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CN-122016744-A - Magnetic control ratio fluorescent biosensor based on RPA-CRISPR/Cas12a and preparation method and application thereof

CN122016744ACN 122016744 ACN122016744 ACN 122016744ACN-122016744-A

Abstract

The invention provides a magnetic control ratio fluorescent biosensor based on RPA-CRISPR/Cas12a, and a preparation method and application thereof, belonging to the technical field of fluorescent biosensor and pathogenic bacteria detection; the magnetic control ratio fluorescent biosensor comprises a fluorescent reference signal element, a fluorescent response signal element and a specific identification element, realizes high-sensitivity and specific detection of staphylococcus aureus and salmonella by the magnetism and fluorescence characteristics of the fluorescent reference signal element, the fluorescence characteristics of the fluorescent response signal element and the nucleic acid cleavage activity of Cas12a, has a built-in calibration function, can reduce interference caused by environment and instruments, has good selectivity on staphylococcus aureus and salmonella, has good anti-interference performance on other bacteria, and can be used for rapid detection in the food safety field.

Inventors

  • SUN ZONGBAO
  • XU HONG
  • GUO WANG
  • ZOU XIAOBO
  • ZHANG SIYU

Assignees

  • 镇江市疾病预防控制中心
  • 江苏大学

Dates

Publication Date
20260512
Application Date
20260130

Claims (10)

