Double-layer filtering device for simultaneously detecting multiple bacteria and detection method

Abstract

The invention discloses a double-layer filtering device for simultaneously detecting various bacteria and a detection method. The double-layer filtering device comprises a liquid input device, a hollow filtering pipe, a liquid output device, a large-aperture fiber membrane and a small-aperture fiber membrane; the liquid input device is connected with a water inlet of the hollow filter tube, and the water inlet is covered with a large-aperture fiber membrane; the liquid output device is connected with the water outlet of the hollow filter tube, and the water outlet is covered with a small-aperture fiber membrane. The double-layer filtering device can be used for rapid water sample treatment and SERS analysis, meanwhile, the provided detection method has the advantages of simplicity, rapidness, cheapness, anti-jamming capability and robustness, can be applied to the field, opens up a new way for promoting development of multifunctional analysis tools in drinking water and food safety monitoring and management, and has a wide application prospect. The method is of great significance to water quality detection and control.

Description

Technical Field

[0001] The present invention belongs to the field of filtration and detection technology, and relates to a double-layer filtration device and a detection method for simultaneously detecting multiple bacteria, and in particular to a portable double-layer filtration and concentration device that can directly use SERS to simultaneously detect and decode multiple bacteria and control water quality, and a method for simultaneously detecting and decoding multiple bacteria in water using SERS. Background Art

[0002] At present, the discussion on water pollution control is very heated worldwide. Water pollution can cause the following hazards. First, it will harm human health. Secondly, it will reduce the yield and quality of crops, affect the yield and quality of fishery production, restrict industrial development, accelerate the degradation and destruction of the ecological environment, cause economic losses, etc. These are the serious water pollution problems we are currently facing. According to a survey by the World Health Organization (WHO), 80% of diseases and 50% of child mortality are related to poor drinking water quality. People who get sick because of pollution account for about half of the number of hospitalizations in hospitals around the world. The hazards caused by water pollution mainly include the following three aspects: water bodies polluted by pathogenic microorganisms (such as bacteria) can cause infectious diseases such as cholera, typhoid, poliomyelitis, hepatitis A, bacillary dysentery, etc.; water bodies polluted by heavy metals (Hg, Pb, Cr, Cd, etc.) and other toxic inorganic substances (such as fluoride, arsenide, nitrite, etc.) can cause various poisonings and diseases; water bodies polluted by organic substances such as phenol, benzene, chloroform, carbon tetrachloride, pesticides, synthetic detergents, etc. can cause various poisonings and diseases, such as blood diseases, cancer, etc., especially organic nitrate compounds and organic halogen compounds in water. They are strong carcinogenic and tumor-causing substances to animals, plants and humans, and have attracted widespread attention and attention from the international scientific and medical communities.

[0003] Pathogenic microorganisms are considered to be a serious threat to human health in developed, developing and underdeveloped countries. There is a growing awareness that drinking water and aquatic environments are easily contaminated by various bacteria, which can lead to outbreaks of waterborne diseases. According to existing evidence, bacteria such as Escherichia coli O157:H7 (E. coli O157:H7), Staphylococcus aureus (S. aureus), Listeria monocytogenes (L. monocytogenes) and Pseudomonas 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, sepsis, etc. through direct drinking of contaminated water or indirect ingestion of fruits and vegetables irrigated with contaminated water. There is a consensus that if bacterial infections can be detected and identified in a timely manner, the control of waterborne diseases will be greatly improved. Therefore, the development of a rapid, accurate, low-cost and simple method to simultaneously detect multiple pathogens in water is of great significance to reduce or avoid the risk of human infection or loss of life.

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

[0005] Therefore, the development of rapid, accurate, low-cost and simple detection methods that can simultaneously detect the presence of multiple pathogens in water is of great significance for reducing or avoiding 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), which can amplify specific regions of nucleic acids, but there are still some performance limitations, such as false positive amplification and cross-contamination, especially when facing multiple pathogens. In addition, the most common PCR and RT-PCR methods also require skilled technicians to handle specialized instruments and a certain level of expertise to interpret the final results, limiting the popularization and application of molecular methods. Methods based on immune principles have also been applied to bacterial detection. Previously reported methods based on immune recognition require complex interface construction and multiple steps of immobilization, recognition and washing. They are not user-friendly in terms of operational simplicity, sensitivity and efficiency. In addition, it is widely recognized that water samples are often contaminated with different pathogens. Current research mainly focuses on detecting a single target species, which cannot meet the needs of accurately assessing the types and concentrations of pathogens in water. Summary of the invention

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

[0007] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0008] An embodiment of the present invention provides a double-layer filtration device for simultaneous detection of multiple bacteria, which 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 water inlet of the hollow filter tube, and the water inlet is covered with a large-pore fiber membrane; the liquid output device is connected to the water outlet of the hollow filter tube, and the water outlet is covered with a small-pore fiber membrane.

