SERS (Surface Enhanced Raman Scattering) rapid detection method for bacterial meningitis pathogenic bacteria in liquid

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) rapid detection method for bacterial meningitis pathogenic bacteria in liquid, and relates to the field of SERS rapid detection of bacteria. The invention aims to solve the problems of complex detection process, long detection period and low positive rate of detection results in the existing common detection method for bacterial meningitis pathogenic bacteria, and the problems of poor salt solution induced particle aggregation effect and poor SERS detection data quality in the existing SERS detection method. The method comprises the following steps: 1, pretreating pathogenic bacteria samples; 2, a rich SERS hot spot area is rapidly formed on the surface of the pathogenic bacteria; 3, preparing a pathogenic bacteria SERS detection sample; and 4, collecting SERS data of the pathogenic bacteria by using a Raman spectrometer. According to the invention, SERS rapid detection of five pathogenic bacteria of common bacterial meningitis of infants can be realized, and compared with the prior art, the enrichment effect of SERS particles on the surfaces of thalli is improved, the SERS data quality of the pathogenic bacteria is improved, and the detection time is shortened.

Description

Technical Field

[0001] The invention relates to the field of bacterial SERS rapid detection, and in particular to a SERS rapid detection method for bacterial meningitis pathogens in liquid. Background Art

[0002] Bacterial meningitis is a very common and serious central nervous system inflammation caused by pathogens crossing the blood-brain barrier and invading the cerebral cortex, causing intracranial infection. It causes nearly one million deaths worldwide each year, with an acute mortality rate of more than 40%. Infants and young children are vulnerable to various conditional pathogens because their central nervous system functions and blood-brain barrier functions are not fully developed, and their immune system is weak, causing serious intracranial infection. Clinical statistics show that more than 12,600 infants and young children died of meningitis within 28 days of birth worldwide, and the total number of deaths from meningitis in infants and young children under 5 years old exceeded 91,100; and more than 21% of infants and young children may have sequelae such as mental retardation and motor development retardation. Among them, Escherichia coli, Haemophilus influenzae, Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus pneumoniae are the five pathogens with the highest clinical incidence in infants and young children with bacterial meningitis. Both China and Europe's clinical practice guidelines for bacterial meningitis clearly state that rapid diagnosis and rapid and accurate antimicrobial treatment are the only way to increase patient survival rates and improve patient prognosis.

[0003] The clinical diagnosis of bacterial meningitis is generally based on clinical symptoms combined with blood biochemical indicators. There is no clear diagnostic basis, which can easily lead to missed diagnoses or misdiagnoses. Pathogen analysis methods such as polymerase chain reaction, metagenomic sequencing, flow cytometry and other methods have complex operating procedures, require professional personnel to operate, and the detection cycle generally takes more than 20 hours. Cerebrospinal fluid pathogen culture is the gold standard for the identification of bacterial meningitis, but it generally requires a culture cycle of two to several days, and the positive rate of cerebrospinal fluid culture is extremely low. Existing methods cannot achieve rapid detection of bacterial meningitis pathogens.

[0004] SERS technology can provide fingerprint information of pathogens, and has the advantages of rapid response and rich information, meeting all the requirements for rapid detection of pathogens of bacterial meningitis in infants and young children. The auxiliary agglutination strategy has the advantages of simple operation process and rapid response, making it the most promising solution for rapid SERS detection of pathogens of bacterial meningitis in infants and young children. However, in existing studies, sodium chloride is used as agglutinating agent, and only cations are used as auxiliary groups to achieve the aggregation of SERS particles on the surface of pathogens. Anions do not play a relevant role, and the enrichment effect of SERS particles on the surface of pathogens is poor. The limited hotspot area leads to the problem of poor quality of the collected pathogen SERS data. Summary of the invention

[0005] The purpose of the present invention is to solve the problems of complex detection process, long detection cycle and low positive rate of detection results in the existing common bacterial meningitis pathogen detection methods, poor salt solution induced particle aggregation effect and poor SERS detection data quality in the existing SERS detection methods, and to provide a SERS rapid detection method for bacterial meningitis pathogens in liquid.

[0006] The present invention proposes sodium citrate as an auxiliary molecule for the rapid and large-scale aggregation of SERS particles on the surface of pathogens. Experimental results show that the combined action of citrate and sodium ions assists Au NPs to rapidly and densely aggregate on the surface of pathogenic bacteria, generating abundant SERS hotspot enhancement areas on the surface of the bacteria. The SERS rapid detection of five common bacterial meningitis pathogens in infants and young children was successfully achieved within 15 minutes.

