Double-layer filtration device and detection method for simultaneous detection of multiple bacteria

Abstract

The application discloses a double-layer filtering device and a detection method for simultaneously detecting multiple bacteria. The double-layer filtering device comprises a liquid input device, a hollow filter pipe, a liquid output device, a large-pore fiber membrane and a small-pore fiber membrane. The liquid input device is connected with a water inlet of the hollow filter pipe, and the water inlet is covered with the large-pore fiber membrane. The liquid output device is connected with a water outlet of the hollow filter pipe, and the water outlet is covered with the small-pore fiber membrane. The double-layer filtering device can be used for rapidly processing water samples and SERS analysis, and the provided detection method has the advantages of simplicity, rapidity, low cost, anti-interference ability and robustness, can be applied to the field, opens up a new way for promoting the development of multifunctional analysis tools in drinking water and food safety monitoring management, and has important significance for water quality detection and control.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of filtration and detection, and relates to a double-layer filtration device and a detection method for simultaneous detection of multiple bacteria, in particular to a portable double-layer filtration and concentration device capable of directly detecting and decoding multiple bacteria using SERS and controlling water quality, and a method for simultaneously detecting and decoding multiple bacteria in water using SERS. BACKGROUND

[0002] At present, the discussion on water pollution treatment is very popular worldwide. Water pollution can cause the following hazards. First, it can harm human health. Second, it can reduce the yield and quality of crops, affect the yield and quality of fishery production, restrict the development of industry, accelerate the degradation and destruction of the ecological environment, and cause economic losses. These are the serious water pollution problems we are currently facing. The World Health Organization (WHO) survey shows that 80% of diseases and 50% of child mortality are related to poor drinking water quality. About half of the hospital inpatients worldwide are due to pollution. The hazards caused by water pollution mainly include the following three aspects: water pollution by pathogenic microorganisms (such as bacteria) can cause cholera, typhoid fever, poliomyelitis, hepatitis A, dysentery, and other infectious diseases; water pollution by heavy metals (Hg, Pb, Cr, Cd, etc.) and other toxic inorganic substances (such as fluorides, arsenic compounds, and nitrite) can cause various poisoning and diseases; water pollution by organic substances such as phenol, benzene, chloroform, carbon tetrachloride, pesticides, and synthetic detergents can cause various poisoning and diseases such as blood diseases and cancer, especially organic nitro compounds and organic halogen compounds in water. They are strong carcinogens and tumor-causing substances for animals, plants, and humans, which have attracted widespread attention and attention from the international scientific and medical communities.

[0003] Pathogenic microorganisms are considered a serious threat to human health in developed, developing and underdeveloped countries. There is an increasing awareness that drinking water and water environments are easily contaminated with a variety of bacteria, leading to outbreaks of waterborne diseases. According to existing evidence, bacteria such as E. coli O157:H7, S. aureus, L. monocytogenes and P. aeruginosa have been found in groundwater, surface water and domestic sewage. These harmful waterborne pathogens can cause a series of life-threatening infectious diseases such as hemorrhagic colitis, hemolytic uremic syndrome, septicemia, etc. through direct drinking of contaminated water or indirect ingestion of fruits and vegetables irrigated with contaminated water. It has been agreed that timely detection and identification of bacterial infection can greatly improve the control of waterborne diseases. Therefore, the development of a rapid, accurate, low-cost and simple method for simultaneous detection of multiple pathogenic bacteria in water is of great significance to reduce or avoid the risk of human infection or loss of life.

[0004] Currently, the most mature technology for pathogenic bacteria detection is traditional culture and molecular amplification-based methods. Although these methods have made remarkable progress in pathogen identification and analysis, traditional culture methods usually require at least 24 hours to several days of long-term culture to obtain the final determination result, which leads to delayed analysis completion time and poses a threat to life safety. In addition, the identification of pathogenic bacteria is usually a single target analysis, which requires the use of specific culture media, and different pathogenic bacteria need to be cultured independently.

[0005] Therefore, the development of a rapid, accurate, low-cost and simple method for simultaneous detection of multiple pathogenic bacteria in water is of great significance to reduce or avoid the risk of human infection or loss of life. Molecular amplification methods include polymerase chain reaction (PCR), real-time polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP) and recombinant polymerase amplification (RPA), etc., which can amplify specific regions of nucleic acids, but still have some performance limitations, such as false positive amplification and cross contamination, especially when faced with multiple pathogens. In addition, the most common PCR and RT-PCR methods also require skilled technicians to handle specialized equipment and a certain level of professional knowledge to interpret the final results, limiting the popular application of molecular methods. Methods based on immunological principles have also been applied to bacterial detection. Previously reported methods based on immunological recognition require complex interface construction and multiple steps of immobilization, recognition and washing. They are not user-friendly in terms of ease of operation, sensitivity and efficiency. In addition, it is widely recognized that water samples are usually contaminated with different pathogenic bacteria. Current research mainly focuses on detecting a single target strain, which cannot meet the demand for accurate assessment of the types and concentrations of pathogenic bacteria in water. SUMMARY

[0006] The main purpose of the present application is to provide a double-layer filtering device and a detection method for simultaneous detection of multiple bacteria to overcome the shortcomings of the prior art.

