Method for rapidly and ultrasensitively detecting escherichia coli O157: H7 based on SERS (Surface Enhanced Raman Scattering)

Abstract

The invention belongs to the technical field of biological detection, and particularly discloses a method for rapidly and ultrasensitively detecting escherichia coli O157: H7 based on SERS (Surface Enhanced Raman Scattering). Comprising the following steps: preparing immunomagnetic bead capture nanoparticles, preparing precious metal or precious metal composite nanoparticle sol, preparing immune precious metal or precious metal composite signal nanoparticles, preparing a sandwich structure, and detecting escherichia coli O157: H7. According to the method, the antibody serves as a specific recognition element and is specifically combined with and used for measuring escherichia coli O157: H7, on one hand, the dispersed state of a nano material in a detection system can be stabilized, on the other hand, target protein can be recognized with high specificity and high affinity, and the method is good in stability, low in preparation cost and suitable for large-scale popularization and application. The detection accuracy is improved to a great extent; the method is higher in detection speed, extremely simple in detection process, short in time consumption, suitable for field rapid detection and batch screening, low in detection limit, ultra-high in detection speed and ultra-high in detection sensitivity, and capable of accurately recognizing trace target thalli.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a method for rapid and ultrasensitive detection of Escherichia coli O157:H7 based on SERS. Background Technology

[0002] Escherichia coli O157:H7 is a serotype of Shiga toxin-producing E. coli (STEC), first identified as a foodborne pathogen in 1982. This strain can be transmitted through contaminated raw produce, undercooked meat products, and the fecal-oral route. Its minimum infectious dose is approximately 10–100 CFU (colony-forming units), which can cause hemorrhagic colitis and progress to hemolytic uremic syndrome (HUS), posing a high pathogenicity and public health risk. Epidemiological data show that this strain accounts for approximately 20% of foodborne outbreaks globally, making it a key target for food safety regulation. Existing detection technologies for Escherichia coli O157:H7 generally have significant shortcomings, namely: low detection sensitivity, making it difficult to capture low pathogenic doses of this bacterium; slow detection speed, failing to meet the needs of rapid on-site screening; poor specificity, easily affected by impurities in complex samples, resulting in false positives or false negatives; weak adaptability to complex matrices, making it difficult to adapt to actual testing scenarios such as food and environment; and the inability to effectively identify biofilms formed by bacteria and the induced VBNC (live but unculturable) state, easily leading to missed detections.

[0003] Surface-enhanced Raman spectroscopy (SERS) is an optical detection technique based on the localized surface plasmon resonance (LSPR) effect on the surface of noble metal nanostructures. It can enhance the Raman scattering signal of molecules adsorbed on the surface of nanostructures by 10-1. 4 -10 11This allows for highly sensitive and specific detection of target molecules. SERS technology can obtain molecular vibrational fingerprint information, enabling specific identification of the chemical structure of substances; the Raman signal of water molecules is extremely weak, making it suitable for direct detection of targets in complex biological matrices; the detection limit can reach the single-molecule level, making it suitable for trace pathogen analysis. Compared to traditional spectroscopic techniques such as UV-Vis spectrophotometry and fluorescence spectroscopy, SERS has significant advantages in terms of anti-interference, sensitivity, and molecular recognition ability, and has been widely used in biomedical detection and food safety monitoring. However, while conventional SERS immunoassay combines the high sensitivity of SERS with the high specificity of antigen-antibody binding, offering certain advantages over traditional detection methods, it still suffers from practical problems such as complex detection systems, poor Raman signal stability, high detection costs, and limited field applications. Ultimately, it cannot meet the demand for rapid, highly sensitive, and highly specific trace detection of E. coli O157:H7, and cannot effectively address the public health and food safety challenges posed by this pathogen.