[0007] The present invention prepares SERS particle colloidal solution for pathogen detection in advance, quickly forms SERS hotspot auxiliary liquid on the bacterial surface, uses simple instruments such as a low-speed centrifuge and a Raman spectrometer, and only performs centrifugation, mixing, SERS detection and other operations to achieve on-site SERS rapid detection of five common bacterial meningitis pathogens in infants and young children within 15 minutes.

[0008] A SERS rapid detection method for bacterial meningitis pathogens in liquid is specifically completed in the following steps:

[0009] 1. Pretreatment of pathogen samples:

[0010] ①, centrifuge the liquid sample of pathogens to be tested and remove the supernatant;

[0011] ②, repeat step 1① 1 to 3 times, and resuspend in ultrapure water of the same volume as the supernatant removed in step 1①, to obtain a pretreated liquid sample of pathogens to be detected;

[0012] 2. Rapid formation of abundant SERS hotspots on the surface of pathogenic bacteria:

[0013] ①. Heat a 0.01 mg / mL to 1 mg / mL chloroauric acid solution to boiling; add a 1 mg / mL to 50 mg / mL sodium citrate solution to the boiling chloroauric acid solution under stirring, continue stirring and heating to maintain boiling for a period of time, and cool to room temperature to obtain an AuNP colloidal solution; centrifuge the AuNP colloidal solution, remove the supernatant, add 1 / 4 of the original solution volume of ultrapure water to resuspend, and store in a refrigerator at 4°C in a sealed state away from light to obtain a SERS particle colloidal solution for pathogen detection;

[0014] ②, prepare sodium citrate solution, store in a refrigerator at 4℃, away from light, and seal to obtain auxiliary solution;

[0015] ③. Mix the pretreated liquid sample of pathogens to be detected with the SERS particle colloidal solution for pathogen detection, shake it thoroughly until it is evenly mixed, then add the auxiliary liquid, shake it thoroughly until it is evenly mixed, and then let it stand. The citrate and sodium ions work together to assist the AuNP particles to quickly and densely aggregate on the surface of the bacteria, so that a rich SERS hotspot enhancement area is generated on the surface of the pathogens to be detected, and the pathogens with SERS hotspot areas on the surface are obtained;

[0016] 3. Preparation of pathogen SERS detection samples:

[0017] The pathogenic bacteria with the SERS hotspot area on the surface are centrifuged, the supernatant is removed, ultrapure water is added and mixed evenly, and a pathogenic bacteria dispersion liquid with the SERS hotspot area on the surface is obtained; the pathogenic bacteria dispersion liquid with the SERS hotspot area on the surface is dripped onto a single crystal silicon wafer, and then evaporated to dryness, and a pathogenic bacteria SERS detection sample is obtained;

[0018] 4. Use Raman spectrometer to collect SERS data of pathogens:

[0019] The Raman spectrum parameters for pathogen collection are set as follows: the excitation wavelength is 785nm, the integration time is 1 to 30s, the average number of times is 1 to 3 times, and 5 to 50 sample points are collected for each pathogen SERS detection sample; if the SERS characteristic peaks of the five pathogens of bacterial meningitis are collected, such as 563cm -1 、663cm -1 、734cm -1 、892cm -1 、928cm -1 、960cm -1 、1002cm -1 、1030cm -1 、1126cm -1 、1243cm -1 、1267cm -1 、1319cm -1 、1470cm -1 、1575cm -1 、1582cm -1 、1597cm -1 , indicating that the pathogen liquid sample to be detected contains bacterial meningitis pathogens; that is, a SERS rapid detection method for bacterial meningitis pathogens in liquid is completed.

[0020] Advantages of the present invention:

[0021] 1. The present invention provides a new method for rapidly generating abundant SERS hotspot enhancement areas on the surface of pathogenic bacteria; the SERS detection interferences generated by the growth and metabolism of pathogenic bacteria are removed through the pathogen sample pretreatment process, and the pre-prepared SERS particle colloid solution and the auxiliary liquid for rapidly forming SERS hotspot areas on the surface of the bacteria are mixed with the pathogenic bacteria to be detected, and the SERS particles on the surface of the pathogenic bacteria are densely enriched for 3 minutes, thereby generating abundant SERS hotspot areas;

[0022] 2. The present invention provides a new SERS rapid detection method for bacterial meningitis pathogens; through experimental research, it is proposed for the first time that citrate and sodium ions work together to assist AuNP particles in rapid, large-scale and dense enrichment on the surface of pathogenic bacteria. Only through simple operations such as centrifugation, mixing, and standing, using small instruments and equipment such as low-speed centrifuges and Raman spectrometers, the SERS on-site rapid detection of five common pathogens in infants and young children was successfully achieved within 15 minutes;