[0009] The embodiment of the present invention also provides a method for simultaneously detecting multiple bacteria in water based on SERS, which comprises:

[0010] providing gold nanoparticles;

[0011] The gold nanoparticles are reacted with Raman signal molecules to obtain gold nanoparticle Raman signal molecules; wherein the Raman signal molecules include any one of acetocresyl violet, rhodamine 6G, and methylene blue; the gold nanoparticle Raman signal molecules include gold nanoparticles acetocresyl violet, gold nanoparticles rhodamine 6G, and nanoparticles methylene blue;

[0012] The gold nanoparticle Raman signal molecule is subjected to a reduction reaction with L-ascorbic acid and Ag salt to obtain a gold nanoparticle Raman signal molecule / Ag complex;

[0013] The gold nanoparticle Raman signal molecule / Ag complex is mixed with a bacterial antibody for reaction, and then blocked with bovine serum albumin to obtain a surface enhanced Raman scattering label; the bacterial antibody includes any one of an anti-Escherichia coli O157:H7 antibody, an anti-Staphylococcus aureus antibody, and an anti-Listeria monocytogenes antibody; 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 monocytogenes surface enhanced Raman scattering label;

[0014] Furthermore, 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 the water sample to be tested, and then the cultured water sample to be tested is treated with the aforementioned double-layer filtration device, and then Raman detection is performed on the small-pore fiber membrane in the double-layer filtration device to determine whether Escherichia coli O157:H7, Staphylococcus aureus or Listeria is present in the water sample to be tested.

[0015] Compared with the prior art, the invention has the following advantages: the portable double-layer filtration and concentration device provided by the invention can directly use SERS to simultaneously detect and decode multiple bacteria and control water quality, and can detect all target bacteria in the target water source within 10 1 -10 6 The detection method provided by the present invention is simple, rapid, cheap, anti-interference and robust, and can be used to simultaneously detect multiple pathogens in water samples. This invention scheme has opened up a new way to promote 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0018] Figure 1B is a graph showing individual Raman spectra of CVa, R6G and MB in a typical embodiment of the present invention;

[0019] Figure 1C-Figure 1D In a typical embodiment of the present invention, Au Tag , Raman spectrum of SERS-Tag;

[0020] Figure 2A-2B They are electron microscope characterization images of Au nanoparticles and Au@Ag in a typical embodiment of the present invention;

[0021] Figure 2C-Figure 2D They are respectively the UV-Vis spectra and photos of Au nanoparticles and Au@Ag in a typical embodiment of the present invention;

[0022] Figure 3A is a SEM image of a clean enrichment membrane in a typical embodiment of the present invention;

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

[0024] Figure 3C is a SEM image of drinking water contaminated by Escherichia coli O157:H7 in a typical embodiment of the present invention;

[0025] Figure 3D is a SEM image of an Escherichia coli O157:H7 cell in a typical embodiment of the present invention;

[0026] Figure 4A-4F The dose-response surface enhanced Raman spectroscopy detection results of Escherichia coli O157:H7, Staphylococcus aureus and Listeria monocytogenes and the logarithmic relationship between SERS intensity and Escherichia coli O157:H7, Staphylococcus aureus and Listeria monocytogenes are shown respectively;

[0027] Figure 5A-5C Representative SERS spectra collected from the target mixtures of Escherichia coli O157:H7 / Staphylococcus aureus, Escherichia coli O157:H7 / Lactobacillus monocytogenes, and Staphylococcus aureus / Listeria monocytogenes, respectively;

[0028] Fig. 6A To simultaneously perform SERS decoding of different concentrations of Escherichia coli O157:H7, Staphylococcus aureus, and Listeria in drinking water;

[0029] Figure 6B The figure shows the gel electrophoresis analysis of the polymerase chain reaction amplification products of the enriched membrane surface bacteria;

[0030] Figure 6C Raman intensity map of characteristic peaks caused by target bacteria and non-target bacteria;

[0031] Fig.6D 10 were detected from drinking water, deionized water and liquid beverages 3 CFU / mL of Escherichia coli O157:H7, Staphylococcus aureus, and Listeria monocytogenes. DETAILED DESCRIPTION

[0032] In view of the defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

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

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

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

[0037] In some preferred embodiments, the pore size of the large-pore fiber membrane is 10-30 μm, such as 30 μm.