  1. 1. A method for preparing a magnetic control ratio fluorescent biosensor based on RPA-CRISPR/Cas12a, which is characterized by comprising the following steps: (1) Preparation of a fluorescent reference Signal element Eu-Fe 3 O 4 @SiO 2 -COOH: Dispersing Fe 3 O 4 in distilled water, adding europium chloride hexahydrate, benzoyl trifluoroacetone, 1, 10-phenanthroline, ammonia water, ethanol and tetraethyl orthosilicate into the distilled water, uniformly mixing, and performing a first stirring reaction to obtain Eu-Fe 3 O 4 @SiO 2 after the reaction is finished; Dispersing Eu-Fe 3 O 4 @SiO 2 in distilled water, adding citric acid into the distilled water, carrying out a second stirring reaction, washing and drying after the reaction is finished to obtain Eu-Fe 3 O 4 @SiO 2 -COOH; Dispersing Eu-Fe 3 O 4 @SiO 2 -COOH in distilled water, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, then carrying out a third stirring reaction, washing after the reaction is finished, and dispersing the product in distilled water to obtain carboxyl-activated Eu-Fe 3 O 4 @SiO 2 -COOH; (2) Preparation of fluorescence response Signal element BQD@ZIF-8-ssDNA 2: Dispersing BQD@ZIF-8, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and ssDNA2 in distilled water, then reacting the mixed solution at room temperature, centrifuging and washing after the reaction is finished to obtain BQD@ZIF-8-ssDNA2; (3) Preparation of a magnetic control ratio fluorescent biosensor: Pre-assembling Cas12a protein and crRNA in NEBuffer2.1 and enzyme-free water, adding an RPA product and ssDNA1 after assembling, mixing and incubating, and obtaining cleaved ssDNA1 after incubating; Mixing the cracked ssDNA1 with carboxyl activated Eu-Fe 3 O 4 @SiO 2 -COOH and BQD@ZIF-8-ssDNA2 for reaction, collecting precipitate by using a magnet after the reaction is finished, and dispersing the precipitate in distilled water to obtain the magnetic control ratio fluorescent biosensor.
  2. 2. The preparation method of claim 1, wherein in the step (1), the mass ratio of Fe 3 O 4 , europium chloride hexahydrate, benzoyl trifluoroacetone, 1, 10-phenanthroline, ammonia water, ethanol and tetraethyl orthosilicate is 40-60 mg:150-250 mg:300-400 mg:90-110 mg:0.1-0.6 mL:5-20 mL:0.5-2 mL; the dosage ratio of Eu-Fe 3 O 4 @SiO 2 to citric acid is 30-100 mg:50-150 mg; The dosage ratio of Eu-Fe 3 O 4 @SiO 2 -COOH, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to N-hydroxysuccinimide is 0.05-0.5 mg:0.1-5 mg; the first stirring reaction and the second stirring reaction are both stirring reactions for 10-20 h at room temperature; And the third stirring reaction is that stirring reaction is carried out for 0.5-2 h at room temperature.
  3. 3. The preparation method according to claim 1, wherein in the step (2), the preparation method of bqd@zif-8 comprises: dissolving citric acid and polyethyleneimine in distilled water, transferring to a reaction kettle for hydrothermal reaction, and obtaining blue quantum dots BQD after the reaction is finished; 2-methylimidazole, nitric acid, BQD and 3-aminopropyl triethoxysilane are dissolved in methanol, stirring reaction is carried out, and freeze drying is carried out after the reaction is finished, so that BQD@ZIF-8 is obtained.
  4. 4. The preparation method of claim 1, wherein in the step (2), the dosage of BQD@ZIF-8 is 0.5-1.5 mg, the dosage ratio of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and ssDNA2 is 0.3~0.5 mg:0.1~0.3 mg:1-10 mu L, and the initial concentration of ssDNA2 is 1-10 mu M; The reaction conditions are that stirring reaction is carried out for 0.5-2 hours at room temperature; the sequence of ssDNA2 is 5'-COOH-AAAAAAAAAAGAGAACCTGGG-3' (SEQ ID No: 6).
  5. 5. The method of claim 1, wherein in step (3), the volume ratio of Cas12a protein, crRNA, RPA product, and ssDNA1 is 0.5-2:0.5-2:1-4:2-6; The volume ratio of NEBuffer2.1 to the non-enzymatic water is 1-5:10-30; the initial concentration of the Cas12a is 0.5-2 mu M; the initial concentration of the crRNA is 0.5-2 mu M; The initial concentration of ssDNA1 is 1-10 mu M.
  6. 6. The preparation method according to claim 1, wherein in the step (3), the pre-assembly condition is that incubation is performed at 20-50 ℃ for 1-20 min; the condition of mixed incubation is that incubation is carried out for 1-50 min at 20-50 ℃; The volume ratio of the cleaved ssDNA1 to the carboxyl-activated Eu-Fe 3 O 4 @SiO 2 -COOH to the BQD@ZIF-8-ssDNA2 is 10-40:100-200:150-250; the concentration of the carboxyl activated Eu-Fe 3 O 4 @SiO 2 -COOH is 400-600 mug/mL; The concentration of the BQD@ZIF-8-ssDNA2 solution is 3-7 mg/mL; The condition of the mixing reaction is that the reaction is carried out for 1-30 min at 20-50 ℃.
  7. 7. The method of claim 1, wherein in step (3), the crRNA comprises staphylococcus aureus crRNA or salmonella crRNA; The sequence of the staphylococcus aureus crRNA is 5'-UAAUUUCUACUAAGUGUAGAUGUUGAAGUUGCACUAUAUAC-3' (SEQ ID No: 3); The sequence of the salmonella crRNA is 5'-UAAUUUCUACUAAGUGUAGAUAAATAGAAGAGTACGCTTAAAAC-3' (SEQ ID No: 4); the RPA product comprises a staphylococcus aureus RPA product or a salmonella RPA product; The sequence of the staphylococcus aureus RPA product is ：5'-GCATCACAAACAGATAATGGCGTAAATAGAAGTGGTTCTGAAGATCCAACAGTATATAGTGCAACTTCAACTAAAAAATTACATAAAGAACCTGCGACATTAATTAAAGCGATTGATGGTGATACGGTTAAATTAATGT-3'（SEQ ID No:1）; The sequence of the salmonella RPA product is ：5'-GCGGCTGCTCGCCTTTGCTGGTTTTAGGTTTGGCGGCGCTACGTTTTGCTTCACGGAATTTAAAATAGAAGAGTACGCTTAAAACCACCGATAAAATAACAAAAACCGGCAGTGGGAATCCCGGCAGAGTTCCCATTGAAATGGTCAAAATAGCCGTAACAACCAATACAAATGGGT-3'（SEQ ID No:2）; The ssDNA1 has the sequence of 5'-HN2-AAAAAAAAACCCAGGTTCTCT-3' (SEQ ID No: 5).
  8. 8. The magnetic control ratio fluorescent biosensor prepared by the preparation method of any one of claims 1-7, which is characterized by comprising a fluorescent reference signal element Eu-Fe 3 O 4 @SiO 2 -COOH, a fluorescent response signal element BQD@ZIF-8-ssDNA2 and a specific recognition element RPA-CRISPR/Cas12a; the Eu-Fe 3 O 4 @SiO 2 -COOH is formed by a Fe 3 O 4 core and a SiO 2 shell layer coated on the core, wherein Eu is doped on the SiO 2 shell layer; the BQD@ZIF-8 presents a block-like structure; The specific recognition element is obtained by mixed incubation of an RPA product, cas12a protein, crRNA and ssDNA1 in base fluid NEBuffer2.1 and enzyme-free water.
  9. 9. Use of a magnetic ratio fluorescent biosensor as claimed in claim 8 for detecting staphylococcus aureus and/or salmonella.
  10. 10. A method of detecting staphylococcus aureus and/or salmonella in a food product, the method comprising: (1) Adding the sample into the magnetic control ratio fluorescent biosensor of claim 8 for reaction, and then placing the sample in a closed camera to shoot and collect fluorescent images; (2) And constructing a deep learning model, inputting the acquired fluorescent image into the deep learning model, modeling and analyzing, and outputting a quantitative detection result.