[0023] 3. The SERS rapid detection method for bacterial meningitis pathogens provided by the present invention can not only be used for SERS rapid detection of bacterial meningitis pathogens in infants and young children, but can also be used for on-site rapid detection of pathogens in clinical bacterial infection analysis, conditional pathogen analysis in food, sterile laboratories and other scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of a SERS rapid detection method for bacterial meningitis pathogens in liquid according to the present invention;

[0025] Figure 2 is the characterization result of the SERS particles prepared in Example 1; wherein a is the SEM photo of the SERS particles; b is the UV-visible absorption spectrum result of the SERS particles; c is the hydrated particle size measurement result of the SERS particles; d is the particle size distribution statistical result of the SERS particles;

[0026] Figure 3 These are the SEM results of pathogens with SERS hotspots on their surfaces: a is a SEM photo of sodium chloride as the auxiliary liquid, which assists the rapid aggregation of SERS particles on the surface of Escherichia coli; b is a SEM photo of sodium citrate as the auxiliary liquid, which assists the rapid aggregation of Au NPs on the surface of Escherichia coli; c is a SEM photo of sodium citrate as the auxiliary liquid, which assists the rapid aggregation of Au NPs on the surface of Haemophilus influenzae; d is a SEM photo of sodium citrate as the auxiliary liquid, which assists the rapid aggregation of Au NPs on the surface of Staphylococcus aureus; e is a SEM photo of sodium citrate as the auxiliary liquid, which assists the rapid aggregation of Au NPs on the surface of Streptococcus agalactiae; f is a SEM photo of sodium citrate as the auxiliary liquid, which assists the rapid aggregation of Au NPs on the surface of Streptococcus pneumoniae;

[0027] Figure 4 These are the SERS detection results of five common pathogens in Examples 1 to 5, which are the Raman spectra of Escherichia coli, Haemophilus influenzae, Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus pneumoniae from top to bottom. DETAILED DESCRIPTION

[0028] Specific implementation method 1: This implementation method is a SERS rapid detection method for bacterial meningitis pathogens in liquid, which is specifically completed according to the following steps:

[0029] 1. Pretreatment of pathogen samples:

[0030] ①, centrifuge the liquid sample of pathogens to be tested and remove the supernatant;

[0031] ②, repeat step 1① 1 to 3 times, and resuspend in ultrapure water of the same volume as the supernatant removed in step 1①, to obtain a pretreated liquid sample of pathogens to be detected;

[0032] 2. Rapid formation of abundant SERS hotspots on the surface of pathogenic bacteria:

[0033] ①. Heat a 0.01 mg / mL to 1 mg / mL chloroauric acid solution to boiling; add a 1 mg / mL to 50 mg / mL sodium citrate solution to the boiling chloroauric acid solution under stirring, continue stirring and heating to maintain boiling for a period of time, and cool to room temperature to obtain an AuNP colloidal solution; centrifuge the AuNP colloidal solution, remove the supernatant, add 1 / 4 of the original solution volume of ultrapure water to resuspend, and store in a refrigerator at 4°C in a sealed state away from light to obtain a SERS particle colloidal solution for pathogen detection;

[0034] ②, prepare sodium citrate solution, store in a refrigerator at 4℃, away from light, and seal to obtain auxiliary solution;

[0035] ③. Mix the pretreated liquid sample of pathogens to be detected with the SERS particle colloidal solution for pathogen detection, shake it thoroughly until it is evenly mixed, then add the auxiliary liquid, shake it thoroughly until it is evenly mixed, and then let it stand. The citrate and sodium ions work together to assist the AuNP particles to quickly and densely aggregate on the surface of the bacteria, so that a rich SERS hotspot enhancement area is generated on the surface of the pathogens to be detected, and the pathogens with SERS hotspot areas on the surface are obtained;

[0036] 3. Preparation of pathogen SERS detection samples:

[0037] The pathogenic bacteria with the SERS hotspot area on the surface are centrifuged, the supernatant is removed, ultrapure water is added and mixed evenly, and a pathogenic bacteria dispersion liquid with the SERS hotspot area on the surface is obtained; the pathogenic bacteria dispersion liquid with the SERS hotspot area on the surface is dripped onto a single crystal silicon wafer, and then evaporated to dryness, and a pathogenic bacteria SERS detection sample is obtained;

[0038] 4. Use Raman spectrometer to collect SERS data of pathogens:

[0039] The Raman spectrum parameters for pathogen collection are set as follows: the excitation wavelength is 785nm, the integration time is 1 to 30s, the average number of times is 1 to 3 times, and 5 to 50 sample points are collected for each pathogen SERS detection sample; if the SERS characteristic peaks of the five pathogens of bacterial meningitis are collected, such as 563cm -1 、663cm -1 、734cm -1 、892cm -1 、928cm -1 、960cm -1 、1002cm -1 、1030cm -1 、1126cm -1 、1243cm -1 、1267cm -1 、1319cm -1 、1470cm -1 、1575cm -1 、1582cm -1 、1597cm -1 , indicating that the pathogen liquid sample to be detected contains bacterial meningitis pathogens; that is, a SERS rapid detection method for bacterial meningitis pathogens in liquid is completed.

