Three-dimensional Raman enhanced membrane and method and system for rapid identification of bacteria

Abstract

The present invention provides a three-dimensional Raman enhanced membrane for rapid identification of bacteria and a method and system thereof, wherein the three-dimensional Raman enhanced membrane for rapid identification of bacteria comprises a polymer material layer; the polymer material layer is provided with a plurality of funnel-shaped through holes, and the polymer material layer and the surfaces of the plurality of funnel-shaped through holes are both provided with Raman enhanced material layers. The three-dimensional Raman enhanced membrane for rapid identification of bacteria described in the present invention has a plurality of funnel-shaped through holes added to the polymer material layer, and during the detection process, the contact area between the pathogenic bacteria and the through holes (i.e., the polymer material layer) is large, which can indirectly improve the sensitivity of the detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of Raman spectrum detection, and in particular relates to a three-dimensional Raman enhancement membrane for rapid bacterial identification and a method and system thereof. Background Art

[0002] Bacterial infections are among the most common clinical diseases and pose a significant threat to human health. Treatment requires identification and analysis of the bacterial species, followed by antibiotic susceptibility testing to help physicians select the type and dosage of antibiotic. Furthermore, the widespread overuse of antibiotics in China has led to increasing resistance among pathogenic microorganisms, necessitating the timely identification and classification of diverse pathogens. Traditional pathogen identification methods rely on phenotypic analysis, primarily employing staining, culture, and simple biochemical tests. Traditional detection methods are suitable for organisms cultured in vitro, but some strains exhibit unique biochemical properties that do not conform to known species patterns. In response, many modern methods, such as PCR, DNA array microarrays, immunology, fatty acid analysis, and metabolite profiling, do not rely on living cultures and can discern subtle differences between organisms that are undetectable using traditional methods.

[0003] However, the detection cycles of these detection methods are relatively long, mainly because the number of pathogens is relatively small. Samples need to be cultured and amplified in vitro before identification. Depending on the type of pathogen, the culture cycle ranges from two to five days, which greatly prolongs the time required to determine the disease, hindering patient treatment and even delaying the optimal treatment window. Although some automated detection instruments can improve the accuracy and efficiency of pathogen identification to a certain extent, they are only applicable to common pathogens that can be cultured in vitro and require a relatively long in vitro culture cycle. In addition, for some areas with poor medical conditions, there is a lack of microbial culture equipment and expensive automated detection equipment, making it impossible to accurately identify and detect pathogens. Therefore, there is an urgent need for a low-cost, reliable, and rapid identification method to meet the needs of portable, rapid, and highly sensitive detection.

[0004] Raman spectroscopy, with its molecular fingerprint properties, enables not only qualitative and quantitative analysis of substances but also functional group and molecular structure analysis. It has broad applications in a wide range of fields, including substance identification, molecular structure research, chemical processes, medicine, biochemistry, archaeology and gem identification, public security and legal sample analysis, counterterrorism technology, food safety, geology, and environmental science. More importantly, Raman spectroscopy is a non-destructive, label-free detection technology. Surface-enhanced Raman spectroscopy (SERS) is a Raman spectroscopy technique based on the nanoscale photoelectric field coupling enhancement effect on the surface of metal nanomaterials. SERS offers ultra-high sensitivity and is a powerful analytical method for trace substance detection, with detection sensitivity reaching the single-molecule level. Currently, Raman spectroscopy has been applied to bacterial identification and analysis. For example, patent CN 109266717 A describes a bacterial detection method based on single-cell analysis, which primarily uses conventional Raman spectroscopy to identify pathogen species. However, this method, which only uses conventional Raman spectroscopy and collects the overall Raman signal of a single cell, is not conducive to identifying pathogen subtypes. Patent CN 107586823 A mentions a pathogen detection method based on SERS technology, which mainly uses a mixture of precious metal nanoparticles and pathogens, dried, and then uses a portable Raman detector to detect pathogens; when this method is used for detection, the pathogens will cause changes in biochemical information of the bacteria during the drying process, which will greatly reduce the accuracy and reliability of identification. Patent CN 109060757 A mentions a SERS technology based on filter paper, in which a solution of precious metal nanoparticles is dried on filter paper to form an SERS substrate, and then the pathogen solution is dripped onto the SERS filter paper substrate; this method also has the problem of irreversible changes in pathogens during the drying process. Patent CN 104458694 A proposes a SERS substrate assembled from nanoparticles of different morphologies. Its detection process also adopts the post-drying detection mode, and there is also the problem of pathogen denaturation.

