluted phosphomolybdic acid solution was added with the Vc mother liquor (1) (about 10 drops), (2) anhydrous glucose solid (0.03G), and (3) AA-2G solid (0.014G) in this order; as shown in FIG. 8, the color of the solution (No. 3) changed from pale yellow to bluish green instantaneously; the glucose and AA-2G systems are unchanged and still in light yellow;
3. the results of heating at 80 ℃ are shown in FIG. 9, in which (1) changed from light yellow to blue-green, (2) remained light yellow, and (3) remained blue-green, and the experimental results show that AA-2G and glucose did not interfere with the reaction system.
The above description is of the preferred embodiment of the present invention, but it is not intended to limit the present invention. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (6)
1. A high-throughput screening method for cyclodextrin transferase for catalyzing and synthesizing L-ascorbic acid-2-glucoside is characterized by comprising the following steps:
adding beta-cyclodextrin and ascorbic acid into a culture system of a strain to be screened, and adjusting the pH value to 5.0-6.0 to perform catalytic reaction;
removing the strain after the catalytic reaction, and adding an oxidant to carry out an oxidation reaction;
after the oxidation reaction, a color-developing agent of L-ascorbic acid-2-glucoside is added for color development.
2. The screening method according to claim 1, wherein the beta-cyclodextrin and the ascorbic acid are added in a weight ratio of 1:1-2.
3. The screening method according to claim 1, wherein the oxidizing agent is hydrogen peroxide, ozone, or Fe 3+ A combination of salt and one or more of air or oxygen.
4. The screening method according to claim 1, wherein the oxidizing agent is hydrogen peroxide.
5. The screening method according to claim 1, wherein the developer is phosphomolybdic acid, and the color is developed by heating to 70-90 ℃.
6. The screening method according to claim 1, wherein after the color reaction, the absorbance is detected by a microplate reader, and the content of the L-ascorbic acid-2-glucoside is quantitatively determined through absorbance data.
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| CN202211723747.0A CN115976157A (en) | 2022-12-30 | 2022-12-30 | High-throughput screening method of cyclodextrin transferase for catalytic synthesis of L-ascorbic acid-2-glucoside |
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| CN202211723747.0A CN115976157A (en) | 2022-12-30 | 2022-12-30 | High-throughput screening method of cyclodextrin transferase for catalytic synthesis of L-ascorbic acid-2-glucoside |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667102A (en) * | 2013-09-23 | 2014-03-26 | 江南大学 | Bacterial strain for cyclodextrin glycosyltransferase production and application thereof |
| CN110734945A (en) * | 2019-10-30 | 2020-01-31 | 安徽泰格生物技术股份有限公司 | method for synthesizing L-ascorbic acid-2-glucoside |
| KR20200081038A (en) * | 2018-12-27 | 2020-07-07 | 건양대학교산학협력단 | The method for producing ascorbic acid-2-glucoside(AA-2G) using the immobilization of recombinant cyclodextrin glucanotransferase (CGTase) |
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- 2022-12-30 CN CN202211723747.0A patent/CN115976157A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667102A (en) * | 2013-09-23 | 2014-03-26 | 江南大学 | Bacterial strain for cyclodextrin glycosyltransferase production and application thereof |
| KR20200081038A (en) * | 2018-12-27 | 2020-07-07 | 건양대학교산학협력단 | The method for producing ascorbic acid-2-glucoside(AA-2G) using the immobilization of recombinant cyclodextrin glucanotransferase (CGTase) |
| CN110734945A (en) * | 2019-10-30 | 2020-01-31 | 安徽泰格生物技术股份有限公司 | method for synthesizing L-ascorbic acid-2-glucoside |
Non-Patent Citations (2)
| Title |
|---|
| 袁普伟等: "环麦芽糊精葡聚糖转移酶高活性的土壤微生物菌种筛选", 食品与发酵工业, vol. 34, no. 10, 30 October 2008 (2008-10-30), pages 67 * |
| 陶秀梅: "Bacillus stearothermophilus NO2环糊精葡萄糖基转移酶的分子改造及制备AA-2G的研究", 中国博士学位论文全文数据库 工程科技Ⅰ辑, no. 1, 15 January 2021 (2021-01-15), pages 1 - 1 * |
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