[0007] To achieve the above-mentioned purposes, the technical solutions adopted by the present application include:

[0008] The double-layer filtering device for simultaneous detection of multiple bacteria provided by the embodiments of the present application comprises a liquid input device, a hollow filter tube, a liquid output device, a large-pore fiber membrane and a small-pore fiber membrane; the liquid input device is connected with a water inlet of the hollow filter tube, and the water inlet is covered with the large-pore fiber membrane; the liquid output device is connected with a water outlet of the hollow filter tube, and the water outlet is covered with the small-pore fiber membrane.

[0009] The embodiments of the present application also provide a method for simultaneous detection of multiple bacteria in water based on SERS, which comprises:

[0010] Providing gold nanoparticles;

[0011] Reacting the gold nanoparticles with Raman signal molecules to obtain gold nanoparticle Raman signal molecules; wherein the Raman signal molecules include any one of methylphenyl acetate, rhodamine 6G and methylene blue; and the gold nanoparticle Raman signal molecules include gold nanoparticle methylphenyl acetate, gold nanoparticle rhodamine 6G and gold nanoparticle methylene blue;

[0012] Carrying out a reduction reaction on the gold nanoparticle Raman signal molecules, L-ascorbic acid and Ag salt to prepare a gold nanoparticle Raman signal molecule / Ag complex;

[0013] Mixing and reacting the gold nanoparticle Raman signal molecule / Ag complex with bacterial antibodies, and then carrying out a bovine serum albumin blocking treatment to prepare a surface-enhanced Raman scattering label; the bacterial antibodies include any one of an anti-Escherichia coli O157:H7 antibody, an anti-Staphylococcus aureus antibody and an anti-Listeria antibody; and the surface-enhanced Raman scattering label includes an Escherichia coli O157:H7 surface-enhanced Raman scattering label, a Staphylococcus aureus surface-enhanced Raman scattering label and a Listeria surface-enhanced Raman scattering label;

[0014] In addition, the Escherichia coli O157:H7 surface-enhanced Raman scattering label, the Staphylococcus aureus surface-enhanced Raman scattering label and the Listeria surface-enhanced Raman scattering label are mixed and cultured with a water sample to be tested, and then the water sample after the culture is treated by the aforementioned double-layer filtering device, and then the small-pore fiber membrane in the double-layer filtering device is subjected to Raman detection, so as to determine whether the water sample to be tested contains Escherichia coli O157:H7, Staphylococcus aureus or Listeria.

[0015] Compared with the prior art, the application has the beneficial effects that the portable double-layer filtering and concentrating device for directly detecting and decoding multiple bacteria and controlling water quality by using SERS can simultaneously and accurately measure all target bacteria in the target water source in the range of 10 1 -10 6 CFU / mL within a short time, the detection limit is 10 CFU / mL, and the traditional pre-culturing procedure is not needed. The detection method has the advantages of simplicity, rapidity, low cost, anti-interference ability and robustness, and can be used for simultaneously detecting multiple pathogens in water samples. The application scheme opens up a new way for promoting the development of multifunctional analysis tools in drinking water and food safety monitoring management, and has important significance for water quality control in developing countries. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0017] FIG. 1A is a typical Raman signal spectrum of CVa, R6G and MB in a typical embodiment of the present application;

[0018] FIG. 1B is a single Raman spectrum result graph of CVa, R6G and MB in a typical embodiment of the present application;

[0019] FIGS. 1C-1D are Raman spectra of Au Tag , SERS-Tag in a typical embodiment of the present application, respectively;

[0020] FIGS. 2A-2B are electron microscope characterization graphs of Au nanoparticles and Au@Ag in a typical embodiment of the present application, respectively;

[0021] FIGS. 2C-2D are UV-Vis spectrum and photo of Au nanoparticles and Au@Ag in a typical embodiment of the present application, respectively;

[0022] FIG. 3A is a SEM image of a clean enrichment membrane in a typical embodiment of the present application;

[0023] FIG. 3B is a SEM image of drinking water without bacterial contamination in a typical embodiment of the present application;

[0024] FIG. 3C is a SEM image of the drinking water contaminated by E. coli O157:H7 in an exemplary embodiment of the present application;

[0025] FIG. 3D is a SEM image of an E. coli O157:H7 cell in an exemplary embodiment of the present application;

[0026] FIGS. 4A-4F are the dose-response SERS detection results of E. coli O157:H7, Staphylococcus aureus and Listeria, respectively, and the SERS intensity versus the logarithm of E. coli O157:H7, Staphylococcus aureus and Listeria;

[0027] FIGS. 5A-5C are the representative SERS spectra collected from the target mixtures of E. coli O157:H7 / Staphylococcus aureus, E. coli O157:H7 / Lactobacillus monocytogenes, and Staphylococcus aureus / Listeria, respectively;

[0028] FIG. 6A are the SERS decoding diagrams for simultaneously detecting E. coli O157:H7, Staphylococcus aureus and Listeria in drinking water at different concentrations;

[0029] FIG. 6B is a gel electrophoresis analysis diagram of the PCR amplification products of the bacteria enriched on the membrane surface;

[0030] FIG. 6C are the Raman intensity diagrams of the characteristic peaks caused by the target bacteria and non-target bacteria;

[0031] FIG. 6D is the detection of 10 3 CFU / mL of E. coli O157:H7, Staphylococcus aureus and Listeria from drinking water, deionized water and liquid beverage. DETAILED DESCRIPTION

[0032] In view of the defects of the prior art, the present inventors have long studied and practiced to propose the technical solution of the present application. The technical solution of the present application will be described clearly and completely below. Obviously, the described embodiments are some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0033] Specifically, as one aspect of the technical scheme of the present application, a double-layer filtering device for simultaneous detection of multiple bacteria comprises a liquid input device, a hollow filter tube, a liquid output device, a large-pore fiber membrane and a small-pore fiber membrane; the liquid input device is connected with a water inlet of the hollow filter tube, and the water inlet is covered with the large-pore fiber membrane; the liquid output device is connected with a water outlet of the hollow filter tube, and the water outlet is covered with the small-pore fiber membrane.