[0005] In these methods, bacteria are attached to the surface of a two-dimensional SERS substrate, and their Raman information mainly comes from the contact surface between the SERS substrate and the bacteria. Since bacterial identification is mainly based on the signals of specific protein molecules on their surface, and these specific protein molecules distributed on the bacterial surface have a certain probability of contact with the SERS substrate, the success rate and accuracy of this bacterial identification method based on two-dimensional SERS substrates are low. In addition, because the preparation method of SERS substrates is not suitable for large-scale production, these methods are not conducive to actual market promotion and application. Summary of the Invention

[0006] In view of this, the present invention aims to propose a three-dimensional Raman enhanced membrane for rapid bacterial identification to overcome the defects of the existing technology. A number of funnel-shaped through holes are added to the polymer material layer. During the detection process, the contact area between the pathogens and the through holes (i.e., the polymer material layer) is large, which can indirectly improve the detection sensitivity.

[0007] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0008] The three-dimensional Raman enhancement membrane for rapid bacterial identification comprises a polymer material layer; the polymer material layer is provided with a plurality of funnel-shaped through holes, and the polymer material layer and the surfaces of the plurality of funnel-shaped through holes are both provided with Raman enhancement material layers.

[0009] Preferably, the polymer material layer is made of PMMA, PS, PC, PE, PDMS or PEGMA.

[0010] Preferably, the material of the Raman enhancement material layer is noble metal nanoparticles; preferably, the material of the Raman enhancement material layer is gold nanoparticles and / or silver nanoparticles.

[0011] Preferably, the thickness of the polymer material layer is 5-30 μm; the upper opening size of the funnel-shaped through hole is 3-30 μm; and the lower opening size is smaller than the size of the pathogen, which is 0.2-3 μm.

[0012] Preferably, the plurality of funnel-shaped through holes are arranged in different regions on the polymer material layer according to their sizes, and the funnel-shaped through holes in the same region have the same size.

[0013] Preferably, a plurality of funnel-shaped through holes are distributed in an array on the polymer material layer; the material of the polymer material layer is PEGMA.

[0014] Another object of the present invention is to provide an analysis and detection system comprising the three-dimensional Raman enhanced membrane for rapid bacterial identification as described above, so as to identify pathogenic bacteria using the three-dimensional Raman enhanced membrane.

[0015] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0016] An analytical detection system including the three-dimensional Raman-enhanced membrane for rapid bacterial identification as described above, further comprising a sample pool, a support structure, and a waste liquid pool arranged in sequence from top to bottom; the three-dimensional Raman-enhanced membrane is arranged between the sample pool and the support structure, and the three-dimensional Raman-enhanced membrane is connected to the sample pool and the waste liquid pool through a plurality of funnel-shaped through holes; the top of the sample pool is open, and a Raman probe is provided above it; the waste liquid pool is provided with a liquid outlet, which can be connected to a negative pressure device.

[0017] Furthermore, the lower surface of the sample pool and the upper surface of the three-dimensional Raman enhancement membrane, the three-dimensional Raman enhancement membrane and the upper surface of the support structure, and the lower surface of the support structure and the upper surface of the waste liquid pool are all tightly fitted and fixedly connected.

[0018] Furthermore, the support structure is a single-sided oxidized silicon wafer provided on the edge of the lower surface of the three-dimensional Raman enhancement film.

[0019] Furthermore, the inner side of the support structure is in the shape of a truncated cone or a prism with a smaller upper portion and a larger lower portion, or is in the shape of a cylinder or a prism, and the inner side of the support structure and the waste liquid pool together constitute a waste liquid cavity; the negative pressure device is a small negative pressure pump or a simple syringe.

[0020] The working principle of the three-dimensional Raman enhanced membrane analysis and detection system for rapid bacterial identification described in the present invention is as follows:

[0021] When a moderate negative pressure is applied, the pathogen liquid begins to flow through the 3D Raman-enhanced membrane, with the water in the solution flowing out of the opening below the funnel. When a pathogen falls into the microfunnel (i.e., the funnel-shaped through-hole), the opening is smaller than the pathogen's, trapping it there and stopping the flow of liquid. By adjusting the pathogen concentration to a slightly lower number than the number of microfunnels in the array, each pathogen is ensured to fall into a specific 3D Raman-enhanced microfunnel. Then, while maintaining the negative pressure, the contact area between the pathogen and the 3D Raman-enhanced microfunnel is greatly increased. A portable or handheld Raman probe is then used to collect data from the arrayed 3D Raman-enhanced membrane, completing the pathogen identification process.

[0022] The present invention also relates to a method for preparing a three-dimensional Raman enhanced membrane for rapid bacterial identification, which is mainly used to prepare the three-dimensional Raman enhanced membrane for rapid bacterial identification as described above. This method can meet the needs of large-scale production of three-dimensional Raman enhanced membranes.