[0040] This embodiment verifies the advancedness of the SERS rapid detection method of bacterial meningitis pathogens in liquids of the present invention based on laboratory subcultured Escherichia coli, Haemophilus influenzae, Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus pneumoniae, five pathogens with the highest clinical incidence in infant bacterial meningitis patients, and verifies the experimental results with the assistance of instruments such as scanning electron microscopes, ultraviolet-visible absorption spectrometers, and nanoparticle size analyzers. The SERS particle colloid solution for pathogen detection and the auxiliary liquid for rapid formation of SERS hotspots on the bacterial surface used in the SERS rapid detection method of pathogens can be prepared in advance, and the storage validity period is more than 3 months.

[0041] Specific implementation method 2: The difference between this implementation method and specific implementation method 1 is that the pathogens to be detected in step 1① are one or more of Escherichia coli, Haemophilus influenzae, Staphylococcus aureus, Streptococcus agalactiae and Streptococcus pneumoniae; in step 1①, 0.1mL to 3mL of the pathogen liquid sample to be detected with an OD value of 0.08 to 0.2 is loaded into a centrifuge tube, and then centrifuged, the centrifuge speed is 2000rpm to 6000rpm, and the centrifuge time is 1min to 10min. The other steps are the same as those in specific implementation method 1.

[0042] Specific implementation method three: This implementation method is different from specific implementation method one or two in that: the method of repeating step ① once in step 1② is: adding ultrapure water of the same volume as the supernatant removed in step 1①, fully oscillating until the pathogens are evenly dispersed, and then centrifuging, the centrifugal speed is 2000rpm / min to 6000rpm / min, and the centrifugal time is 1min to 10min. The other steps are the same as those in specific implementation method one or two.

[0043] Specific embodiment 4: This embodiment differs from specific embodiments 1 to 3 in that the volume ratio of the chloroauric acid solution with a concentration of 0.01 mg / mL to 1 mg / mL and the sodium citrate with a concentration of 1 mg / mL to 50 mg / mL described in step 2① is (50 mL to 100 mL): (3 mL to 8 mL). The other steps are the same as those of specific embodiments 1 to 3.

[0044] Specific embodiment 5: This embodiment differs from specific embodiments 1 to 4 in that: the stirring speed in step 2① is 700 rpm to 1100 rpm; in step 2①, stirring is continued and heating is maintained to maintain boiling for 30 min to 40 min. The other steps are the same as those in specific embodiments 1 to 4.

[0045] Specific embodiment 6: This embodiment differs from specific embodiments 1 to 5 in that: the centrifugal speed in step 2 ① is 6000 rpm to 12000 rpm; the concentration of the sodium citrate solution in step 2 ② is 50 mmol / L to 1000 mmol / L. The other steps are the same as those in specific embodiments 1 to 5.

[0046] Specific embodiment 7: The difference between this embodiment and specific embodiments 1 to 6 is that: the volume ratio of the pre-treated liquid sample of pathogens to be detected and the SERS particle colloidal solution for pathogen detection described in step 2 ③ is (0.1mL~1mL):(0.1mL~1mL); the volume ratio of the pre-treated liquid sample of pathogens to be detected and the auxiliary liquid described in step 2 ③ is (0.1mL~1mL):(10μL~200μL); the standing time described in step 2 ③ is 1min~10min. The other steps are the same as specific embodiments 1 to 6.

[0047] Specific embodiment eight: This embodiment differs from specific embodiments one to seven in that: the centrifugal speed in step three is 2000rpm~6000rpm, and the centrifugal time is 1min~10min; the volume ratio of the ultrapure water in step three to the pre-treated liquid sample of pathogens to be detected in step two (3) is (0.1mL~1mL):(0.1mL~1mL). The other steps are the same as specific embodiments one to seven.

[0048] Specific embodiment 9: This embodiment differs from specific embodiments 1 to 8 in that: in step 3, 0.01 mL to 1 mL of pathogen dispersion liquid with a surface SERS hotspot is dripped onto a single crystal silicon wafer with a thickness of 100 μm to 1000 μm, and then naturally evaporated to obtain a pathogen SERS detection sample. The other steps are the same as those of specific embodiments 1 to 8.