[0004] Therefore, there is an urgent need to establish an analytical method that can quickly, sensitively, and specifically detect this strain, in order to overcome the limitations of traditional culture and molecular biology methods in terms of detection timeliness and trace identification. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a rapid and ultrasensitive method for detecting Escherichia coli O157:H7 based on SERS. Using IR-808 as the signal molecule, carboxyl-modified magnetic beads as a capture antibody carrier and noble metal nanoparticles or noble metal composite nanoparticles as detection antibody sensing probes, a highly sensitive SERS immunoassay system is constructed. Through magnetic bead enrichment and concentration combined with dual signal enhancement from noble metal nanoparticles or noble metal composite nanoparticles, along with a one-step mixing incubation and magnetic separation process, rapid and ultrasensitive detection of the target bacteria is achieved.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: a method for rapid and ultrasensitive detection of Escherichia coli O157:H7 based on SERS, comprising the following steps:

[0007] S1: Preparation of immunomagnetic bead capture nanoparticles: The capture antibody combination of Escherichia coli O157:H7 was modified onto carboxyl magnetic beads through coupling to form immunomagnetic bead capture nanoparticles.

[0008] S2: Preparation of noble metal or noble metal composite nanoparticle sol;

[0009] S3: Preparation of immunonoble metal or noble metal composite signal nanoparticles: The noble metal or noble metal composite nanoparticle sol prepared in step S2 is mixed with Raman reporter molecule IR-808 to obtain noble metal or noble metal composite signal nanoparticles; then mixed with detection antibody of Escherichia coli O157:H7; the active site is blocked to obtain immunonoble metal or noble metal composite signal nanoparticles.

[0010] S4: Preparation of sandwich structure: Immunomagnetic bead capture nanoparticles and immunometal signal nanoparticles are mixed with the sample to be tested to obtain a sandwich structure;

[0011] S5: Detection of Escherichia coli O157:H7: The precipitate with the sandwich structure was taken out and dropped onto a substrate with a gold film on the surface for Raman detection to obtain the Raman spectrum of Escherichia coli O157:H7.

[0012] In a preferred embodiment of the present invention, in step S1, the immunomagnetic beads capturing nanoparticles are magnetic and modified with capture antibodies that specifically recognize Escherichia coli O157:H7.

[0013] In a preferred embodiment of the present invention, step S1 specifically includes taking 200-800 μL of carboxyl-modified magnetic beads with a concentration of 1-10 μg / μL, adding washing buffer, vortexing to fully suspend the magnetic beads, and magnetically removing the supernatant; resuspending the magnetic beads in washing buffer, adding 6-10 μL of 8-12 mg / mL EDC and NHS to activate for 20-40 min, then adding 2-20 μL of 200 μg / mL capture antibody, incubating at 4°C for 20-28 h, magnetically removing the supernatant, washing with washing buffer, resuspending in PBS solution, then adding 100-5000 μL of 0.5-1.5 wt% blocking solution, reacting at room temperature for 1-5 h to block the remaining binding sites on the surface of the magnetic beads, magnetic separation, washing with washing buffer, and finally redispersing in washing buffer to obtain immunomagnetic bead capture nanoparticles.

[0014] More preferably, the magnetic beads have a particle size of 0.2-5 μm, and more preferably 1-2 μm.

[0015] More preferably, the washing buffer is prepared from 1-200mM inorganic salt buffer with pH 5.5-8, 1-10wt% surfactant, and 15-100mM inorganic chloride salt. The inorganic salt buffer includes, but is not limited to, one of phosphate buffer (PB), citrate-sodium citrate buffer (CPBS), borate-borax buffer (BB), and citrate-borax buffer (CBS). The surfactant includes, but is not limited to, one of TW-20, TW-60, TW-80, CHAPS, and TRITON-X100. The inorganic chloride salt includes, but is not limited to, one of NaCl, KCl, and MgCl2.

[0016] More preferably, the blocking solution is prepared from 1-20 wt% BSA, 1-200 mM phosphate buffer (PB) pH 5.5-8, 15-100 mM inorganic chloride salt and 1-10 wt% surfactant. The BSA includes, but is not limited to, one of casein, glycine and skim milk. The surfactant includes, but is not limited to, one of Triton-X400, TW-60, TW-80, CHAPS and TRITON-X100. The inorganic chloride salt includes, but is not limited to, one of NaCl, KCl and MgCl2.