[0023] A method for preparing a three-dimensional Raman enhanced membrane for rapid bacterial identification comprises the following steps:

[0024] (1) Using nanoimprinting technology to prepare a polymer material layer containing several funnel-shaped through holes;

[0025] (2) Selecting a single-sided oxidized silicon wafer, sequentially performing the steps of spin coating, photolithography, etching, stripping, dry etching or wet etching, and removing the protective layer to prepare a support structure;

[0026] (3) Spin coating or spray coating a layer of 1 to 20 μm of alcohol-soluble polymer on the surface of the support structure. The polymer can be an alcohol-soluble resin, polyacrylate, polyvinyl butyral, polyethylphenol, or other similar polymers;

[0027] (4) immersing the polymer material layer containing a plurality of funnel-shaped through holes in ethanol, taking it out and spreading it flat on the surface of the support structure, and drying it at 20-80°C;

[0028] (5) immersing the overall structure obtained in step (4) in an HF solution for 1-5 minutes to remove the support film at the support window, and then immersing it in ethanol for 1-10 minutes to remove the alcohol-soluble polymer at the support window;

[0029] (6) The structure treated in step (5) is treated with oxygen plasma for 2-10 minutes, immersed in a 5 wt% APTES ethanol solution for reaction for 15-30 minutes, and then immersed in a Raman enhancement material solution for reaction for 2-8 hours, and rinsed with clean water to obtain a three-dimensional Raman enhancement film.

[0030] Compared with the prior art, the three-dimensional Raman enhanced membrane for rapid bacterial identification described in the present invention has the following advantages:

[0031] The funnel-shaped cavity (i.e., the funnel-shaped through-holes) of the three-dimensional Raman-enhanced membrane for rapid bacterial identification described herein significantly increases the contact area between pathogens trapped within the cavity and the surrounding area, thereby obtaining more Raman information about the pathogens and improving the accuracy of pathogen identification. Furthermore, when the funnel-shaped through-holes in the polymer layer are arranged in zones of different sizes, the identification and detection of multiple pathogens can be achieved.

[0032] The method for preparing a three-dimensional Raman enhanced film for rapid bacterial identification of the present invention adopts continuous nanoimprinting technology and can realize large-scale preparation of three-dimensional funnel-shaped Raman enhanced films.

[0033] The three-dimensional Raman enhanced membrane analysis and detection system for rapid bacterial identification described in the present invention can achieve rapid capture, fixation and Raman detection of single pathogenic bacteria cells.

[0034] The three-dimensional Raman enhanced membrane analysis and detection system for rapid bacterial identification of the present invention can realize the identification and detection of various pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a simplified longitudinal cross-sectional view of a three-dimensional Raman enhanced membrane for rapid bacterial identification according to an embodiment of the present invention;

[0036] Figure 2 A top view of a three-dimensional Raman enhanced membrane for rapid bacterial identification according to one embodiment of the present invention (the plurality of funnel-shaped through-holes are not arranged in zones according to size, and the through-holes are of uniform size);

[0037] Figure 3 A top view of a three-dimensional Raman enhanced membrane for rapid bacterial identification according to one embodiment of the present invention (several funnel-shaped through holes are arranged in different areas according to their sizes);

[0038] Figure 4 This is a schematic diagram of the simple structure of an analysis and detection system containing a three-dimensional Raman enhancement membrane for rapid bacterial identification according to an embodiment of the present invention;

[0039] Figure 5 This is a flow chart for preparing a support structure in an analysis and detection system containing a three-dimensional Raman enhanced membrane for rapid bacterial identification according to an embodiment of the present invention;

[0040] Figure 6 This is a flow chart for preparing a polymer material layer in a three-dimensional Raman enhanced membrane for rapid bacterial identification according to an embodiment of the present invention;

[0041] Figure 7 This is a flow chart for preparing a three-dimensional Raman enhanced membrane for rapid bacterial identification according to an embodiment of the present invention.

[0042] Reference numerals:

[0043] 1-three-dimensional Raman enhancement membrane; 101-polymer material layer; 102-funnel-shaped through hole; 103-Raman enhancement material layer; 4-sample pool; 5-support structure; 6-waste liquid pool; 601-liquid outlet; 7-Raman probe; 8-Raman detector; 9-pathogen. DETAILED DESCRIPTION

[0044] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods described, unless otherwise specified, are conventional methods.

[0045] The present invention will be described in detail below with reference to the embodiments and accompanying drawings.