[0038] In some preferred embodiments, the pore size of the small-pore fiber membrane is 200-220 nm, such as 200 nm.

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

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

[0041] Another aspect of the embodiments of the present invention further provides a method for simultaneously detecting multiple bacteria in water based on SERS, which comprises:

[0042] providing gold nanoparticles;

[0043] The gold nanoparticles are reacted with Raman signal molecules to obtain gold nanoparticle Raman signal molecules; wherein the Raman signal molecules include any one of acetocresyl violet, rhodamine 6G, and methylene blue; the gold nanoparticle Raman signal molecules include gold nanoparticles acetocresyl violet, gold nanoparticles rhodamine 6G, and nanoparticles methylene blue;

[0044] The gold nanoparticle Raman signal molecule is subjected to a reduction reaction with L-ascorbic acid and Ag salt to obtain a gold nanoparticle Raman signal molecule / Ag complex;

[0045] The gold nanoparticle Raman signal molecule / Ag complex is mixed with a bacterial antibody for reaction, and then blocked with bovine serum albumin to obtain a surface enhanced Raman scattering label; the bacterial antibody includes any one of an anti-Escherichia coli O157:H7 antibody, an anti-Staphylococcus aureus antibody, and an anti-Listeria monocytogenes antibody; 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 monocytogenes surface enhanced Raman scattering label;

[0046] Furthermore, 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 the water sample to be tested, and then the cultured water sample to be tested is treated with the aforementioned double-layer filtration device, and then Raman detection is performed on the small-pore fiber membrane in the double-layer filtration device to determine whether Escherichia coli O157:H7, Staphylococcus aureus or Listeria is present in the water sample to be tested.

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

[0048] In some preferred embodiments, the method specifically includes: mixing acetocresyl violet, rhodamine 6G, and methylene blue with gold nanoparticles respectively and reacting them at room temperature for 10 to 30 minutes, and then purifying them to obtain gold nanoparticles acetocresyl violet, gold nanoparticles rhodamine 6G, and nanoparticles methylene blue.

[0049] In some preferred embodiments, the method specifically includes: ultrasonically reacting L-ascorbic acid and Ag salt with gold nanoparticles acetic acid cresyl violet, gold nanoparticles rhodamine 6G and nanoparticles methylene blue, respectively, and then centrifuging to obtain gold nanoparticles acetic acid cresyl violet / Ag complex, gold nanoparticles rhodamine 6G / Ag complex and nanoparticles methylene blue / Ag complex.

[0050] In some preferred embodiments, the method specifically includes: dispersing gold nanoparticle acetocresyl violet / Ag complex, gold nanoparticle rhodamine 6G / Ag complex and nanoparticle methylene blue / Ag complex in sterile water and adjusting the pH value to 8.0-10.0, then adding anti-Escherichia coli O157:H7 antibody, anti-Staphylococcus aureus antibody and anti-Listeria monocytogenes antibody to react at room temperature, and then blocking with bovine serum albumin and centrifuging to obtain Escherichia coli O157:H7 surface enhanced Raman scattering label, Staphylococcus aureus surface enhanced Raman scattering label and Listeria monocytogenes surface enhanced Raman scattering label.

[0051] In some preferred embodiments, the method specifically includes: mixing the Escherichia coli O157:H7 surface enhanced Raman scattering label, the Staphylococcus aureus surface enhanced Raman scattering label, and the Listeria monocytogenes surface enhanced Raman scattering label to obtain a surface enhanced Raman scattering label mixture, and then adding it to the water sample to be tested and culturing it for 5-10 minutes.

[0052] Furthermore, the volume ratio of 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 in the surface-enhanced Raman scattering label mixture is 0.5-1.2: 0.8-1.4: 0.4-1.8.