[0034] In some preferred embodiments, the liquid input device comprises a syringe, and is not limited thereto.

[0035] In some preferred embodiments, the liquid output device comprises a needle, and is not limited thereto.

[0036] In some preferred embodiments, the hollow filter tube comprises a hollow polystyrene filter tube, and is not limited thereto.

[0037] In some preferred embodiments, the large-pore fiber membrane has a pore size of 10-30 μm, for example, 30 μm.

[0038] In some preferred embodiments, the small-pore fiber membrane has a pore size of 200-220 nm, for example, 200 nm.

[0039] In some preferred embodiments, the large-pore fiber membrane and the small-pore fiber membrane both have a diameter of 5-15 mm, for example, a diameter of 15 mm.

[0040] Specifically, the frame of the double-layer filtering device for detection of multiple bacteria is composed of a syringe, a hollow polystyrene filter tube and cellulose membranes with different pore sizes (10-30 μm and 200-220 nm). The pore sizes of the different membranes are determined according to the actual use conditions and the existing commercial filter membranes. The filter membrane with a pore size of 10-30 μm can ensure the retention of most large particles, and the lower-layer filter membrane with a pore size of 200-220 nm can retain most bacteria on the membrane. In order to make the device, both of the cellulose membranes are cut into disc-shaped pieces with a diameter of 5-15 mm to cover the water inlet (with a diameter of 6 mm) and the water outlet (with a diameter of 4 mm). The hollow polystyrene tube is mechanically installed between the syringe and the needle.

[0041] Another aspect of the embodiment of the present application further provides a method for simultaneous detection of multiple bacteria in water based on SERS, which comprises:

[0042] providing gold nanoparticles;

[0043] reacting the gold nanoparticles with a Raman signal molecule to obtain gold nanoparticle Raman signal molecules; wherein the Raman signal molecule comprises any one of acetoxy methyl phenol purple, rhodamine 6G, methylene blue; and the gold nanoparticle Raman signal molecules comprise gold nanoparticle acetoxy methyl phenol purple, gold nanoparticle rhodamine 6G, and nanoparticle methylene blue.

[0044] reducing the gold nanoparticle Raman signal molecules with L-ascorbic acid and Ag salt to obtain gold nanoparticle Raman signal molecule / Ag complexes;

[0045] mixing the gold nanoparticle Raman signal molecule / Ag complexes with bacterial antibodies, and then blocking with bovine serum albumin to obtain surface-enhanced Raman scattering labels; wherein the bacterial antibodies comprise any one of anti-Escherichia coli O157:H7 antibody, anti-Staphylococcus aureus antibody, and anti-Listeria antibody; and the surface-enhanced Raman scattering labels comprise Escherichia coli O157:H7 surface-enhanced Raman scattering labels, Staphylococcus aureus surface-enhanced Raman scattering labels, and Listeria surface-enhanced Raman scattering labels.

[0046] Further, the Escherichia coli O157:H7 surface-enhanced Raman scattering labels, Staphylococcus aureus surface-enhanced Raman scattering labels, and Listeria surface-enhanced Raman scattering labels are mixed with a water sample to be tested, and then the water sample to be tested after the mixing is treated by the aforementioned double-layer filter device, and then the small-pore-size fiber membrane in the double-layer filter device is subjected to Raman detection, so as to determine whether the water sample to be tested contains Escherichia coli O157:H7, Staphylococcus aureus, or Listeria.

[0047] In some preferred embodiments, the method specifically comprises: adding chloroauric acid into heated trisodium citrate and mixing and reacting to obtain the gold nanoparticles.

[0048] In some preferred embodiments, the method specifically comprises: mixing and reacting acetoxy methyl phenol purple, rhodamine 6G, and methylene blue with gold nanoparticles respectively at room temperature for 10-30 min, and then purifying to obtain gold nanoparticle acetoxy methyl phenol purple, gold nanoparticle rhodamine 6G, and nanoparticle methylene blue.

[0049] In some preferred embodiments, the method specifically comprises: ultrasonically reacting L-ascorbic acid and Ag salt with gold nanoparticle acetoxy methyl phenol purple, gold nanoparticle rhodamine 6G, and nanoparticle methylene blue respectively, and then centrifuging to obtain gold nanoparticle acetoxy methyl phenol purple / Ag complexes, gold nanoparticle rhodamine 6G / Ag complexes, and nanoparticle methylene blue / Ag complexes.