[0017] In a preferred embodiment of the present invention, step S3 specifically includes mixing 200-800 μL of noble metal or noble metal composite nanoparticles with 2-12 μL of 10-100 μM Raman reporter molecule IR-808, incubating at room temperature for 1-15 min, centrifuging, discarding the supernatant, redispersing in PBS-T solution, adding 2-20 μL of 200 μg / mL detection antibody at -4℃ and incubating for 20-28 h, washing with PBS-T, adding blocking solution to block the remaining active sites, incubating at room temperature, centrifuging, washing with PBS-T solution, finally discarding the supernatant, redispersing in blocking solution, and obtaining immunonoble metal or noble metal composite signal nanoparticles.

[0018] More preferably, the volume ratio of the metal nanoparticles to the Raman reporter molecule is (50-500):(1-30), the concentration of the Raman reporter molecule IR-808 is 40-60 μM, and the incubation time at room temperature is 5-30 min.

[0019] More preferably, the PBS-T solution is a PBS solution with the addition of TW-20 at a concentration of 1-10 wt%.

[0020] In a preferred embodiment of the present invention, step S4 specifically includes mixing immunomagnetic bead-capturing nanoparticles and Escherichia coli O157:H7, incubating at room temperature for 0.2-5 h, washing with PBS-T solution after magnetic separation, and then mixing with immunoCu2O@Ag signaling nanoparticles, incubating at room temperature for 0.2-5 h, washing with PBS-T solution after magnetic separation, to obtain a sandwich-structured precipitate.

[0021] In a preferred embodiment of the present invention, the substrate in step S5 includes, but is not limited to, a silicon wafer and an aluminum foil.

[0022] In a preferred embodiment of the present invention, the Raman spectroscopy test in step S5 is performed using a portable Raman spectrometer or other type of Raman spectrometer, and the excitation wavelength is preferably 785 nm.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. This invention selects IR-808 with a large Raman cross section as the exclusive signal molecule, which can stably output a clear and highly recognizable positive signal, thus establishing a reliable signal benchmark for the detection of target bacteria, avoiding signal ambiguity and distortion from the source, and ensuring the stability and effectiveness of the detection signal.

[0025] 2. This invention uses surface carboxyl-modified magnetic beads as the SERS substrate and antibody capture carrier. After carboxyl activation, the antibodies can be efficiently coupled, ensuring the stability of immune binding. The magnetic beads have magnetic attraction properties, which can quickly separate and concentrate the target complex, amplify the detection signal, and eliminate impurity interference. The large specific surface area brought by the three-dimensional structure can also significantly improve the antibody loading rate, optimize incubation efficiency, and shorten the incubation time.

[0026] 3. This invention selects noble metal or noble metal composite nanoparticle sol nanoparticles with excellent SERS enhancement activity and antibody loading capacity as detection antibody-coupled probes. This can not only stably carry the detection antibody and ensure the accuracy of immune recognition, but also strongly amplify the IR-808 Raman signal, greatly improve the detection sensitivity of the system, and easily achieve the accurate capture of trace targets.

[0027] 4. This invention uses antibodies as the core element for specific recognition. Relying on the high specificity and high affinity of antibodies, they can target and bind to Escherichia coli O157:H7, effectively isolating non-specific interference from bacteria and impurities. At the same time, it stabilizes the dispersion of nanomaterials, avoids particle aggregation and sedimentation, and greatly improves the accuracy and reliability of detection results.

[0028] 5. The detection process of this invention is extremely simple, requiring only four steps: incubation, washing, magnetic adsorption, and detection. Pretreatment can be completed in one step, which is time-saving and suitable for on-site rapid testing and batch screening. At the same time, it has a low detection limit and combines ultra-fast detection speed with ultra-high detection sensitivity, which can accurately identify trace target bacteria. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the detection principle of the present invention;

[0030] Figure 2 This is the ultraviolet absorption spectrum of the immune Cu2O@Ag signal nanoparticles in Example 1 of the present invention;

[0031] Figure 3 This is a TEM image of the structure of the immune Cu2O@Ag signaling nanoparticles in Example 1 of this invention;

[0032] Figure 4 The images show surface-enhanced Raman spectra of different concentrations of Escherichia coli O157:H7 in Example 1 of this invention. Detailed Implementation

[0033] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in more detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0034] A rapid and ultrasensitive method for detecting Escherichia coli O157:H7 based on SERS includes the following steps:

[0035] S1: Preparation of immunomagnetic bead capture nanoparticles: The capture antibody combination of Escherichia coli O157:H7 was modified onto carboxyl magnetic beads through coupling to form immunomagnetic bead capture nanoparticles.

