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CN-121995052-A - Method for rapidly and ultrasensitively detecting escherichia coli O157:H27 based on SERS

CN121995052ACN 121995052 ACN121995052 ACN 121995052ACN-121995052-A

Abstract

The invention belongs to the technical field of biological detection, and particularly discloses a rapid ultrasensitive detection method for escherichia coli O157 based on SERS: the method for H7 comprises the following steps of preparing immunomagnetic bead capture nano particles, preparing noble metal or noble metal composite nano particle sol, preparing immunonoble metal or noble metal composite signal nano particles, preparing sandwich structure, and detecting escherichia coli O157: H7. The method uses the antibody as a specific recognition element, and simultaneously specifically binds and determines the escherichia coli O157H 7, so that on one hand, the dispersion state of the nano material in a detection system can be stabilized, on the other hand, the target protein can be recognized with high specificity and high affinity, the stability is good, the preparation cost is low, the detection accuracy is improved to a great extent, the detection speed is faster, the detection flow is extremely simple, the time is short, the field rapid detection and batch screening are adapted, meanwhile, the detection limit is low, the ultra-rapid detection speed and the ultra-high detection sensitivity are realized, and trace target bacteria can be accurately recognized.

Inventors

  • LI JIANFENG
  • JIANG HAO
  • ZHANG YUEJIAO

Assignees

  • 厦门大学

Dates

Publication Date
20260508
Application Date
20260318

Claims (10)

  1. 1. A method for rapidly and ultra-sensitively detecting escherichia coli O157:H27 based on SERS is characterized by comprising the following steps: S1, preparing immune magnetic bead capturing nano particles, namely modifying a capture antibody combination of escherichia coli O157:H27 on carboxyl magnetic beads through coupling action to form the immune magnetic bead capturing nano particles; s2, preparing noble metal or noble metal composite nanoparticle sol; S3, preparing immune noble metal or noble metal composite signal nano particles, namely mixing the noble metal or noble metal composite nano particle sol prepared in the step S2 with a Raman reporter molecule IR-808 to prepare the noble metal or noble metal composite signal nano particles, and then mixing the noble metal or noble metal composite signal nano particles with a detection antibody of escherichia coli O157:H27; s4, preparing a sandwich structure, namely mixing immune magnetic bead capturing nano particles, immune metal signal nano particles and a sample to be tested to obtain the sandwich structure; S5, detecting the escherichia coli O157H 7, namely taking out the sediment with the sandwich structure, and dripping the sediment on a substrate with a gold film plated on the surface for Raman detection to obtain a Raman spectrum result of the escherichia coli O157H 7.
  2. 2. The method for rapid ultrasensitive detection of escherichia coli O157: H7 based on SERS according to claim 1, wherein the immunomagnetic bead capture nanoparticle in step S1 is magnetic and modifies a capture antibody capable of specifically recognizing escherichia coli O157: H7.
  3. 3. The method for rapid ultrasensitive detection of escherichia coli O157: H7 based on SERS according to claim 1, wherein step S1 specifically comprises taking 200-800 μl of carboxyl-modified magnetic beads with a concentration of 1-10 μg/μl, adding a washing buffer, vortexing to suspend the magnetic beads sufficiently, magnetically discarding the supernatant, resuspending the magnetic beads in the washing buffer, adding 6-10 μl of EDC 8-12 mg/mL and NHS activation 20-40 min, adding 2-20 μl of 200 μg/mL capture antibody, incubating 20-28H at 4 ℃, magnetically discarding the supernatant, washing with the washing buffer, resuspending in PBS solution, then adding 100-5000 μl of 0.5-1.5 wt% blocking solution, reacting 1-5H at room temperature to block the remaining binding sites on the surface of the magnetic beads, magnetically separating, washing with the washing buffer, and finally redispersing the beads in the washing buffer to obtain immunomagnetic capture nanoparticles.
  4. 4. The method for rapid ultrasensitive detection of Escherichia coli O157:H7 based on SERS according to claim 3, wherein the magnetic beads have a particle size of 0.2-5. Mu.m.
  5. 5. The SERS based rapid ultrasensitive detection method of e.coli O157:h7 according to claim 3, wherein the wash buffer is formulated from 1-200 mM inorganic salt buffer at pH 5.5-8, including but not limited to one of phosphate buffer, citric acid-sodium citrate buffer, boric acid-borax buffer, citric acid-borax buffer, 1-10 wt% surfactant, including but not limited to one of TW-20, TW-60, TW-80, CHAPS, TRITON-X100, and 15-100 inorganic chloride salt, including but not limited to one of NaCl, KCl, mgCl 2 .
  6. 6. A SERS based rapid ultrasensitive detection method of e.coli O157:h7 according to claim 3, wherein said blocking solution is formulated from 1-20 wt% BSA, 1-200mm phosphate buffer at pH 5.5-8, 15-100 mM inorganic chloride salt including but not limited to one of casein, glycine and skim milk, and 1-10 wt% surfactant including but not limited to one of Triton-X400, TW-60, TW-80, CHAPS, triton-X100, including but not limited to one of NaCl, KCl, mgCl 2 .
  7. 7. The method for rapid ultrasensitive detection of escherichia coli O157: H7 based on SERS according to claim 1, wherein said step S3 specifically comprises mixing 200-800 μl of noble metal or noble metal composite nanoparticles with 2-12 μl of 10-100 μΜ raman reporter IR-808, incubating at room temperature for 1-15 min, centrifuging, discarding the supernatant, redispersing in PBS-T solution, adding 10-60 μg detection antibody for 20-28H under-4 ℃ condition, washing with PBS-T, adding a blocking solution to block the remaining active sites, incubating at room temperature, centrifuging, washing with PBS-T solution, discarding the supernatant, and redispersing in the blocking solution to obtain immunonoble metal or noble metal composite signal nanoparticles.
  8. 8. The method for rapid ultrasensitive detection of escherichia coli O157: H7 based on SERS according to claim 7, wherein the volume ratio of the metal nanoparticle to the raman reporter is (50-500): (1-30), the concentration of the raman reporter IR-808 is 40-60 μm, the incubation time at room temperature is 5-30 min, and the PBS-T solution is TW-20 added to the PBS solution at a concentration of 1-10 wt%.
  9. 9. The method for rapid ultrasensitive detection of escherichia coli O157: H7 based on SERS according to claim 1, wherein step S4 specifically comprises mixing the immunomagnetic bead capture nanoparticle with escherichia coli O157: H7, incubating at room temperature for 0.2-5H, washing with PBS-T solution after magnetic separation, then mixing with immunocu 2 o@ag signal nanoparticle, incubating at room temperature for 0.2-5H, washing with PBS-T solution after magnetic separation, and obtaining a sandwich-structured precipitate.
  10. 10. The method for rapid ultrasensitive detection of escherichia coli O157: H7 based on SERS according to claim 1, wherein the substrate in step S5 comprises but is not limited to one of a silicon wafer and an aluminum foil, and the raman spectroscopy is performed by a portable raman spectrometer or other type of raman spectrometer, preferably having an excitation wavelength of 785 nm.