[0049] Specific embodiment 10: The difference between this embodiment and specific embodiments 1 to 9 is that: in step 4, the typing of bacterial meningitis pathogens can be achieved according to the peak position attribution of the SERS characteristic peaks;

[0050] For example, the collected Raman data contains 663cm -1 、892cm -1 、928cm -1 、1030cm -1 、1319cm -1 、1582cm -1 The characteristic peak near the wave number is bacterial meningitis caused by Escherichia coli;

[0051] For example, the collected Raman data contains 663cm -1 、960cm -1 、1002cm -1 、1030cm -1 、1126cm -1 、1470cm -1 、1575cm -1The characteristic peak near the wave number is bacterial meningitis caused by Haemophilus influenzae;

[0052] For example, the collected Raman data contains 563cm -1 、663cm -1 、734cm -1 、928cm -1 、960cm -1 、1267cm -1 、1319cm -1 、1470cm -1 、1575cm -1 The characteristic peak near the wave number is bacterial meningitis caused by Staphylococcus aureus;

[0053] For example, the collected Raman data contains 663cm -1 、734cm -1 、960cm -1 、1002cm -1 、1030cm -1 、1319cm -1 、1470cm -1 、1575cm -1 The characteristic peak near the wave number is bacterial meningitis caused by Streptococcus agalactiae;

[0054] For example, the collected Raman data contains 663cm -1 、734cm -1 、960cm -1 、1030cm -1 、1126cm -1 、1319cm -1 、1470cm -1 、1582cm -1 The characteristic peak near the wave number is bacterial meningitis caused by Streptococcus pneumoniae. The other steps are the same as those in the first to ninth embodiments.

[0055] The following examples are used to verify the beneficial effects of the present invention:

[0056] Example 1: A SERS rapid detection method for bacterial meningitis pathogens in liquid is specifically completed according to the following steps:

[0057] 1. Pretreatment of pathogen samples:

[0058] ①. Load 2 mL of the pathogen liquid sample to be tested with an OD value of 0.1 into a centrifuge tube, set the centrifugal speed to 5000 rpm, and the centrifugal time to 2 min; remove the supernatant after centrifugation;

[0059] The pathogen to be detected in step 1① is Escherichia coli;

[0060] ②, repeat step 1① twice, and resuspend in ultrapure water of the same volume as the supernatant removed in step 1①, to obtain a pretreated liquid sample of pathogens to be detected;

[0061] The method of repeating step ① once in step ② is: adding ultrapure water of the same volume as the supernatant removed in step ①, fully shaking until the pathogens are evenly dispersed, and then centrifuging at a speed of 5000 rpm / min for 2 min;

[0062] 2. Rapid formation of abundant SERS hotspots on the surface of pathogenic bacteria:

[0063] ①. Heat a 0.1 mg / mL chloroauric acid solution to boiling; add a 10 mg / mL sodium citrate solution to the boiling chloroauric acid solution under stirring, continue stirring and heating to maintain boiling for a period of time, and cool to room temperature to obtain an AuNP colloidal solution; centrifuge the AuNP colloidal solution, remove the supernatant, add 1 / 4 of the original solution volume of ultrapure water to resuspend, and store in a refrigerator at 4°C in a sealed state away from light to obtain a SERS particle colloidal solution for pathogen detection;

[0064] The volume ratio of the 0.1 mg / mL chloroauric acid solution to the 10 mg / mL sodium citrate solution described in step 2① is 30:1;

[0065] The stirring speed in step 2① is 900 rpm;

[0066] In step 2①, continue stirring and heating to maintain boiling for 40 minutes;

[0067] The centrifugal speed in step 2① is 8000 rpm;

[0068] ②, prepare sodium citrate solution, store in a refrigerator at 4℃, away from light, and seal to obtain auxiliary solution;

[0069] The concentration of the sodium citrate solution described in step 2② is 100mmol / L;

[0070] ③. Place 1 mL of the pretreated liquid sample of the pathogen to be detected in a 5 mL centrifuge tube, add 1 mL of the SERS particle colloid solution for pathogen detection to the centrifuge tube, mix it, shake it for 5 seconds, add 60 μL of auxiliary liquid, shake it for 5 seconds, and let it stand for 3 minutes. The citrate and sodium ions work together to assist the AuNP particles to quickly and densely aggregate on the surface of the bacteria, so that a rich SERS hotspot enhancement area is generated on the surface of the pathogen to be detected, and the pathogen with a SERS hotspot area on the surface is obtained;

[0071] 3. Preparation of pathogen SERS detection samples:

[0072] ① Centrifuge the pathogens with SERS hotspots on the surface, remove the supernatant, add 1 mL of ultrapure water and mix well to obtain a pathogen dispersion with SERS hotspots on the surface; drop 20 μL of the pathogen dispersion with SERS hotspots on the surface on a single crystal silicon wafer with a size of 0.6 cm × 0.6 cm and a thickness of 450 μm, and then place it on a sample drying table and dry it at 50 ° C to obtain a pathogen SERS detection sample;

[0073] The centrifugal speed in step 3① is 5000 rpm and the centrifugal time is 2 min;

[0074] 4. Use Raman spectrometer to collect SERS data of pathogens:

[0075] The Raman spectrum parameters for pathogen collection were set as follows: the excitation light wavelength was 785 nm, the integration time was 5 s, the average number was 1, 30 sample points were collected for each pathogen SERS detection sample, and the SERS characteristic peaks were collected, thus completing a SERS rapid detection method for bacterial meningitis pathogens in liquids.