[0050] In some preferred embodiments, the method specifically comprises: dispersing the gold nanoparticle methylphenol purple acetate / Ag complex, the gold nanoparticle rhodamine 6G / Ag complex and the nanoparticle methylene blue / Ag complex in sterile water respectively and adjusting the pH value to 8.0-10.0, then adding the anti-E. coli O157:H7 antibody, the anti-S. aureus antibody and the anti-Listeria antibody respectively, reacting at room temperature, then blocking with bovine serum albumin and centrifuging to obtain the E. coli O157:H7 surface-enhanced Raman scattering label, the S. aureus surface-enhanced Raman scattering label and the Listeria surface-enhanced Raman scattering label.

[0051] In some preferred embodiments, the method specifically comprises: mixing the E. coli O157:H7 surface-enhanced Raman scattering label, the S. aureus surface-enhanced Raman scattering label and the Listeria surface-enhanced Raman scattering label to obtain a surface-enhanced Raman scattering label mixture, and then adding the surface-enhanced Raman scattering label mixture to the water sample to be tested and incubating for 5-10 min.

[0052] Further, the volume ratio of the E. coli O157:H7 surface-enhanced Raman scattering label, the S. aureus surface-enhanced Raman scattering label and the Listeria surface-enhanced Raman scattering label in the surface-enhanced Raman scattering label mixture is 0.5-1.2:0.8-1.4:0.4-1.8.

[0053] Further, the mass ratio of the surface-enhanced Raman scattering label mixture to the water sample to be tested is 1-2:25-50.

[0054] In some preferred embodiments, the detection limit of E. coli O157:H7, S. aureus and Listeria in the method for simultaneously detecting multiple bacteria in water based on SERS is 10 CFU / mL.

[0055] In some more specific embodiments, the method for simultaneously detecting multiple bacteria in water based on SERS specifically comprises the following steps:

[0056] (1) Preparation of gold nanoparticles AuNPs: 5 g / L chloroauric acid 750-1000 μL was added to 50-100 mL of deionized water and stirred vigorously, after heating and boiling, 1-3% trisodium citrate 800-1000 μL was immediately added, and after boiling for 15 min, it was naturally cooled to room temperature to prepare gold nanoparticles AuNPs;

[0057] (2) Preparation of different types of gold nanoparticle Raman signal molecules Au Tag (Au CVa , Au R6G , Au MBRaman signaling molecules (Tag): 10 μL of 100 μM acetocresol purple (CVa), 10 μL of 100 μM rhodamine 6G (R6G), and 50 μL of 10 μM methylene blue (MB) were mixed with 1-3 mL of AuNPs. The mixture was then shaken at room temperature for 10-30 min to allow the Tag molecules to completely bind to the AuNPs, thus obtaining various Au... Tag (Au CVa Au R6G Au MB After purification by centrifugation at 8000-8500g, it is then suspended in 1-5mL of sterile water;

[0058] (3) Preparation of Au Tag @Ag (i.e., the aforementioned gold nanoparticle Raman signaling molecule / Ag complex): based on AgNO3 in Au Tag Surface reduction reaction, in 1-3 mL of various Au Tag Add 100-500 μL of L-ascorbic acid to the solution, stir for 10-30 min, and then quickly add 300-500 μL of 1% AgNO3 under sonication. Stop sonicating when the color of the reaction solution changes from purple-red to orange-yellow. Centrifuge completely at 8000-10000 g and then suspend in 1-3 mL of sterile water.

[0059] (4) Preparation of surface-enhanced Raman scattering (SERS-Tag) tags: Synthesized SERS-Tags (Escherichia coli O157:H7 surface-enhanced Raman scattering tag SERS-TagCVa, Staphylococcus aureus surface-enhanced Raman scattering tag SERS-TagR6G, and Listeria monocytogenes surface-enhanced Raman scattering tag SERS-TagMB), namely anti-Escherichia coli O157:H7 and Au cVa @Ag conjugate antibody (Au CVa @Ag@Anti-E-Ab), anti-Staphylococcus aureus and Au R6G @Ag conjugate antibody (Au R6G @Ag@Anti-S-Ab) and anti-Listeria with Au MB @Ag conjugate antibody (Au MB @Ag@Anti-L-Ab); First, use K2CO3 to react Au CVa @Ag、Au R6G @Ag or Au MB Adjust the pH of GAg to 8.0-10.0. Then, take 4 μL of 1 mg / mL anti-Escherichia coli O157:H7 antibody, anti-Staphylococcus aureus antibody, and anti-Listeria antibody and add them to 1-3 mL of GAg solution. CVa @Ag、Au R6G @Ag or Au MBAg mixing. After 1-2 h of reaction at room temperature, the free antibody and bovine serum albumin were removed by centrifugation at 8000-10000 g for 10-30 min after blocking with 20-50 μL of 10% bovine serum albumin for 1-5 h. Finally, the final product was resuspended in 1 mL of stock solution (10 mM PBS) for subsequent use.

[0060] (5) A mixture of different surface-enhanced Raman scattering tags (SERS-Tag) was added to the water sample for co-culture for 10 min. The water sample after co-culture was quickly treated by using the double-layer filter device constructed in the foregoing, and then Raman detection was performed, so as to determine whether the water sample to be tested contains E. coli O157:H7, Staphylococcus aureus, or Listeria.

[0061] As preferred, in step (1), 750 μL of chloroauric acid was added to 5 g / L of deionized water and stirred vigorously.