[0053] Furthermore, 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 the method for simultaneously detecting multiple bacteria in water based on SERS for Escherichia coli O157:H7, Staphylococcus aureus, and Listeria monocytogenes 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: 750-1000 μL of 5 g / L chloroauric acid was added to 50-100 mL of deionized water and stirred vigorously. After heating to boil, 800-1000 μL of 1-3% trisodium citrate was immediately added. After boiling for 15 minutes, the mixture was naturally cooled to room temperature to obtain gold nanoparticles AuNPs.

[0057] (2) Preparation of various gold nanoparticle Raman signal molecules Au corresponding to different types of bacteria Tag (Au CVa 、Au R6G 、Au MB): Mix Raman signal molecules Tag: 10 μL 100 μM acetic acid cresyl violet (CVa), 10 μL 100 μM rhodamine 6G (R6G), 50 μL 10 μM methylene blue (MB) with 1-3 mL of AuNPs, and then shake the mixture at room temperature for 10-30 min to allow the Tag molecules to completely bind to the AuNPs. Tag (Au CVa 、Au R6G 、Au MB ) was purified by centrifugation at 8000-8500 g and then suspended in 1-5 mL of sterile water;

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

[0059] (4) Preparation of surface enhanced Raman scattering tag SERS-Tag: Synthesized SERS-Tag (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), 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 @Ag@Anti-S-Ab) and anti-Listeria and Au MB @Ag conjugated antibody (Au MB @Ag@Anti-L-Ab); first, Au CVa @Ag,Au R6G @Ag or Au MB The pH of GAg was adjusted to 8.0-10.0, and then 4 μL of 1 mg / mL anti-E. coli O157:H7 antibody, anti-Staphylococcus aureus antibody, and anti-Listeria monocytogenes antibody were mixed with 1-3 mL Au CVa @Ag,Au R6G @Ag or Au MB@Ag mixed. After reacting at room temperature for 1-2 hours, block with 20-50μL 10% bovine serum albumin for 1-5 hours, centrifuge at 8000-10000g for 10-30 minutes to remove free antibodies and bovine serum albumin. Finally, resuspend the final product in 1mL stock solution (10MM PBS) for subsequent use.

[0060] (5) A mixture of different surface enhanced Raman scattering labels (SERS-Tags) was added to a water sample and co-cultured for 10 min. The water sample after co-culture was quickly processed using the double-layer filtration device constructed above, and then Raman detection was performed to determine whether Escherichia coli O157:H7, Staphylococcus aureus, or Listeria monocytogenes was present in the water sample.

[0061] Preferably, in step (1), 750 μL of 5 g / L chloroauric acid is added to 50 mL of deionized water and stirred vigorously.

[0062] Preferably, in step (1), after heating to boiling, 800 μL of 1% trisodium citrate is immediately added, and the mixture is boiled for another 15 minutes and then naturally cooled to room temperature.

[0063] Preferably, the amount of AuNPs in step (2) is 1 mL, and the mixture is shaken at room temperature for 10 min.

[0064] Preferably, the various Au Tag The solution was purified by centrifugation at 8000 g and resuspended in 1 mL of sterile water.

[0065] Preferably, in step (3), in 1 mL of various Au Tag Add 100 μL of L-ascorbic acid.

[0066] Preferably, after stirring for 10 min in step (3), 300 μL 1% AgNO3 is quickly added under ultrasonic conditions, and the ultrasonic wave is stopped when the color of the reaction solution changes from purple-red to orange-yellow. After complete centrifugation at 8000-10000 g, the mixture is suspended in 1-3 mL sterile water.

[0067] Preferably, in step (3), the mixture is completely centrifuged at 8000 g and then suspended in 1 mL of sterile water.

[0068] Preferably, in step (4), Au is treated with K2CO3 CVa @Ag,Au R6G @Ag or Au MB @Ag pH was adjusted to 8.0.

[0069] Preferably, in step (4), after reacting at room temperature for 1 hour, the mixture is blocked with 20 μL of 10% bovine serum albumin for 1 hour and centrifuged at 10,000 g for 10 minutes to remove free antibodies and bovine serum albumin.