[0046] 1. 3D Raman Enhanced Membrane for Rapid Bacterial Identification

[0047] like Figure 1As shown, a three-dimensional Raman enhancement film for rapid identification of bacteria includes a polymer material layer 101, on which a plurality of funnel-shaped through holes 102 are arrayed. The funnel-shaped through holes 102 can also be referred to as funnel cavities. Each funnel-shaped through hole 102 can be a truncated cone or a prism-shaped one with a larger top and a smaller bottom, with an upper opening size of 5 to 30 μm and a lower opening size of 0.2 to 3 μm. Preferably, each funnel-shaped through hole 102 can be a prism-shaped one with a larger top and a smaller bottom, and more preferably a truncated cone. The thickness of the entire polymer material layer 101 is 5 to 30 μm, and the material can be selected from PMMA (polymethyl methacrylate), PS (polystyrene), PC (polycarbonate), PE (polyethylene), PDMS (polydimethylsiloxane) or PEGMA (polyethylene glycol methacrylate) and other types of nanoimprint glue, but PEGMA (polyethylene glycol methacrylate) is preferred. A Raman enhancement material layer 103 is provided on the surfaces of the polymer material layer 101 and the plurality of funnel-shaped through holes 102 . The material of the Raman enhancement material layer 103 can be selected from precious metal nanoparticles such as nano-gold and / or nano-silver.

[0048] As a preferred embodiment of the present invention, Figure 2 As shown, there are 100 funnel-shaped through-holes 102 arranged in a 10×10 array. Each funnel-shaped through-hole 102 is a truncated cone with a larger top and smaller bottom, and an upper opening size of 20 μm and a lower opening size of 0.5 μm. The entire polymer material layer 101 is 15 μm thick. Nano-gold is used as the material for the Raman enhancement material layer 103.

[0049] As another preferred embodiment of the present invention, the plurality of funnel-shaped through holes 102 can be arranged in different regions on the polymer material layer 101 according to their sizes. The sizes of the multiple funnel-shaped through holes in the same region are the same. According to the requirements of detection and identification and industrial scale, the number of regions can be multiple, and the number of hole-shaped through holes in each region can be the same or different. Figure 2 As shown, for example Figure 3As shown, the number of the funnel-shaped through holes 102 is 100 (all in the shape of a truncated cone), and they are distributed in a 10×10 array, which can be divided into four areas, namely the upper left corner area, the upper right corner area, the lower left corner area, and the lower right corner area. The number of funnel-shaped through holes in these four areas can be evenly divided, or the number can be more or less as needed, but the sizes of the funnel-shaped through holes in the four areas can be decreasing in sequence from the upper left corner area to the upper right corner area, the lower left corner area, and the lower right corner area. For example: the size of the funnel-shaped through hole in the upper left corner area is 30 μm for the upper opening and 3 μm for the lower opening; the size of the funnel-shaped through hole in the upper right corner area is 20 μm for the lower opening and 2 μm for the lower opening; the size of the funnel-shaped through hole in the lower left corner area is 10 μm for the lower opening and 1 μm for the lower opening; the size of the funnel-shaped through hole in the lower right corner area is 5 μm for the lower opening and 0.5 μm for the lower opening. Alternatively, the sizes of the funnel-shaped through holes in the four regions can also adopt other forms of pore size distribution, as long as the sizes of the funnel-shaped through holes in each region are consistent. This can be used for industrial pathogen detection, and a three-dimensional Raman enhanced film can be used to detect and identify four different types and sizes of pathogens.

[0050] 2. 3D Raman-enhanced membrane analysis and detection system for rapid bacterial identification

[0051] like Figure 4 As shown, an analysis and detection system of a three-dimensional Raman enhanced membrane for rapid identification of bacteria includes the above-mentioned three-dimensional Raman enhanced membrane 1 for rapid identification of bacteria, a sample pool 4 is provided above the three-dimensional Raman enhanced membrane 1, and the sample pool 4 itself is a shell with only a peripheral wall, with an open top and a Raman probe 7 provided above. The sample pool 4 has the three-dimensional Raman enhanced membrane 1 as its bottom, and the edge of its lower surface is tightly fitted and connected to the three-dimensional Raman enhanced membrane 1 to prevent liquid leakage. A support structure 5 is provided below the three-dimensional Raman enhanced membrane 1, and the edge of the upper surface of the support structure 5 is tightly fitted and fixed to the edge of the lower surface of the three-dimensional Raman enhanced membrane 1. The support structure 5 itself is shaped like a cylinder, and together with the waste liquid pool 6 provided below it, it constitutes a waste liquid cavity. The waste liquid pool 6 is connected to the sample pool 4 through a number of funnel-shaped through holes 102 on the three-dimensional Raman enhanced membrane. It should be noted that the lower surface of the support structure 5 is also tightly fitted and fixedly connected to the upper surface of the waste liquid pool 6. The waste liquid pool 6 is provided with a liquid outlet (not shown), which can be used to connect an external negative pressure device.