[0036] S2: Preparation of noble metal or noble metal composite nanoparticle sol;

[0037] S3: Preparation of immunonoble metal or noble metal composite signal nanoparticles: The noble metal or noble metal composite nanoparticle sol prepared in step S2 is mixed with Raman reporter molecule IR-808 to obtain noble metal or noble metal composite signal nanoparticles; then mixed with detection antibody of Escherichia coli O157:H7; the active site is blocked to obtain immunonoble metal or noble metal composite signal nanoparticles.

[0038] S4: Preparation of sandwich structure: Immunomagnetic bead capture nanoparticles and immunometal signal nanoparticles are mixed with the sample to be tested to obtain a sandwich structure;

[0039] S5: Detection of Escherichia coli O157:H7: The precipitate with the sandwich structure was taken out and dropped onto a substrate with a gold film on the surface for Raman detection to obtain the Raman spectrum of Escherichia coli O157:H7.

[0040] In step S1, the immunomagnetic beads capture nanoparticles are magnetic and modified with capture antibodies that specifically recognize Escherichia coli O157:H7.

[0041] Step S1 specifically includes taking 200-800 μL of carboxyl-modified magnetic beads with a concentration of 1-10 μg / μL, adding washing buffer, vortexing to fully suspend the magnetic beads, and magnetically removing the supernatant; resuspending the magnetic beads in washing buffer, adding 6-10 μL of 8-12 mg / mL EDC and NHS to activate for 20-40 min, then adding 2-20 μL of 200 μg / mL capture antibody, incubating at 4℃ for 20-28 h, magnetically removing the supernatant, washing with washing buffer, resuspending in PBS solution, then adding 100-5000 μL of 0.5-1.5 wt% blocking solution, reacting at room temperature for 1-5 h to block the remaining binding sites on the surface of the magnetic beads, magnetic separation, washing with washing buffer, and finally redispersing in washing buffer to obtain immunomagnetic bead capture nanoparticles.

[0042] The magnetic beads have a particle size of 0.2-5 μm, more preferably 1-2 μm.

[0043] The washing buffer is prepared from 1-200 mM inorganic salt buffer (pH 5.5-8), 1-10 wt% surfactant, and 15-100 mM inorganic chloride salt. The inorganic salt buffer includes, but is not limited to, one of phosphate buffer (PB), citrate-sodium citrate buffer (CPBS), borate-borax buffer (BB), and citrate-borax buffer (CBS). The surfactant includes, but is not limited to, one of TW-20, TW-60, TW-80, CHAPS, and TRITON-X100. The inorganic chloride salt includes, but is not limited to, one of NaCl, KCl, and MgCl2.

[0044] The blocking solution is prepared from 1-20 wt% BSA, 1-200 mM pH 5.5-8 phosphate buffer (PB), 15-100 mM inorganic chloride salt, and 1-10 wt% surfactant. The BSA includes, but is not limited to, one of casein, glycine, and skim milk. The surfactant includes, but is not limited to, one of Triton-X400, TW-60, TW-80, CHAPS, and TRITON-X100. The inorganic chloride salt includes, but is not limited to, one of NaCl, KCl, and MgCl2.

[0045] Step S3 specifically includes mixing 200-800 μL of noble metal or noble metal composite nanoparticles with 2-12 μL of 10-100 μM Raman reporter molecule IR-808, incubating at room temperature for 1-15 min, centrifuging, discarding the supernatant, redispersing in PBS-T solution, adding 10-60 μg of detection antibody at -4℃ and incubating for 20-28 h, washing with PBS-T, adding blocking solution to block the remaining active sites, incubating at room temperature, centrifuging, washing with PBS-T solution, finally discarding the supernatant, redispersing in blocking solution, and obtaining immunonoble metal or noble metal composite signal nanoparticles.