[0070] The portable double-layer filtration and concentration device made of a common syringe in the present invention is used for rapid processing of water samples and SERS analysis. The core components of the device are two filter membranes with different pore sizes, which are used for their respective functions. The upper filter membrane is used to initially intercept large interferences (interception membrane), while the lower filter membrane is used to collect multiple target pathogens (enrichment membrane) for analysis. This combination can allow contaminated environmental water (taking surface water as an example) to be quickly filtered through the device, and only target bacteria such as Escherichia coli O157: H7 (E. coli O157: H7), Staphylococcus aureus (S. aureus) and Listeria monocytogenes (L. monocytogenes) are retained on the lower enrichment membrane. Combined with surface enhanced Raman spectroscopy (SERS) decoding of SERS-Tags (SERS-TagCVa, SERS-TagR6G and SERS-TagMB) that have been labeled on the enriched bacteria by the immune recognition effect, rapid separation, concentration and detection of multiple pathogenic bacteria from a large amount of contaminated environmental water are achieved. The results showed that within a short period of time, all the target bacteria in the lake water could be 1 -10 6 CFU / mL range, with a detection limit of 10 CFU / mL, and no traditional pre-culture procedure is required. The present invention emphasizes the simplicity, rapidity, cheapness, anti-interference ability and robustness of the constructed method, which can be used to simultaneously detect multiple pathogens in water samples. The technical scheme of the present invention is further described in detail below in conjunction with several preferred embodiments and the accompanying drawings. This embodiment is implemented on the premise of the technical scheme of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0071] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0072] Example

[0073] Preparation of double-layer filtration device: The framework of the device consists of a syringe, a hollow polystyrene filter tube and cellulose membranes of different pore sizes (30 μm and 200 nm). Both cellulose membranes were cut into discs with a diameter of 15 mm to cover the water inlet (6 mm in diameter) and the water outlet (4 mm in diameter). The hollow polystyrene tube was mechanically mounted between the syringe and the needle.

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

[0075] (2) Preparation of various gold nanoparticle Raman signal molecules Au corresponding to different types of bacteria Tag (Au cVa 、Au R6G 、Au MB ): Mix the Raman signal molecule Tag: 10 μL 100 μM acetic acid cresyl violet (CVa), 10 μL 100 μM rhodamine 6G (R6G), 50 μL 10 μM methylene blue (MB) with 1 mL of AuNPs, and then shake the mixture at room temperature for 10-30 minutes to allow the Tag molecule to completely bind to the AuNPs. Tag (Au CVa 、Au R6G 、Au MB ) was purified by centrifugation at 8000 g and then suspended 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 AgNO3 in Au Tag Surface reduction reaction, in 1mL of various Au Tag 100 μL of L-ascorbic acid was added, and after stirring for 10 min, 300 μL of 1wt% AgNO3 was quickly added under ultrasonic conditions. When the color of the reaction solution changed from purple-red to orange-yellow, ultrasonication was stopped. After complete centrifugation at 8000 g, the solution was resuspended in 1 mL of sterile water.

[0077] (4) Preparation of surface enhanced Raman scattering tag SERS-Tag: Synthesized SERS-Tag (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), 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 MB@Ag@Anti-L-Ab); first, Au CVa GAg、Au R6G GAg or Au MB The pH of GAg was adjusted to 8.0, and then 4 μL of 1 mg / mL anti-E. coli O157:H7 antibody, anti-Staphylococcus aureus antibody, and anti-Listeria monocytogenes antibody were mixed with 1-3 mL Au CVa GAg、Au R6G @Ag or Au MB After reacting for 1 h at room temperature, the cells were blocked with 20 μL 10 wt% bovine serum albumin for 1 h and centrifuged at 10000 g for 10 min to remove free antibodies and bovine serum albumin. The final product was resuspended in 1 mL stock solution (10 MM PBS) for subsequent use.

[0078] (5) A mixture of different surface enhanced Raman scattering labels (SERS-Tags) was added to a water sample and co-cultured for 10 min. The water sample after co-culture was quickly processed using the double-layer filtration device constructed as described above, and then Raman detection was performed to determine whether Escherichia coli O157:H7, Staphylococcus aureus, or Listeria monocytogenes was present in the water sample.

