Preparation method and application of fluorescent biosensor for detecting transgenic protein

A biosensor and transgenic technology, applied in the field of preparation of fluorescent biosensors, can solve problems such as low sensitivity, and achieve the effects of high sensitivity, easy operation and low experimental cost

Active Publication Date: 2022-04-15
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The miniaturization of photoelectric detection equipment has become a hot spot of concern, but the miniaturization of equipment often brings low sensitivity. In order to improve this problem, if the performance of photoelectric materials can be further improved, a photoelectric sensor that can realize transgenic crops can be developed. Sensing microdevices for rapid and sensitive on-site detection of genetically modified products

Method used

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  • Preparation method and application of fluorescent biosensor for detecting transgenic protein
  • Preparation method and application of fluorescent biosensor for detecting transgenic protein
  • Preparation method and application of fluorescent biosensor for detecting transgenic protein

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0032] A method for preparing a fluorescent biosensor for detecting transgenic proteins, such as figure 1 shown, including the following steps:

[0033] (1) Synthesis of 3,4,9,10-perylenetetracarboxylic acid (PTCA)

[0034] Add 9 mg of 3,4,9,10 perylenetetracarboxylic dianhydride (PTCDA) to 11 mL of 1 mol / L NaOH solution, sonicate at an ultrasonic frequency of 90 kHz for 15 min, and then heat the solution until PTCDA is completely Dissolve, after the solution color turns yellow-green, add 1 mol / L HCl solution in the solution until the mixture color turns red completely, centrifuge and wash, and wash the collected precipitate alternately with absolute ethanol and ultrapure water, that is, The PTCA precipitate was obtained, dried in vacuum at 40°C for 3 hours, and dispersed in ultrapure water;

[0035] (2) Synthesis of silver nanoclusters

[0036]Add 4.3 mg of lipoic acid powder to 2.0 mL of ultrapure water, then add 0.012 mL of sodium borohydride solution with a concentratio...

Embodiment 2

[0042] Same as above-mentioned Example 1, the difference is: in the synthesis of step (1) 3,4,9,10-perylenetetracarboxylic acid (PTCA): 8mg 3,4,9,10 perylenetetracarboxylic dianhydride (PTCDA ) was added to 10 mL of NaOH solution with a concentration of 1 mol / L, and sonicated at an ultrasonic frequency of 80 kHz for 15 min; step (2) in the synthesis of silver nanoclusters: 4.2 mg of lipoic acid powder was added to 2.0 mL of ultrapure water , then add 0.01 mL of sodium borohydride solution with a concentration of 2 mol / L, and after stirring vigorously for 30 min, the solution becomes clear and transparent, then slowly add 0.04 mL of a concentration of 0.05 mol / L silver nitrate solution and 0.03 Sodium borohydride solution with a concentration of 2 mol / L in mL, continued to react for 90 minutes and then stopped stirring to obtain a silver nanocluster solution, which was stored in a refrigerator at 4°C for more than 24 hours.

Embodiment 3

[0044] Same as above-mentioned Example 1, the difference is: in the synthesis of step (1) 3,4,9,10-perylenetetracarboxylic acid (PTCA): 10mg 3,4,9,10 perylenetetracarboxylic dianhydride (PTCDA ) was added to 12 mL of NaOH solution with a concentration of 1 mol / L, and sonicated at an ultrasonic frequency of 100 kHz for 15 min; step (2) in the synthesis of silver nanoclusters: 4.5 mg of lipoic acid powder was added to 2.0 mL of ultrapure water , then add 0.015mL of sodium borohydride solution with a concentration of 2 mol / L, and after stirring vigorously for 30 min, the solution becomes clear and transparent, then slowly add 0.05mL of a concentration of 0.05mol / L silver nitrate solution and 0.04 Sodium borohydride solution with a concentration of 2 mol / L in mL, continued to react for 90 minutes and then stopped stirring to obtain a silver nanocluster solution, which was stored in a refrigerator at 4°C for more than 24 hours.

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Abstract

The invention discloses a preparation method and application of a fluorescent biosensor for detecting transgenic protein, and the preparation method is characterized by comprising the following steps: mixing a 1 mg/mL 3, 4, 9, 10-perylenetetracarboxylic acid solution and a silver nano-cluster solution according to a volume ratio of 5: 1, and stirring at normal temperature for 4 hours to obtain an AgNCs/PTCA solution; adding the EDC/NHS mixed solution into the AgNCs/PTCA solution in equal volume, keeping out of light at room temperature, continuously stirring for 6 hours, adding a second antibody Ab2 of the transgenic protein to be detected, which is equal to the EDC/NHS mixed solution in volume, incubating at 37 DEG C for 2 hours, centrifuging to remove the free second antibody Ab2, washing the product with ultrapure water for three times, re-dispersing into the PBS buffer solution, which is equal to the EDC/NHS mixed solution in volume, and centrifuging to remove the free second antibody Ab2; the supermolecular high-fluorescence material has the advantages of strong specificity, high sensitivity and good accuracy.

Description

technical field [0001] The invention relates to a fluorescent immunosensor, in particular to a preparation method and application of a fluorescent biosensor for detecting transgenic proteins. Background technique [0002] PAT protein is a homodimer encoded by the bar gene with a molecular weight of about 23 KDa, consisting of 183 amino acids. PAT protein belongs to the family of acetyltransferases. It has relatively the same structure and function as acetyltransferases. Its physiological function is to acetylate the herbicide glufosinate, inhibit the activity of glufosinate, and detoxify glufosinate. . With the widespread use of genetically modified products, people are increasingly concerned about some potential dangers that genetically modified products may bring. Therefore, the development of accurate, sensitive and convenient detection methods for genetically modified crops and foods is urgently needed. [0003] At present, the most mature detection method for GMO pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/543G01N33/573G01N33/68
CPCG01N33/533G01N33/543G01N33/573G01N33/68G01N2333/91051G01N2333/325
Inventor 郭智勇陈小双郝婷婷王照亮郭文博曹国洲邬杨波王忠琦
Owner NINGBO UNIV
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