Description

Magnetic control ratio fluorescent biosensor based on RPA-CRISPR/Cas12a and preparation method and application thereof Technical Field The invention belongs to the technical field of fluorescent biosensors and pathogen detection, and particularly relates to a magnetic control ratio fluorescent biosensor based on RPA-CRISPR/Cas12a, and a preparation method and application thereof. Background Eggs are one of the globally important nutritional foods that are susceptible to microbial contamination leading to food safety issues. Staphylococcus aureus (Staphylococcus aureus) and Salmonella (Salmonella) are common pathogens in chicken eggs that can cause a variety of diseases, ranging from minor skin infections to severe life threatening diseases sepsis and pneumonia. Therefore, the method has important clinical and public health significance for rapid and accurate detection of staphylococcus aureus and salmonella. Currently, methods for detecting staphylococcus aureus and salmonella mainly include culture methods, molecular biological methods and immunological methods. The culture method has high specificity, but needs long culture time and has insufficient sample detection sensitivity with low initial bacterial load, the molecular biological method such as Polymerase Chain Reaction (PCR) and nucleic acid hybridization technology has high equipment cost, complex operation and easy influence by pollutants in a sample, and the immunological method depends on specific antibodies, and has complex preparation and high cost and can influence the accuracy of results due to cross reaction. The method is difficult to be widely applied in the actual detection process, so that a novel food-borne pathogenic bacteria detection method with high sensitivity, good specificity and strong stability needs to be developed. Disclosure of Invention Aiming at some defects existing in the prior art, the invention provides a magnetic control ratio fluorescent biosensor based on an RPA-CRISPR/Cas12a, a preparation method and application thereof, wherein the magnetic control ratio fluorescent biosensor comprises a fluorescent reference signal element Eu-Fe 3O4@SiO2 -COOH, a fluorescent response signal element BQD@ZIF-8-ssDNA2 and a specific recognition element RPA-CRISPR/Cas12a, the magnetic control ratio fluorescent biosensor realizes high-sensitivity and specific detection of staphylococcus aureus and salmonella through magnetic and fluorescent characteristics of the fluorescent reference signal element, fluorescent characteristics of the fluorescent response signal element and nucleic acid cleavage activity of the Cas12a, photographs detection results by using a smart phone, and performs systematic modeling analysis on photographed images by introducing a deep learning method, has a calibration function, can reduce interference brought by environment and built-in instruments, has good selectivity on staphylococcus aureus and salmonella, has good anti-interference performance on other bacteria, and can be used in the field of fast detection of food safety. In order to achieve the technical purpose, the invention adopts the following technical means: The invention firstly provides a preparation method of a magnetic control ratio fluorescent biosensor based on RPA-CRISPR/Cas12a, which comprises the following steps: (1) Preparation of a fluorescent reference Signal element Eu-Fe 3O4@SiO2 -COOH: Dispersing Fe 3O4 in distilled water, adding europium chloride hexahydrate, benzoyl trifluoroacetone, 1, 10-phenanthroline, ammonia water, ethanol and tetraethyl orthosilicate into the distilled water, uniformly mixing, and performing a first stirring reaction to obtain Eu-Fe 3O4@SiO2 after the reaction is finished; Dispersing Eu-Fe 3O4@SiO2 in distilled water, adding citric acid into the distilled water, carrying out a second stirring reaction, washing and drying after the reaction is finished to obtain Eu-Fe 3O4@SiO2 -COOH; Dispersing Eu-Fe 3O4@SiO2 -COOH in distilled water, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, then carrying out a third stirring reaction, washing after the reaction is finished, and dispersing the product in distilled water to obtain carboxyl-activated Eu-Fe 3O4@SiO2 -COOH. Preferably, in the step (1), the mass ratio of Fe 3O4, europium chloride hexahydrate, benzoyl trifluoroacetone, 1, 10-phenanthroline, ammonia water, ethanol and tetraethyl orthosilicate is 40-60 mg:150-250 mg:300-400 mg:90-110 mg:0.1-0.6 mL:5-20 mL:0.5-2 mL; the dosage ratio of Eu-Fe 3O4@SiO2 to citric acid is 30-100 mg:50-150 mg; The dosage ratio of Eu-Fe 3O4@SiO2 -COOH, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to N-hydroxysuccinimide is 0.05-0.5 mg:0.1-5 mg; the first stirring reaction and the second stirring reaction are both stirring reactions for 10-20 h at room temperature; And the third stirring reaction is that stirring reaction is carried out for 0.5-2 h at room temperature. (2) P