[0079] Figure 1A The typical Raman signal spectra of CVa, R6G and MB, with characteristic peaks at 586 cm -1 、1501cm -1 、1613cm -1 (The illustrations in the figure are their structural formulas respectively). Figure 1B are the individual Raman spectra of CVa, R6G and MB. Figure 1C and Figure 1D Au Tag and Raman spectra of SERS-Tag. Figure 1A -As shown in the result of Figure 1D, the step-enhanced Raman intensity shows a good Raman enhancement effect. The present invention is used for Raman spectroscopy research and Raman enhancement effect of multiple analysis. Selecting a group of Raman spectroscopy molecules without spectral interference is a prerequisite for achieving SERS simultaneous multiple detection. Figure 1A As shown, CVa, R6G and MB are at 586 cm -1 、1501cm -1 and 1614cm -1 Since these Raman spectra have their own characteristic spectra, the distance between them is at least 100cm -1, so it is technically possible to achieve simultaneous multi-channel detection with these recorders. The illustrations show the structural models of CVa, R6G and MB respectively. Secondly, the SERS-Tag should have a strong Raman enhancement effect so that its enhanced Raman signal is conducive to improving sensitivity. Figure 1B The results in the present invention verify the SERS effect. The direct Raman test of CVa can only produce a weak signal, while when CVa is replaced by Au CVa When the Au@Ag surface is attached in the form of R6G@Ag, a significant Raman signal enhancement can be observed. The extraordinary enhancement effect lays the foundation for accurate and sensitive detection of target bacteria. Similarly, the verification results of the enhancement effect of R6G and MB are as follows: Figure 1C and Figure 1D These results lay a solid foundation for the simultaneous detection of multiple pathogenic bacteria in drinking water using the designed detection method.

[0080] TEM and UV-Vis spectroscopy were used to characterize the AuNPs and Au Tag @Ag(Au CVa The size and morphology of Ag were characterized. Figure 2A The spherical AuNPs with a highly uniform dispersion and an average size of 20 nm are shown in Fig. Figure 2B In the figure, it can be clearly observed that the surface of AuNPs is uniformly covered with a silver shell with a thickness of about 2 nm. Figure 2C The corresponding UV-Vis measurements in Figure 5 show that AgNO3 treatment of AuNPs leads to a blue shift of the absorption peak from 524 nm to 403 nm, indicating the formation of a silver shell. Figure 2D The dark (wine red) AuNPs turned into light (orange yellow) Au CVa @Ag. All these comparison results indicate that the functional AuNPs and Au@Ag for SERS measurements were successfully prepared. Tag @Ag.

[0081] The morphology of the enrichment membrane (small pore size cellulose membrane) was characterized by SEM. In order to verify the enrichment of the lower enrichment membrane, the morphology of the enrichment membrane before and after enrichment of the target bacteria was observed by scanning electron microscopy. Figure 3A-3D shown. Figure 3A The results of this study indicate the high porosity of the original enrichment membrane before use. Figure 3B The results shown show that despite the attachment of some tiny particles, the pore structure of the membrane is still good. 5 The results of filtration of contaminated water with CFU / mL E. coli O157:H7 are as follows Figure 3CAs shown, a large number of bacteria are wrapped on the surface of the enrichment membrane. A TEM magnified a collected bacterial cell. Figure 3D In the figure, it can be clearly seen that many small-sized SERS-TagCVa are closely distributed on the surface of E. coli O157: H7 through antigen-antibody recognition. These morphological characterization results strongly prove the feasibility of the double-layer filtration and concentration device of the present invention, which can effectively separate and concentrate target pathogens and perform multiple tests on the membrane at the same time.

[0082] Figure 4A , Figure 4C , Figure 4E The following are the dose-response surface enhanced Raman spectroscopy detection results of Escherichia coli O157:H7, Staphylococcus aureus and Listeria monocytogenes. The target concentrations are 0, 1×10 0 , 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 and 1×10 6 CFU / mL. 586cm -1 ( Figure 4B )、1501cm -1 ( Figure 4D ) and 1614cm -1 ( Figure 4F ) peak and the logarithmic relationship between the SERS intensity of Escherichia coli O157:H7, Staphylococcus aureus and Listeria monocytogenes. The sensing ability of the designed filter device to a pathogen was tested respectively. Figure 4A-4F The detailed recorded Raman signal and the corresponding linear response to the target bacterial concentration are depicted. Figure 4A In the detection of E. coli O157:H7, typically as the concentration of E. coli O157:H7 in the water sample increases, the number of bacteria collected on the enrichment membrane also increases accordingly. The characteristic peak of CVa (586cm -1 ) also increases with the increase of E. coli O157:H7 content in water ( Figure 4A ). The linear response relationship is Y CVa =1515.961gx-21.72, the correlation coefficient is R 2 =0.9929, where Y cVa and X represent CVa at 586 cm -1 The Raman signal intensity and the logarithmic concentration of the target E. coli O157:H7 ( Figure 4B ). Similarly, the results of Staphylococcus aureus and Listeria monocytogenes were also shown in Figure 4C-4F The linear regression equations for Staphylococcus aureus and Listeria monocytogenes are YR6G =609.99X+9.94(R 2 =0.9854) and Y MB =981.61X+79.89(R 2 =0.9942). For all the detection results of these target bacteria, 1×10 1 The signal of CFU / mL can be well distinguished from the blank group, indicating that the method has good sensitivity for the detection of bacteria in water. The good detection performance is attributed to the effective removal of potential interference from the upper large-pore membrane and the effective retention of the target bacteria on the lower small-pore membrane for SERS measurement. Compared with other measurement methods, the present invention is significantly superior to other methods in terms of sensitivity, linear range, target species, detection time and even detection cost.