[0046] The volume ratio of the metal nanoparticles to the Raman reporter molecule is (50-500):(1-30), the concentration of the Raman reporter molecule IR-808 is 40-60 μM, and the incubation time at room temperature is 5-30 min.

[0047] The PBS-T solution is a PBS solution with 1-10 wt% TW-20 added.

[0048] Step S4 specifically includes mixing immunomagnetic bead-capturing nanoparticles and E. coli O157:H7, incubating at room temperature for 0.2-5 h, washing with PBS-T solution after magnetic separation, and then mixing with immunoCu2O@Ag signaling nanoparticles, incubating at room temperature for 0.2-5 h, washing with PBS-T solution after magnetic separation, to obtain a sandwich-structured precipitate.

[0049] The substrate in step S5 includes, but is not limited to, silicon wafers and aluminum foil.

[0050] In step S5, the Raman spectroscopy test is performed using a portable Raman spectrometer or other types of Raman spectrometers, with the excitation wavelength preferably being 785 nm.

[0051] Figure 1This is a schematic diagram illustrating the detection principle of the present invention. Immunomagnetic beads capture nanoparticles that undergo a specific immune reaction with E. coli O157:H7. Through incubation with immunoprecipitated noble metal or noble metal composite signal nanoparticles, a sandwich-like structure is constructed. Rapid magnetic separation is then performed, and rapid virus detection is achieved using a portable Raman spectroscopy instrument.

[0052] In the following examples, both the capture and detection antibodies were provided by Shanghai Bohu Biotechnology Co., Ltd.

[0053] In the following examples, the washing buffer was prepared from 20 mM pH 7 phosphate buffer (PB), 2 wt% surfactant and 20 mM inorganic chloride salt. The surfactant was TW-20 and the inorganic chloride salt was NaCl.

[0054] In the following examples, the blocking solution was prepared from 4 wt% BSA, 20 mM pH 7 phosphate buffer (PB), 20 mM inorganic chloride salt and 4 wt% surfactant. BSA is skim milk, surfactant is Triton-X400 and inorganic chloride salt is NaCl.

[0055] Example 1

[0056] (1) Preparation of immunomagnetic bead capture nanoparticles: Take 200 μL of 1 μm carboxyl-modified magnetic beads with a concentration of 10 μg / μL, add 1 mL of washing buffer, vortex to mix and fully suspend the magnetic beads, magnetically remove the supernatant, and wash twice with 200 μL of washing buffer; resuspend the treated magnetic beads in washing buffer, add 10 μL of 10 mg / mL EDC and NHS for 30 min activation, wash with washing buffer, resuspend in PBS buffer, add 10 μL of 200 μg / mL capture antibody, incubate overnight at 4℃, magnetically remove the supernatant, wash with washing buffer, resuspend in PBS buffer, add 200 μL of 1 wt% blocking solution BSA, react at room temperature for 8 h to block the remaining binding sites on the surface of the magnetic beads, magnetically separate, wash with washing buffer, and finally redisperse in washing buffer to obtain the immunomagnetic bead capture nanoparticles.

[0057] (2) Preparation of nano-Cu2O@Ag sol: 4.5 mg Cu2O was dispersed in 100 mL of ultrapure water by ultrasonic vibration within 3 min, and then 1.05 mL of sodium citrate (30 mM) and 1.05 mL of NaBH4 (100 mM) aqueous solution were slowly added dropwise. After stirring for 5 min, 1.05 mL of AgNO3 solution (10 mM) was introduced, and a visible pale yellow color shift was initiated with the formation of Ag-NP. The reaction was carried out under continuous stirring for 1 h, and then the solution was purified by washing with ultrapure water and ethanol sequentially; the ultraviolet absorption wavelength of the Cu2O@Ag nanoparticles was 646 nm.