[0062] As preferred, in step (1), after the heating and boiling, 800 μL of 1% trisodium citrate was immediately added, and after boiling for 15 min, the solution was naturally cooled to room temperature.

[0063] As preferred, in step (2), the AuNPs were 1 mL, and the mixture was shaken at room temperature for 10 min.

[0064] As preferred, in step (2), the AuNPs were 1 mL, and the mixture was shaken at room temperature for 10 min. Tag After purification by centrifugation at 8000 g, the AuNPs were resuspended in 1 mL of sterile water.

[0065] As preferred, in step (3), 100 μL of L-ascorbic acid was added to 1 mL of each type of AuNPs. Tag

[0066] As preferred, in step (3), after stirring for 10 min, 300 μL of 1% AgNO3 was quickly added under ultrasonic conditions, and the ultrasonic treatment was stopped when the color of the reaction solution changed from purple red to orange yellow. After complete centrifugation at 8000-10000 g, the AuNPs were resuspended in 1-3 mL of sterile water.

[0067] As preferred, in step (3), after complete centrifugation at 8000 g, the AuNPs were resuspended in 1 mL of sterile water.

[0068] As preferred, in step (4), the Au@Ag, Au@Ag, or Au@Ag was adjusted to pH 8.0 by using K2CO3. CVa @Ag, Au R6G @Ag, or Au MB @Ag.

[0069] ​As preferred, after the reaction at room temperature for 1 h, 20 μL of 10% bovine serum albumin is used for blocking for 1 h, and 10000 g centrifugation for 10 min is used to remove free antibodies and bovine serum albumin.

[0070] The portable double-layer filtration and concentration device made of a common syringe is used for rapid processing of water samples and SERS analysis in the present application. The core components of the device are two filters with different pore sizes, which are used for respective functions. The upper filter is used for preliminary interception of large interfering substances (interception membrane), and the lower filter is used for collection of various target pathogens (enrichment membrane) for analysis. This combination can make the contaminated environmental water (for example, surface water) quickly pass through the device for filtration, and only the target bacteria such as E. coli O157:H7, S. aureus and L. monocytogenes are retained on the lower enrichment membrane. Combined with surface-enhanced Raman spectroscopy (SERS), SERS-Tag (SERS-Tag CVa, SERS-Tag R6G and SERS-Tag MB) labeled on the enriched bacteria by an immune recognition effect is decoded, so that the rapid separation, concentration and detection of various pathogenic bacteria from a large amount of contaminated environmental water are realized. The results show that all target bacteria in the lake water can be simultaneously and accurately measured in the range of 10 1 -10 6 CFU / mL in a short time, the detection limit is 10 CFU / mL, and the traditional pre-culture procedure is not required. The present application emphasizes the simplicity, rapidity, inexpensiveness, anti-interference ability and robustness of the constructed method, and can be used for simultaneous detection of various pathogens in water samples. The technical solutions of the present application are further described in detail in combination with several preferred embodiments and the accompanying drawings. The embodiments are implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0071] In the following examples, the experimental materials used in the examples are commercially available from conventional biochemical reagent companies unless otherwise specified.

[0072] Embodiment

[0073] Preparation of the double-layer filtration device: The frame of the device is composed of a syringe, a hollow polystyrene filter tube and cellulose membranes with different pore sizes (30 μm and 200 nm). Both of the cellulose membranes are cut into disc shapes with a diameter of 15 mm to cover the water inlet (diameter 6 mm) and the water outlet (diameter 4 mm). The hollow polystyrene tube is mechanically installed between the syringe and the needle.

[0074] (1) Preparation of gold nanoparticles AuNPs: 5 g / L of chloroauric acid 750 μL was added into 50 mL of deionized water and stirred vigorously, after heating to boiling, 1 wt% of trisodium citrate 800 μL was immediately added, and after boiling for 15 minutes, it was naturally cooled to room temperature to prepare gold nanoparticles AuNPs;

[0075] (2) Preparation of different types of gold nanoparticles Raman signal molecules Au Tag (Au cVa , Au R6G , Au MB ) corresponding to different types of bacteria: Raman signal molecules Tag: 10 μL of 100 μM cresyl violet acetate (CVa), 10 μL of 100 μM rhodamine 6G (R6G), 50 μL of 10 μM methylene blue (MB) were mixed with 1 mL of AuNPs, and the mixture was shaken at room temperature for 10-30 min to allow the Tag molecules to fully bind to AuNPs. After purification by centrifugation at 8000 g, each type of Au Tag (Au CVa , Au R6G , Au MB ) was resuspended in 1 mL of sterile water.

[0076] (3) Preparation of Au Tag @Ag (i.e. the aforementioned gold nanoparticle Raman signal molecule / Ag complex): based on the reduction reaction of AgNO3 on the surface of Au Tag , 100 μL of L-ascorbic acid was added to 1 mL of each type of Au Tag , stirred for 10 min, then 300 μL of 1 wt% AgNO3 was quickly added under ultrasonic conditions, and the ultrasonic was stopped when the color of the reaction solution changed from purple red to orange yellow. After centrifugation at 8000 g, it was resuspended in 1 mL of sterile water.