[0083] Figure 5A Escherichia coli O157:H7 / Staphylococcus aureus, Figure 5B Escherichia coli O157:H7 / Lactobacillus monocytogenes and Figure 5C Representative SERS spectra collected from a target mixture of S. aureus / Listeria. To detect the combination of E. coli O157:H7 and S. aureus in water samples, Figure 5A The 586cm of CVa can be observed simultaneously in the Raman results. -1 (for E. coli O157:H7) and 1501cm for R6G -1 (For Staphylococcus aureus). It is worth noting that the intensity of these two peaks shows that the corresponding target bacteria have obvious concentration dependence. Figure 4A-4F The linear response relationship constructed in the above method can be used to calculate the recovery rate. Without interference from other coexisting bacteria, the number of target bacteria can be determined well and accurately, and satisfactory recovery rate and RSD can be obtained in complex samples. These studies strongly prove that the filtration device of the present invention is fully capable of simultaneously determining multiple bacteria in the same water sample.

[0084] Fig. 6A To simultaneously perform SERS decoding of different concentrations of Escherichia coli O157:H7, Staphylococcus aureus and Listeria in drinking water. Figure 6B The polymerase chain reaction amplification products of the enriched membrane surface bacteria were analyzed by gel electrophoresis. The concentration of each bacterium was 10 3 CFU / mL; Figure 6C Figure D is the Raman intensity of the characteristic peaks caused by target bacteria and non-target bacteria. Figure D is the detection of 10 3 CFU / mL of Escherichia coli O157:H7, Staphylococcus aureus, and Listeria monocytogenes.

[0085] Figure 6A-6D A sample containing a mixture of three bacteria was evaluated using the filter-assisted SERS method. Fig. 6A The results in the present invention show that the portable double-layer filtration and concentration device that can directly use 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. Figure 6B The results in the study further confirmed the accuracy of the test results. The specificity of the portable double-layer filtration and concentration device that can directly use SERS to simultaneously detect and decode multiple bacteria and control water quality was evaluated. Figure 6C As shown in the figure, only when the target E. coli O157:H7, Staphylococcus aureus and Listeria exist, positive Raman signals can be observed on their characteristic peaks. Figure 6C ), the Raman intensity results of the other four non-target bacteria including Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aeruginosa 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 monocytogenes, the obvious Raman signals can be easily distinguished compared with the non-target bacteria and the blank control group, indicating that the device of the present invention has good specificity and can be used for rapid and multiple bacterial detection and identification. Not only that, we also took advantage of the versatility of the present invention by comparing the multiple pathogen map results of drinking water from deionized water and liquid beverage samples with the same concentration of various target bacteria. The Raman signal value in drinking water is Fig.6D The calculated relative response values ​​of the Raman signals in deionized water and liquid beverages are very consistent with the relative response values ​​of the Raman signals in drinking water, indicating that the portable double-layer filtration and concentration device of the present invention that can directly use SERS to simultaneously detect and decode multiple bacteria and control water quality has universality and good anti-matrix effect.

[0086] The above results show that the present invention can be used for direct and simultaneous analysis of multiple target bacteria in water samples. All target bacteria in the target water source can be detected within 10 1 -10 6 The method can be used to simultaneously detect multiple pathogens in water samples. This invention scheme has opened up a new way to promote 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 this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0088] It should be understood that the technical solution of the present invention is not limited to the above-mentioned specific implementation cases. Any technical deformation made according to the technical solution of the present invention without departing from the scope of protection of the purpose of the present invention and the claims shall fall within the protection scope of the present invention.