[0058] 400 μL of Cu2O@Ag nanoparticles were mixed with 10 μL of 50 μM Raman reporter molecule and incubated at room temperature for 10 min. After centrifugation, the supernatant was discarded, and the mixture was redispersed in PBS-T solution. 10 μL of 200 μg / mL detection antibody was added at -4℃ and incubated for 20 h. After washing with PBS-T, the remaining active sites were blocked with blocking solution. The mixture was incubated at room temperature, centrifuged, washed with PBS-T solution, and finally the supernatant was discarded. The mixture was redispersed in blocking solution to obtain immune Cu2O@Ag signaling nanoparticles.

[0059] (3) Preparation of immunogenic Cu2O@Ag signaling nanoparticles: 400 μL of Cu2O@Ag nanoparticles were mixed with 8 μL of 30 μM Raman reporter molecule IR-808, incubated at room temperature for 15 min, centrifuged, and the supernatant was discarded. The nanoparticles were then redispersed in PBS-T (PBS solution with 4% TW-20 solution added). 20 μL of 200 μg / mL detection antibody was added at -4℃ and incubated for 20 h. After washing with PBS-T, the remaining active sites were blocked with blocking solution, incubated at room temperature for 20 min, centrifuged, washed with PBS-T solution, and finally the supernatant was discarded. The nanoparticles were then redispersed in the blocking solution to obtain immunogenic Cu2O@Ag signaling nanoparticles. The UV absorption spectrum and TEM image are shown below. Figure 2 and Figure 3 As shown, from Figure 2 It can be seen that Cu₂O NPs exhibit a maximum absorption band at 646 nm, and after successful modification with Ag NPs, this band redshifts to 654 nm. From... Figure 3 It can be seen that the synthesized Cu2O NPs exhibit a regular polyhedral structure, and Ag NPs are uniformly distributed on its surface.

[0060] (4) Preparation of sandwich structure: 20 μL of the prepared immunomagnetic bead-capturing nanoparticles and 200 μL of Escherichia coli O157:H7 at different concentrations (1 CFU / ml, 5 CFU / ml, 10 ... 2 CFU / ml, 10 3 CFU / ml, 10 4 CFU / ml, 10 5 After mixing with CFU / ml, the mixture was incubated at room temperature for 20 min, magnetically separated, washed once with 200 μL PBS-T solution, and then mixed with 40 μL of immunogenic Cu2O@Ag signaling nanoparticles. The mixture was then incubated at room temperature for 20 min to construct a sandwich structure.

[0061] (5) Detection of Escherichia coli O157:H7: The precipitate forming a sandwich structure was taken out and dropped onto an aluminum foil coated with a gold film. After drying, Raman detection was performed directly using a portable Raman spectrometer (laser wavelength 785 nm). Rapid detection of Escherichia coli O157:H7 was achieved within 22 min. The surface-enhanced Raman spectra of different concentrations of Escherichia coli O157:H7 are shown below. Figure 4 As shown, from Figure 4 It can be seen that the SERS intensity of IR-808 shows a concentration-dependent increase, with a LOD as low as 1 CFU / mL.

[0062] Example 2

[0063] (1) Preparation of immunomagnetic bead capture nanoparticles: Take 200 μL of 1 μm carboxyl-modified magnetic beads with a concentration of 10 μg / μL, add 1 mL of washing buffer, vortex to mix and fully suspend the magnetic beads, magnetically remove the supernatant, and wash twice with 200 μL of washing buffer; resuspend the treated magnetic beads in washing buffer, add 10 μL of 10 mg / mL EDC and NHS for 30 min activation, wash with washing buffer, resuspend in PBS buffer, add 10 μL of 200 μg / mL capture antibody, incubate overnight at 4℃, magnetically remove the supernatant, wash with washing buffer, resuspend in PBS buffer, add 200 μL of 1 wt% blocking solution BSA, react at room temperature for 8 h to block the remaining binding sites on the surface of the magnetic beads, magnetically separate, wash with washing buffer, and finally redisperse in washing buffer to obtain the immunomagnetic bead capture nanoparticles.