[0076] Example 2: The difference between this example and Example 1 is that the pathogen to be detected in step 1① is Haemophilus influenzae. The other steps and parameters are the same as those in Example 1.

[0077] Example 3: The difference between this example and Example 1 is that the pathogen to be detected in step 1① is Staphylococcus aureus. The other steps and parameters are the same as those in Example 1.

[0078] Example 4: This example is different from Example 1 in that the pathogen to be detected in step 1① is Streptococcus agalactiae. Other steps and parameters are the same as those in Example 1.

[0079] Example 5: The difference between this example and Example 1 is that the pathogen to be detected in step 1① is Streptococcus pneumoniae. The other steps and parameters are the same as those in Example 1.

[0080] Comparative Example: The difference between this example and Example 1 is that in step 2②, a sodium chloride solution is prepared and placed in a refrigerator at 4°C in a sealed state and protected from light to obtain an auxiliary liquid; the concentration of the sodium chloride solution in step 2② is 100 mmol / L. The other steps and parameters are the same as those in Example 1.

[0081] Figure 2is the characterization result of the SERS particles prepared in Example 1; wherein a is the SEM photo of the SERS particles; b is the UV-visible absorption spectrum result of the SERS particles; c is the hydrated particle size measurement result of the SERS particles; d is the particle size distribution statistical result of the SERS particles;

[0082] from Figure 2 From the SEM photo of the SERS particles in Figure a, it can be seen that the prepared SERS particles are spherical particles of uniform size; from the UV-vis results in Figure b, it can be seen that the SERS particle colloidal solution has only one extremely narrow absorption peak, indicating that the SERS particles are uniform in size and have good dispersion; the hydrated particle size measurement result in Figure c is 85nm; Figure d is the particle size statistics result of the SERS particles in the SEM photo, and the SERS particles are uniform in size with an average particle size of 80.83nm.

[0083] Figure 3 These are the SEM results of pathogens with SERS hotspots on their surfaces: a is a SEM photo of sodium chloride as the auxiliary liquid, which assists the rapid aggregation of SERS particles on the surface of Escherichia coli; b is a SEM photo of sodium citrate as the auxiliary liquid, which assists the rapid aggregation of Au NPs on the surface of Escherichia coli; c is a SEM photo of sodium citrate as the auxiliary liquid, which assists the rapid aggregation of Au NPs on the surface of Haemophilus influenzae; d is a SEM photo of sodium citrate as the auxiliary liquid, which assists the rapid aggregation of Au NPs on the surface of Staphylococcus aureus; e is a SEM photo of sodium citrate as the auxiliary liquid, which assists the rapid aggregation of Au NPs on the surface of Streptococcus agalactiae; f is a SEM photo of sodium citrate as the auxiliary liquid, which assists the rapid aggregation of Au NPs on the surface of Streptococcus pneumoniae;

[0084] contrast Figure 3 In Figures a and b, under the same conditions, sodium chloride and sodium citrate rapidly form SERS hotspots on the surface of Escherichia coli. In Figure a, sodium chloride is used as an auxiliary agent, and the SERS particles are scattered around the Escherichia coli, with very few particles gathering on the surface of the bacteria. In Figure b, sodium citrate is used as an auxiliary agent, and the number of SERS particles gathering on the surface of the bacteria is larger, and the particles on the surface of the bacteria are more tightly gathered. From the SERS particle aggregation effects of the two auxiliary agents in Figures a and b, it can be seen that the effect of citrate and sodium ions jointly assisting the rapid and large-scale aggregation of AuNPs on the surface of the bacteria is significantly better than the effect of sodium ions alone as auxiliary agents assisting the aggregation of AuNPs on the surface of the bacteria. From Figures c to f, the SEM results of the rapid aggregation of SERS particles on the surfaces of the other four pathogens show that sodium citrate as an auxiliary agent has good versatility in the rapid aggregation of SERS particles on the surface of pathogens, and a large number of SERS particles are generated on the surfaces of the four pathogens.

[0085] Figure 4These are the SERS detection results of five common pathogens in Examples 1 to 5, which are the Raman spectra of Escherichia coli, Haemophilus influenzae, Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus pneumoniae from top to bottom.