Claims

1. A double-layer filtration device for simultaneous detection of multiple bacteria, characterized in that: It 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 water inlet of the hollow filter tube, and the water inlet is covered with a large-pore fiber membrane; the liquid output device is connected to the water outlet of the hollow filter tube, and the water outlet is covered with a small-pore fiber membrane.

2. The double-layer filtering device according to claim 1, characterized in that: The liquid input device comprises a syringe; and / or, the liquid output device comprises a needle; and / or, the hollow filter tube comprises a hollow polystyrene filter tube; And / or, the pore size of the large-pore fiber membrane is 10-30 μm; and / or, the pore size of the small-pore fiber membrane is 200-220 nm; and / or, the diameters of the large-pore fiber membrane and the small-pore fiber membrane are both 5-15 mm.

3. A method for simultaneously detecting multiple bacteria in water based on SERS, characterized in that: include: providing gold nanoparticles; The gold nanoparticles are reacted with Raman signal molecules to obtain gold nanoparticle Raman signal molecules; wherein the Raman signal molecules include any one of acetocresyl violet, rhodamine 6G, and methylene blue; the gold nanoparticle Raman signal molecules include gold nanoparticles acetocresyl violet, gold nanoparticles rhodamine 6G, and nanoparticles methylene blue; The gold nanoparticle Raman signal molecule is subjected to a reduction reaction with L-ascorbic acid and Ag salt to obtain a gold nanoparticle Raman signal molecule / Ag complex; The gold nanoparticle Raman signal molecule / Ag complex is mixed with a bacterial antibody for reaction, and then blocked with bovine serum albumin to obtain a surface enhanced Raman scattering label; the bacterial antibody includes any one of an anti-Escherichia coli O157:H7 antibody, an anti-Staphylococcus aureus antibody, and an anti-Listeria antibody; 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; Furthermore, 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 the water sample to be tested, and then the cultured water sample to be tested is treated with the double-layer filtration device described in claim 1 or 2, and then the small-pore fiber membrane in the double-layer filtration 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.

4. The method according to claim 3, characterized in that Specifically include: Chloroauric acid is added to trisodium citrate under heating conditions, mixed and reacted to obtain the gold nanoparticles.

5. The method according to claim 3, characterized in that: Specifically include: Acetocetacetocresyl violet, rhodamine 6G and methylene blue were mixed with gold nanoparticles respectively and reacted at room temperature for 10 to 30 minutes, and then purified to obtain gold nanoparticles acetocetacetocresyl violet, gold nanoparticles rhodamine 6G and nanoparticles methylene blue.

6. The method according to claim 5, characterized in that Specifically, the method comprises: subjecting L-ascorbic acid and Ag salt to ultrasonic reaction with gold nanoparticles acetic acid cresyl violet, gold nanoparticles rhodamine 6G and nanoparticles methylene blue respectively, and then subjecting the mixture to centrifugal treatment to obtain gold nanoparticles acetic acid cresyl violet / Ag complex, gold nanoparticles rhodamine 6G / Ag complex and nanoparticles methylene blue / Ag complex.

7. The method according to claim 5, characterized in that Specifically include: Gold nanoparticle acetocresyl violet / Ag complex, gold nanoparticle rhodamine 6G / Ag complex and nanoparticle methylene blue / Ag complex were dispersed in sterile water and the pH value was adjusted to 8.0-10.0, and then anti-Escherichia coli O157∶H7 antibody, anti-Staphylococcus aureus antibody and anti-Listeria monocytogenes antibody were added to react at room temperature, and then blocked with bovine serum albumin and centrifuged to obtain Escherichia coli O157∶H7 surface enhanced Raman scattering label, Staphylococcus aureus surface enhanced Raman scattering label and Listeria monocytogenes surface enhanced Raman scattering label.

8. The method according to claim 7, characterized in that Specifically include: 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 to obtain a surface enhanced Raman scattering label mixture, which is then added to a water sample to be tested and cultured for 5-10 minutes.

9. The method according to claim 8, characterized in that: The volume ratio of 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 in the surface-enhanced Raman scattering label mixture is 0.5-1.2:0.8-1.4:0.4-1.8; and / or the mass ratio of the surface-enhanced Raman scattering label mixture to the water sample to be tested is 1-2:25-50.

10. The method according to claim 3, characterized in that: The detection limit of the method for simultaneously detecting multiple bacteria in water based on SERS for Escherichia coli O157:H7, Staphylococcus aureus and Listeria monocytogenes is 10 CFU / mL.

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