[0064] (2) Preparation of silver nanoparticle sol: 200 ml of 1 mM AgNO3 aqueous solution was heated to boiling, and 6 ml of 1% sodium citrate aqueous solution was added. The solution color gradually changed from colorless and transparent to milky white with a slight green tinge. The mixture was kept at a gentle boil for 3 h, then the reaction was stopped and cooled in a water bath. Ag nanoparticles with a particle size of approximately 80 nm were obtained and stored away from light. To synthesize silver nanoparticles of different sizes, the amount of sodium citrate added can be changed.

[0065] (3) Preparation of immunosilver signal nanoparticles: 800 μL of silver nanoparticles were mixed with 8 μL of 30 μM Raman reporter molecule IR-808, incubated at room temperature for 15 min, centrifuged, and the supernatant was discarded. The mixture was then redispersed in PBS-T (PBS solution with 4% TW-20 solution added). 20 μL of 200 μg / mL detection antibody was added at -4℃ and incubated for 20 h. After washing with PBS-T, the remaining active sites were blocked with blocking solution, incubated at room temperature, centrifuged, washed with PBS-T solution, and finally the supernatant was discarded. The mixture was then redispersed in blocking solution to obtain immunosilver signal nanoparticles.

[0066] (4) Preparation of sandwich structure: 20 μL of the prepared immunomagnetic bead-capturing nanoparticles and 200 μL of Escherichia coli O157:H7 at different concentrations (1 CFU / ml, 5 CFU / ml, 10 ... 2 CFU / ml, 10 3 CFU / ml, 10 4 CFU / ml, 10 5 After mixing with CFU / ml, the mixture was incubated at room temperature for 20 min, magnetically separated, washed once with 200 μL PBS-T solution, and then mixed with 40 μL of immunosilver signaling nanoparticles. The mixture was then incubated at room temperature for 20 min to form a sandwich structure.

[0067] (5) Detection of Escherichia coli O157:H7: The precipitate that forms the Meiji sandwich structure was taken out and dropped onto the gold-coated aluminum foil. After drying, Raman detection was performed directly using a portable Raman spectrometer (laser wavelength 785nm). The Raman spectrum results of Escherichia coli O157:H7 were rapidly detected within 22 minutes.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A rapid and ultrasensitive method for detecting Escherichia coli O157:H7 based on SERS, characterized in that, Includes the following steps: S1: Preparation of immunomagnetic bead capture nanoparticles: The capture antibody combination of Escherichia coli O157:H7 was modified onto carboxyl magnetic beads through coupling to form immunomagnetic bead capture nanoparticles. S2: Preparation of noble metal or noble metal composite nanoparticle sol; S3: Preparation of immunonoble metal or noble metal composite signal nanoparticles: The noble metal or noble metal composite nanoparticle sol prepared in step S2 is mixed with Raman reporter molecule IR-808 to obtain noble metal or noble metal composite signal nanoparticles; then mixed with detection antibody of Escherichia coli O157:H7; the active site is blocked to obtain immunonoble metal or noble metal composite signal nanoparticles. S4: Preparation of sandwich structure: Immunomagnetic bead capture nanoparticles and immunometal signal nanoparticles are mixed with the sample to be tested to obtain a sandwich structure; S5: Detection of Escherichia coli O157:H7: The precipitate with the sandwich structure was taken out and dropped onto a substrate with a gold film on the surface for Raman detection to obtain the Raman spectrum of Escherichia coli O157:H7.

2. The method for rapid and ultrasensitive detection of Escherichia coli O157:H7 based on SERS as described in claim 1, characterized in that, In step S1, the immunomagnetic beads capture nanoparticles are magnetic and modified with capture antibodies that specifically recognize Escherichia coli O157:H7.