[0077] (4) Preparation of surface enhanced Raman scattering tag SERS-Tag: synthesis of SERS-Tag (Escherichia coli O157:H7 surface enhanced Raman scattering tag SERS-Tag CVa, Staphylococcus aureus surface enhanced Raman scattering tag SERS-Tag R6G and Listeria surface enhanced Raman scattering tag SERS-Tag MB), i.e. anti-E. coli O157:H7 and Au CVa @Ag conjugated antibody (Au CVa @Ag@Anti-E-Ab), anti-Staphylococcus aureus and Au R6G @Ag conjugated antibody (Au R6G GAgGAnti-S-Ab) and anti-Listeria and Au MB @Ag conjugated antibody (Au MBAg, Anti-L-Ab); Au was first coated with K2CO3 CVa GAg, Au R6G GAg or Au MB GAg was pH adjusted to 8.0, then, 4 μL of 1 mg / mL of Anti-E. coli O157:H7 antibody, Anti-S. aureus antibody, and Anti-Listeria antibody were added to 1-3 mL of Au CVa GAg, Au R6G @Ag or Au MB GAg was mixed. After h of reaction at room temperature, 20 μL of 10 wt% bovine serum albumin was added to block for 1 h, and centrifuged at 10,000 g for 10 min to remove free antibody and bovine serum albumin. Finally, the final product was resuspended in 1 mL of stock solution (10 mM PBS) for later use.

[0078] (5) A mixture of different SERS-Tag was added to the water sample and incubated for 10 min. The water sample after incubation was quickly treated by the double-layer filter device constructed above, and then Raman detection was performed to determine whether the water sample contained E. coli O157:H7, S. aureus, or Listeria.

[0079] FIG. 1A The typical Raman spectra of CVa, R6G, and MB are shown in the figure, and their characteristic peaks are located at 586 cm -1 , 1501 cm -1 , and 1613 cm -1 , respectively (the insets in the figure are their structural formulas). FIG. 1B The Raman spectra of CVa, R6G, and MB are shown in the figure, FIG. 1C and FIG. 1D are the Raman spectra of Au Tag and SERS-Tag, respectively. In combination with FIG. 1A the results in 1D of the figure, the Raman intensity of the step enhancement shows a better Raman enhancement effect. The present application is used for Raman spectral research and Raman enhancement effect for multiplex analysis. Selecting a group of Raman spectral molecules without spectral interference is a prerequisite for realizing SERS simultaneous multiplex detection. As shown in FIG. 1A , CVa, R6G, and MB have obvious characteristic peaks at 586 cm -1 , 1501 cm -1 , and 1614 cm -1 , respectively. Since these Raman spectral results have their own characteristic spectra, the distance between them is at least 100 cm -1Therefore, simultaneous multi-channel detection with these recorders is technically possible. The illustrations show the structural models of CVa, R6G, and MB, respectively. Secondly, the SERS-Tag should possess a strong Raman enhancement effect, so that its enhanced Raman signal is beneficial for improving sensitivity. FIG. 1B The results show that this invention verifies the SERS effect; direct Raman testing of CVA only produces a weak signal, while when CVA is at Au... CVa When @Ag adheres to the Au@Ag surface, significant Raman signal enhancement can be observed. This remarkable enhancement lays the foundation for accurate and sensitive detection of target bacteria. Similarly, the validation results for the enhancement effects of R6G and MB are as follows: FIG. 1C and FIG. 1D As shown in the figure, these results lay a solid foundation for the simultaneous detection of multiple pathogenic bacteria in drinking water using the designed detection method.

[0080] AuNPs and Au were analyzed using TEM and UV-Vis. Tag @Ag(with Au) CVa The size and morphology of (taking @Ag as an example) were characterized. FIG. 2A The image shows highly uniformly dispersed spherical AuNPs with an average size of 20 nm. After reduction of their surface with AgNO3, [the following text appears to be incomplete and requires further context: "in..."] FIG. 2B In the study, a uniform silver shell with a thickness of approximately 2 nm can be clearly observed covering the surface of AuNPs. Meanwhile, FIG. 2C The corresponding UV-Vis measurements showed that AgNO3 treatment of AuNPs caused the absorption peak to blue shift from 524 nm to 403 nm, indicating the formation of a silver shell. FIG. 2D The display shows that the dark (burgundy) AuNPs have turned into light (orange-yellow) AuNPs. CVa @Ag. All these comparative results demonstrate the successful fabrication of functional AuNPs and Au for SERS measurements. Tag @Ag.

[0081] The morphology of the enrichment membrane (small-pore cellulose membrane) was characterized using SEM. To verify the enrichment performance of the lower enrichment membrane, the morphology of the enrichment membrane before and after enrichment of the target bacteria was observed using scanning electron microscopy, as shown below. FIGS. 3A-3D As shown. FIG. 3A The results demonstrate the high porosity of the original enrichment membrane before use. When treating sterile water with this enrichment membrane, such as... FIG. 3B The results show that despite the adhesion of some tiny particles, the membrane's pore structure remains excellent. Conversely, membranes containing 1×10⁻⁶ particles exhibit poor pore structure. 5 The filtration results of contaminated water with CFU / mL E. coli O157:H7 are as follows: FIG. 3CAs shown, a large number of bacteria were wrapped on the surface of the enrichment membrane. One collected bacterial cell was magnified by TEM, and FIG. 3D In the middle, it can be clearly seen that many small size SERS-Tag CVa were tightly distributed on the surface of E. coli O157:H7 by antigen-antibody recognition. These morphological characterization results strongly proved the feasibility of the double-layer filtration enrichment device of the present application, which can effectively separate and concentrate the target pathogenic bacteria on the membrane while performing multiple detections.