Description

Method for rapidly and ultrasensitively detecting escherichia coli O157:H27 based on SERS Technical Field The invention relates to the technical field of biological detection, in particular to a method for rapidly and ultra-sensitively detecting escherichia coli O157:H27 based on SERS. Background Coli (ESCHERICHIA COLI) O157: H7 was a serotype of Shiga toxin-producing E.coli, STEC, first identified in 1982 as a food-borne pathogen. The strain can be transmitted through contaminated fresh agricultural products, inadequately heated livestock meat products and manure-mouth pathway, has a minimum infection dose of about 10-100 CFU (colony forming units), can induce hemorrhagic colitis, can progress to hemolytic uremic syndrome (Hemolytic Uremic Syndrome, HUS), and has higher pathogenicity and public health risks. Epidemiological data shows that the strain accounts for about 20% of food-borne outbreaks worldwide, and is an important prevention and control object in food safety supervision. The existing escherichia coli O157-H7 detection technology has obvious defects commonly, and is characterized by low detection sensitivity, difficulty in capturing low pathogenic dosage of the pathogenic bacteria, slow detection speed, incapability of meeting the on-site rapid screening requirement, poor specificity, easiness in false positive or false negative caused by impurity interference in complex samples, weak suitability of complex matrixes, difficulty in adapting to actual detection scenes such as foods and environments, and incapability of effectively identifying biomembrane formed by thalli and the induced VBNC (viable but non-culturable) state, and easiness in missed detection. Surface Enhanced Raman Spectroscopy (SERS) is an optical detection technique based on the effect of localized Surface plasmon resonance (Localized Surface Plasmon Resonance, LSPR) of noble metal nanostructures, which can enhance the raman scattering signal of molecules adsorbed on the nanostructure Surface by 10 4–1011 times, thereby realizing high-sensitivity and high-specificity detection of target molecules. The SERS technology can obtain molecular vibration fingerprint information to realize the specific recognition of the chemical structure of a substance, has extremely weak Raman signal of water molecules, is suitable for directly detecting target objects in complex biological matrixes, has a detection limit up to a single molecular level, and is suitable for analyzing trace pathogens. Compared with the traditional spectrum technologies such as ultraviolet-visible spectrophotometry and fluorescence spectrometry, SERS has remarkable advantages in anti-interference performance, sensitivity and molecular recognition capability, and has been widely applied to the fields of biomedical detection and food safety monitoring. However, although the conventional SERS immunodetection technology combines the high sensitivity of the SERS technology and the high specificity of antigen-antibody combination, the conventional SERS immunodetection technology has certain advantages compared with the conventional detection means, the conventional SERS immunodetection technology still has the realistic problems of complex detection system, poor Raman signal stability, higher detection cost, limited field application and the like, and finally cannot meet the trace detection requirements of fast, high sensitivity and high specificity of the escherichia coli O157:H7, and cannot effectively challenge public health and food safety brought by the pathogenic bacteria. Therefore, there is a need to establish an analytical method capable of rapidly, sensitively and specifically detecting the strain, so as to overcome the limitations of the conventional culture and molecular biology methods in terms of detection timeliness and trace identification. Disclosure of Invention The invention aims to overcome the defects of the prior art and provides a method for rapidly and ultra-sensitively detecting escherichia coli O157:H7 based on SERS, which uses IR-808 as a signal molecule, uses carboxyl modified magnetic beads as a capture antibody carrier, precious metal nano particles or precious metal composite nano particles as a detection antibody sensing probe to construct a high-sensitivity SERS immune detection system, combines magnetic bead enrichment concentration and dual signal enhancement of the precious metal nano particles or the precious metal composite nano particles, and combines one-step mixed incubation and magnetic attraction separation processes to realize rapid and ultra-sensitively detecting target bacteria. In order to achieve the above purpose, the technical scheme of the invention is that a method for rapidly and ultra-sensitively detecting escherichia coli O157:H27 based on SERS comprises the following steps: S1, preparing immune magnetic bead capturing nano particles, namely modifying a capture antibody combination of escherichia coli O157:H27 on carboxy