3. The method for rapid and ultrasensitive detection of Escherichia coli O157:H7 based on SERS as described in claim 1, characterized in that, Step S1 specifically includes taking 200-800 μL of carboxyl-modified magnetic beads with a concentration of 1-10 μg / μL, adding washing buffer, vortexing to fully suspend the magnetic beads, and magnetically removing the supernatant; resuspending the magnetic beads in washing buffer, adding 6-10 μL of 8-12 mg / mL EDC and NHS to activate for 20-40 min, then adding 2-20 μL of 200 μg / mL capture antibody, incubating at 4℃ for 20-28 h, magnetically removing the supernatant, washing with washing buffer, resuspending in PBS solution, then adding 100-5000 μL of 0.5-1.5 wt% blocking solution, reacting at room temperature for 1-5 h to block the remaining binding sites on the surface of the magnetic beads, magnetic separation, washing with washing buffer, and finally redispersing in washing buffer to obtain immunomagnetic bead capture nanoparticles.

4. The method for rapid and ultrasensitive detection of Escherichia coli O157:H7 based on SERS as described in claim 3, characterized in that, The magnetic beads have a particle size of 0.2-5 μm.

5. The method for rapid and ultrasensitive detection of Escherichia coli O157:H7 based on SERS as described in claim 3, characterized in that, The washing buffer is prepared from 1-200 mM inorganic salt buffer with pH 5.5-8, 1-10 wt% surfactant, and 15-100 mM inorganic chloride salt. The inorganic salt buffer includes, but is not limited to, one of phosphate buffer, citrate-sodium citrate buffer, borate-borax buffer, and citrate-borax buffer. The surfactant includes, but is not limited to, one of TW-20, TW-60, TW-80, CHAPS, and TRITON-X100. The inorganic chloride salt includes, but is not limited to, one of NaCl, KCl, and MgCl2.

6. The method for rapid and ultrasensitive detection of Escherichia coli O157:H7 based on SERS as described in claim 3, characterized in that, The blocking solution is prepared from 1-20 wt% BSA, 1-200 mM phosphate buffer (pH 5.5-8), 15-100 mM inorganic chloride salt, and 1-10 wt% surfactant. The BSA includes, but is not limited to, one of casein, glycine, and skim milk. The surfactant includes, but is not limited to, one of Triton-X400, TW-60, TW-80, CHAPS, and TRITON-X100. The inorganic chloride salt includes, but is not limited to, one of NaCl, KCl, and MgCl2.

7. The method for rapid and ultrasensitive detection of Escherichia coli O157:H7 based on SERS as described in claim 1, characterized in that, Step S3 specifically includes mixing 200-800 μL of noble metal or noble metal composite nanoparticles with 2-12 μL of 10-100 μM Raman reporter molecule IR-808, incubating at room temperature for 1-15 min, centrifuging, discarding the supernatant, redispersing in PBS-T solution, adding 10-60 μg of detection antibody at -4℃ and incubating for 20-28 h, washing with PBS-T, adding blocking solution to block the remaining active sites, incubating at room temperature, centrifuging, washing with PBS-T solution, finally discarding the supernatant, redispersing in blocking solution, and obtaining immunonoble metal or noble metal composite signal nanoparticles.

8. The method for rapid and ultrasensitive detection of Escherichia coli O157:H7 based on SERS as described in claim 7, characterized in that, The volume ratio of the metal nanoparticles to the Raman reporter molecule is (50-500):(1-30), the concentration of the Raman reporter molecule IR-808 is 40-60 μM, the incubation time at room temperature is 5-30 min, and the PBS-T solution is PBS solution with 1-10 wt% TW-20 added.

9. The method for rapid and ultrasensitive detection of Escherichia coli O157:H7 based on SERS as described in claim 1, characterized in that, Step S4 specifically includes mixing immunomagnetic bead-capturing nanoparticles and E. coli O157:H7, incubating at room temperature for 0.2-5 h, washing with PBS-T solution after magnetic separation, and then mixing with immunoCu2O@Ag signaling nanoparticles, incubating at room temperature for 0.2-5 h, washing with PBS-T solution after magnetic separation, to obtain a sandwich-structured precipitate.

10. The method for rapid and ultrasensitive detection of Escherichia coli O157:H7 based on SERS as described in claim 1, characterized in that, The substrate in step S5 includes, but is not limited to, silicon wafers and aluminum foil. Raman spectroscopy testing is performed using a portable Raman spectrometer or other types of Raman spectrometers, with the excitation wavelength preferably being 785 nm.

Images