[0082] FIG. 4A 、 FIG. 4C 、 FIG. 4E The dose-response surface-enhanced Raman spectroscopy detection results of E. coli O157:H7, Staphylococcus aureus and Listeria, respectively, are shown in FIGS. 7, 8 and 9. The target concentrations from low to high are 0, 1 x 10 0 , 1 x 10 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 and 1 x 10 6 CFU / mL. The logarithmic relationship diagrams of the SERS intensity of the 586 cm -1 ( FIG. 4B ), 1501 cm -1 ( FIG. 4D ) and 1614 cm -1 ( FIG. 4F ) peaks and E. coli O157:H7, Staphylococcus aureus and Listeria, respectively, are shown in FIGS. 7, 8 and 9. The transmission ability of the designed filtration device to one pathogenic bacteria was tested. FIGS. 4A-4F The detailed recorded Raman signals and the corresponding linear responses to the target bacterial concentrations are depicted. In FIG. 4A , for the detection of E. coli O157:H7, typically, as the E. coli O157:H7 concentration in the water sample increased, the number of bacteria collected on the enrichment membrane also increased accordingly. The intensity of the characteristic peak (586 cm -1 ) of CVa also increased ( FIG. 4A ) as the E. coli O157:H7 content in the water increased. The linear response relationship was Y CVa = 1515.961gx-21.72, and the correlation coefficient was R 2 = 0.9929, where Y cVa and X represent the Raman signal intensity of CVa at 586 cm -1 and the logarithmic concentration of the target E. coli O157:H7, respectively ( FIG. 4B ). Similarly, the detection results of Staphylococcus aureus and Listeria are shown in FIGS. 4C-4F . The linear regression equations for Staphylococcus aureus and Listeria were YR6G =609.99X + 9.94(R) 2 =0.9854) and Y MB =981.61X + 79.89(R) 2 =0.9942). For the detection results of all these target bacteria, 1×10 1 The CFU / mL signal was well distinguishable from the blank control, indicating that this method has good sensitivity for detecting bacteria in water. The excellent detection performance is attributed to the effective removal of potential interference from the upper large-pore membrane and the efficient retention of target bacteria on the lower small-pore membrane for SERS measurements. Compared with other measurement methods, this invention significantly outperforms them in terms of sensitivity, linear range, target species, detection time, and even detection cost.

[0083] FIG. 5A Escherichia coli O157:H7 / Staphylococcus aureus FIG. 5B Escherichia coli O157:H7 / Lactobacillus mononucleosis and FIG. 5C Representative SERS spectra collected from a target mixture of Staphylococcus aureus / Listeria. To detect the combination of Escherichia coli O157:H7 and Staphylococcus aureus in water samples, in... FIG. 5A The Raman results show that CVa at 586 cm⁻¹ can be observed simultaneously. -1 (For E. coli O157:H7) and R6G 1501cm -1 (For Staphylococcus aureus) Two characteristic peaks. It is noteworthy that the intensity of these two peaks indicates a significant concentration dependence for the corresponding target bacteria. According to... FIGS. 4A-4F The linear response relationship constructed in the model was used to calculate the recovery rate. This method can accurately and reliably determine the number of target bacteria without interference from other coexisting bacteria, achieving satisfactory recovery rates and RSDs even in complex samples. These studies strongly demonstrate that the filtration device of this invention is fully capable of simultaneously determining multiple bacteria in the same water sample.

[0084] FIG. 6A To simultaneously perform SERS decoding on different concentrations of Escherichia coli O157:H7, Staphylococcus aureus, and Listeria in drinking water. FIG. 6B To analyze the polymerase chain reaction amplification products of bacteria enriched on the membrane surface using gel electrophoresis. The concentration of each bacterium was 10. 3 CFU / mL; FIG. 6C Figure D shows the Raman intensities of characteristic peaks caused by target bacteria and non-target bacteria. Figure D is a sample of 10 samples detected from drinking water, deionized water, and liquid beverages. 3 CFU / mL of Escherichia coli O157:H7, Staphylococcus aureus, and Listeria.

[0085] FIGS. 6A-6D The filtration-assisted SERS method was used to evaluate samples containing a mixture of three bacteria. FIG. 6A The results show that the portable dual-layer filtration and concentration device of the present invention, which can directly utilize SERS to simultaneously detect and decode multiple bacteria and control water quality, can accurately decode and measure the corresponding characteristic peaks of each target bacteria for quantitative analysis. FIG. 6B The results further confirmed the accuracy of the detection. The specificity of a portable dual-layer filtration and concentration device that can directly utilize SERS to simultaneously detect and decode multiple bacteria and control water quality was evaluated. For example... FIG. 6C As shown, a positive Raman signal can only be observed on its characteristic peaks when the target *Escherichia coli* O157:H7, *Staphylococcus aureus*, and *Listeria* are present. Compared with the blank control sample ( FIG. 6C Compared to the Raman signal intensity results of the control group, the Raman signal intensity results of the other four non-target bacteria, including *Escherichia coli*, *Pseudomonas aeruginosa*, and *Staphylococcus enteritidis*, were also the same as those of the blank control, indicating that these non-target bacteria did not produce Raman signals. For the three target bacteria, *Escherichia coli* O157:H7, *Staphylococcus aureus*, and *Listeria*, the obvious Raman signals could be easily distinguished compared with the non-target bacteria and the blank control group, indicating that the device of this invention has good specificity and can be used for rapid and multi-bacterial detection and identification. Furthermore, utilizing the versatility of this invention, we compared the multi-pathogen spectra of drinking water samples from deionized water and liquid beverages at the same concentration of various target bacteria. The Raman signal values ​​in drinking water were... FIG. 6D The value is defined as 100%. The calculated relative response values ​​of Raman signals in deionized water and liquid beverages are very consistent with the relative response values ​​of Raman signals in drinking water, indicating that the portable dual-layer filtration and concentration device of the present invention, which can directly utilize SERS to simultaneously detect and decode multiple bacteria and control water quality, has universality and good reverse matrix effect.