[0086] from Figure 4 The SERS rapid detection results of five common clinical pathogens of bacterial meningitis obtained in Examples 1 to 5 show that the SERS characteristic peaks of pathogens such as 563 cm -1 、663cm -1 、734cm -1 、892cm -1 、928cm -1 、960cm -1 、1002cm -1 、1030cm -1 、1126cm -1 、1243cm -1 、1267cm -1 、1319cm -1 、1470cm -1 、1575cm -1 、1582cm -1 、1597cm -1 The method of the present invention has successfully realized the SERS rapid detection of five pathogens; the SERS characteristic peaks of different pathogens are not exactly the same, which is conducive to the rapid typing of five common pathogens of bacterial meningitis in infants and young children.

[0087] It can be seen from the results of the above test examples that the SERS rapid detection method for bacterial meningitis pathogens in liquid of the present invention can achieve SERS rapid detection of five common bacterial meningitis pathogens in infants and young children within 15 minutes through simple operations such as centrifugal washing, mixing and oscillation, and SERS collection. The SERS characteristic peaks of different types of pathogens are not exactly the same, which is conducive to the rapid typing of pathogens.

[0088] The present invention proposes a SERS rapid detection method for bacterial meningitis pathogens in liquid, which combines a pre-prepared SERS particle colloidal solution and an auxiliary liquid for rapidly forming a SERS hotspot area on the bacterial surface. Through simple operations such as centrifugal washing, mixing and oscillation, and SERS collection, the SERS rapid detection of five common pathogens can be achieved within 15 minutes.

[0089] In addition to being suitable for the rapid detection of five common bacterial meningitis pathogens in infants and young children, the present invention is also suitable for on-site rapid detection of pathogens in scenarios such as clinical bacterial infection pathogen analysis, conditional pathogen analysis in food, and sterile laboratories.

Claims

1. A SERS rapid detection method for bacterial meningitis pathogens in liquid, characterized in that The method is specifically completed according to the following steps:

1. Pretreatment of pathogen samples: ①, centrifuge the liquid sample of pathogens to be tested and remove the supernatant; ②, repeat step 1① 1 to 3 times, and resuspend in ultrapure water of the same volume as the supernatant removed in step 1①, to obtain a pretreated liquid sample of pathogens to be detected; 2. Rich SERS hotspots are rapidly formed on the surface of pathogenic bacteria: ①. Heat a 0.01 mg / mL to 1 mg / mL chloroauric acid solution to boiling; add a 1 mg / mL to 50 mg / mL sodium citrate solution to the boiling chloroauric acid solution under stirring, continue stirring and heating to maintain boiling for a period of time, and cool to room temperature to obtain an AuNP colloidal solution; centrifuge the AuNP colloidal solution, remove the supernatant, add 1 / 4 of the original solution volume of ultrapure water to resuspend, and store in a refrigerator at 4°C in a sealed state away from light to obtain a SERS particle colloidal solution for pathogen detection; ②, prepare sodium citrate solution, store in a refrigerator at 4℃, away from light, and seal to obtain auxiliary solution; ③. Mix the pretreated liquid sample of pathogens to be detected with the SERS particle colloidal solution for pathogen detection, shake it thoroughly until it is evenly mixed, then add the auxiliary liquid, shake it thoroughly until it is evenly mixed, and then let it stand. The citrate and sodium ions work together to assist the AuNP particles to quickly and densely aggregate on the surface of the bacteria, so that a rich SERS hotspot enhancement area is generated on the surface of the pathogens to be detected, and the pathogens with SERS hotspot areas on the surface are obtained; 3. Preparation of pathogen SERS detection samples: The pathogenic bacteria with the SERS hotspot area on the surface are centrifuged, the supernatant is removed, ultrapure water is added and mixed evenly, and a pathogenic bacteria dispersion liquid with the SERS hotspot area on the surface is obtained; the pathogenic bacteria dispersion liquid with the SERS hotspot area on the surface is dripped onto a single crystal silicon wafer, and then evaporated to dryness, and a pathogenic bacteria SERS detection sample is obtained; 4. Use Raman spectrometer to collect SERS data of pathogens: The Raman spectrum parameters for pathogen collection are set as follows: the excitation wavelength is 785nm, the integration time is 1 to 30s, the average number of times is 1 to 3 times, and 5 to 50 sample points are collected for each pathogen SERS detection sample; if the SERS characteristic peaks of the five pathogens of bacterial meningitis are collected, such as 563cm -1 、663cm -1 、734cm -1 、892cm -1 、928cm -1 、960cm -1 、1002cm -1 、1030cm -1 、1126cm -1 、1243cm -1 、1267cm -1 、1319cm -1 、1470cm -1 、1575cm -1 、1582cm -1 、1597cm -1 , indicating that the pathogen liquid sample to be detected contains bacterial meningitis pathogens; that is, a SERS rapid detection method for bacterial meningitis pathogens in liquid is completed.