[0086] The results in summary demonstrate that this invention can be used for the direct and simultaneous analysis of multiple target bacteria in water samples. It can analyze all target bacteria in a target water source within a short time (10 minutes). 1 -10 6 This invention enables simultaneous and accurate measurement within the CFU / mL range, with a detection limit of 10 CFU / mL, and eliminates the need for traditional pre-culture procedures. The invention emphasizes the simplicity, speed, low cost, interference resistance, and robustness of the developed method, which can be used for the simultaneous detection of multiple pathogens in water samples. This invention opens new avenues for promoting the development of multifunctional analytical tools in drinking water and food safety monitoring and management, and is of great significance for water quality control in developing countries.

[0087] In addition, the inventors of the present application also refer to the foregoing examples, and other raw materials, process operations, process conditions described in the specification are tested, and ideal results are obtained.

[0088] It should be understood that the technical solutions of the present application are not limited to the specific implementation cases described above, and any technical modification made according to the technical solutions of the present application without departing from the purpose of the present application and the scope protected by the claims falls within the protection scope of the present application.

Claims

1. A method for simultaneous detection of multiple bacteria in water based on SERS, characterized in that, include: Provides gold nanoparticles; Acetyl cresol purple, rhodamine 6G, and methylene blue were mixed with gold nanoparticles and reacted at room temperature for 10-30 min. After purification, gold nanoparticles containing acetyl cresol purple, rhodamine 6G, and methylene blue were obtained. L-ascorbic acid and Ag salt were subjected to ultrasonic reactions with gold nanoparticles acetocresol purple, gold nanoparticles rhodamine 6G and gold nanoparticles methylene blue, respectively, and then centrifuged to obtain gold nanoparticle acetocresol purple / Ag complex, gold nanoparticles rhodamine 6G / Ag complex and gold nanoparticles methylene blue / Ag complex. The gold nanoparticle acetocresol purple / Ag complex, the gold nanoparticle rhodamine 6G / Ag complex, and the gold nanoparticle methylene blue / Ag complex were dispersed in sterile water and the pH was adjusted to 8.0-10.

0. Then, anti-Escherichia coli O157:H7 antibody, anti-Staphylococcus aureus antibody, and anti-Listeria antibody were added and reacted at room temperature. After blocking with bovine serum albumin and centrifugation, surface-enhanced Raman scattering (SERS) tags for Escherichia coli O157:H7, Staphylococcus aureus, and Listeria were obtained. Furthermore, the surface-enhanced Raman scattering (SMR) tags of *E. coli* O157:H7, *S. aureus*, and *Listeria* are mixed to obtain a SMR tag mixture. This mixture is then added to the water sample to be tested and incubated for 5-10 minutes. The incubated water sample is then treated using a double-layer filtration device, and Raman spectroscopy is performed on the small-pore fiber membrane in the double-layer filtration device to determine the presence of *E. coli* O157:H7, *S. aureus*, or *Listeria* in the water sample. The volume ratio of the *E. coli* O157:H7 SMR tag, *S. aureus* SMR tag, and *Listeria* SMR tag mixture is 0.5~1.2:0.8~1.4:0.4~1.8; the mass ratio of the SMR tag mixture to the water sample is 1~2:25~50. The detection limit for Escherichia coli O157:H7, Staphylococcus aureus, and Listeria in the method for simultaneous detection of multiple bacteria in water based on SERS is 10 CFU / mL. The dual-layer filtration device includes a liquid input device, a hollow filter tube, a liquid output device, a large-pore fiber membrane, and a small-pore fiber membrane. The liquid input device is connected to the inlet of the hollow filter tube, and the inlet is covered with a large-pore fiber membrane. The liquid output device is connected to the outlet of the hollow filter tube, and the outlet is covered with a small-pore fiber membrane. The pore size of the large-pore fiber membrane is 10-30 μm, and the pore size of the small-pore fiber membrane is 200-220 nm. The diameters of both the large-pore and small-pore fiber membranes are 5-15 mm.

2. The method according to claim 1, characterized in that: The liquid input device includes a syringe; the liquid output device includes a needle; and the hollow filter tube includes a hollow polystyrene filter tube.

3. The method according to claim 1, characterized in that, Specifically, it includes: The gold nanoparticles were prepared by mixing and reacting chloroauric acid with trisodium citrate under heating conditions.

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