2. The SERS rapid detection method for bacterial meningitis pathogens in liquid according to claim 1, characterized in that The pathogen to be detected described in step 1① is one or more of Escherichia coli, Haemophilus influenzae, Staphylococcus aureus, Streptococcus agalactiae and Streptococcus pneumoniae; in step 1①, 0.1mL~3mL of the liquid sample of the pathogen to be detected with an OD value of 0.08~0.2 is loaded into a centrifuge tube, and then centrifuged, the centrifugal speed is 2000rpm~6000rpm, and the centrifugal time is 1min~10min.

3. The SERS rapid detection method for bacterial meningitis pathogens in liquid according to claim 1, characterized in that The method of repeating step ① once in step ② is: adding ultrapure water of the same volume as the supernatant removed in step ①, fully oscillating until the pathogens are evenly dispersed, and then centrifuging, the centrifugal speed is 2000rpm / min~6000rpm / min, and the centrifugal time is 1min~10min.

4. The SERS rapid detection method for bacterial meningitis pathogens in liquid according to claim 1, characterized in that The volume ratio of the chloroauric acid solution with a concentration of 0.01 mg / mL to 1 mg / mL and the sodium citrate solution with a concentration of 1 mg / mL to 50 mg / mL described in step 2① is (50 mL to 100 mL): (3 mL to 8 mL).

5. The SERS rapid detection method for bacterial meningitis pathogens in liquid according to claim 1, characterized in that The stirring speed in step 2① is 700 rpm to 1100 rpm; in step 2①, stirring is continued and heating is maintained to maintain boiling for 30 min to 40 min.

6. The SERS rapid detection method for bacterial meningitis pathogens in liquid according to claim 1, characterized in that The centrifugal speed described in step 2① is 6000rpm~12000rpm; The concentration of the sodium citrate solution described in step 2② is 50mmol / L~1000mmol / L.

7. The SERS rapid detection method for bacterial meningitis pathogens in liquid according to claim 1, characterized in that The volume ratio of the pretreated liquid sample of pathogens to be detected described in step 2③ to the SERS particle colloidal solution for pathogen detection is (0.1mL~1mL):(0.1mL~1mL); the volume ratio of the pretreated liquid sample of pathogens to be detected described in step 2③ to the auxiliary liquid is (0.1mL~1mL):(10μL~200μL); the standing time described in step 2③ is 1min~10min.

8. The SERS rapid detection method for bacterial meningitis pathogens in liquid according to claim 1, characterized in that The centrifugal speed described in step 3 is 2000rpm~6000rpm, and the centrifugal time is 1min~10min; The volume ratio of the ultrapure water described in step 3 and the pretreated liquid sample of pathogens to be detected described in step 2③ is (0.1mL~1mL):(0.1mL~1mL).

9. The SERS rapid detection method for bacterial meningitis pathogens in liquid according to claim 1, characterized in that In step three, 0.01 mL to 1 mL of pathogen dispersion with a surface SERS hotspot area is dropped onto a single crystal silicon wafer with a thickness of 100 μm to 1000 μm, and then naturally evaporated to obtain a pathogen SERS detection sample.

10. The SERS rapid detection method for bacterial meningitis pathogens in liquid according to claim 1, characterized in that In step 4, the typing of bacterial meningitis pathogens can be achieved according to the peak position attribution of the SERS characteristic peaks; For example, the collected Raman data contains 663cm -1 、892cm -1 、928cm -1 、1030cm -1 、1319cm -1 、1582cm -1 The characteristic peak near the wave number is bacterial meningitis caused by Escherichia coli; For example, the collected Raman data contains 663cm -1 、960cm -1 、1002cm -1 、1030cm -1 、1126cm -1 、1470cm -1 、1575cm -1 The characteristic peak near the wave number is bacterial meningitis caused by Haemophilus influenzae; For example, the collected Raman data contains 563cm -1 、663cm -1 、734cm -1 、928cm -1 、960cm -1 、1267cm -1 、1319cm -1 、1470cm -1 、1575cm -1 The characteristic peak near the wave number is bacterial meningitis caused by Staphylococcus aureus; For example, the collected Raman data contains 663cm -1 、734cm -1 、960cm -1 、1002cm -1 、1030cm -1 、1319cm -1 、1470cm -1 、1575cm -1 The characteristic peak near the wave number is bacterial meningitis caused by Streptococcus agalactiae; For example, the collected Raman data contains 663cm -1 、734cm -1 、960cm -1 、1030cm -1 、1126cm -1 、1319cm -1 、1470cm -1 、1582cm -1 The characteristic peak near the wave number is bacterial meningitis caused by Streptococcus pneumoniae.

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