[0052] As a preferred embodiment of the present invention, the support structure 5 is a single-sided oxidized silicon wafer bonded to the edge of the lower surface of the three-dimensional Raman enhancement film 1, and its thickness is between 2 and 10 μm.

[0053] As a preferred embodiment of the present invention, the lower surface of the sample pool 4 and the upper surface of the three-dimensional Raman enhancement film 1, the three-dimensional Raman enhancement film 1 and the upper surface of the support structure 5, and the lower surface of the support structure 5 and the upper surface of the waste liquid pool 6 are all tightly fitted and fixedly connected by adhesive.

[0054] In a preferred embodiment of the present invention, the inner side of the support structure 5 is shaped like a truncated cone or prism, or a cylinder or prism, with a smaller upper portion and a larger lower portion. The negative pressure device can be a small negative pressure pump or a simple syringe connected thereto, used to provide a negative pressure environment for the waste liquid pool, sample pool, etc. during the detection process. Further preferably, the inner side of the support structure 5 is shaped like a truncated cone, with a smaller upper portion and a larger lower portion, and the negative pressure device is a small negative pressure pump.

[0055] During use, the Raman probe 7 is connected to the existing Raman detector 8. When an appropriate negative pressure is applied, the pathogen liquid begins to flow through the three-dimensional Raman enhanced membrane 1, and the water in the solution flows out from the funnel-shaped through-hole 102. When a pathogen 9 falls into the micron funnel (i.e., the funnel-shaped through-hole 102), since the lower opening size of the funnel-shaped through-hole 102 is smaller than the size of the pathogen 9, the pathogen 9 is stuck at the lower opening of the funnel-shaped through-hole 102, and no liquid flows out of the funnel-shaped through-hole 102 at this location; by adjusting the concentration of the pathogens 9, the number of pathogens 9 is slightly lower than the number of arrayed three-dimensional Raman enhanced micron funnels (funnel-shaped through-holes 102) to ensure that each pathogen 9 can fall into a three-dimensional Raman enhanced micron funnel (funnel-shaped through-hole 102). Then, while maintaining the negative pressure, the contact area between the pathogen 9 and the three-dimensional Raman enhanced micron funnel (funnel-shaped through hole 102) is greatly increased. At this time, the portable Raman probe 7 or the handheld Raman probe 7 is used to collect data from the arrayed three-dimensional Raman enhanced film once to complete the identification of the pathogen 9.

[0056] 3. Preparation of 3D Raman-enhanced membranes and support structures for rapid bacterial identification

[0057] like Figure 5-7 As shown, the method for three-dimensional Raman enhanced membrane for rapid identification of bacteria comprises the following steps:

[0058] (1) A polymer material layer 101 containing a plurality of funnel-shaped through holes 102 is prepared using nanoimprinting technology;

[0059] (2) Selecting a single-sided oxidized silicon wafer, and sequentially performing the steps of spin coating, photolithography, etching, stripping, dry etching or wet etching, and removing the protective layer to prepare a support structure 5;

[0060] (3) Spin-coat or spray a layer of 1-20 μm (preferably 10 μm) of alcohol-soluble polymer on the surface of the support structure 5. The polymer can be an alcohol-soluble resin, polyacrylate, polyvinyl butyral, polyethylphenol, etc. and other similar polymers, preferably an alcohol-soluble resin, such as thermoplastic phenolic resin.

[0061] (4) Immerse the polymer material layer 101 containing the plurality of funnel-shaped through holes 102 in ethanol, remove it, spread it flat on the surface of the support structure 5, and dry it at 20-80°C;

[0062] (5) immersing the overall structure obtained in step (4) in an HF solution for 1-5 minutes to remove the support membrane at the support window (i.e., the location of the funnel-shaped through hole), and then immersing it in ethanol for 1-10 minutes to remove the alcohol-soluble polymer at the support window;

[0063] (6) The structure treated in step (5) is treated with oxygen plasma for 2-10 minutes, immersed in a 5 wt% APTES ethanol solution for reaction for 15-30 minutes, and then immersed in a Raman enhancement material solution for reaction for 2-8 hours, and rinsed with clean water to obtain a three-dimensional Raman enhancement film.

[0064] Wherein, in step (1), Figure 5 As shown, the method for preparing a polymer material layer 101 containing a plurality of funnel-shaped through-holes 102 using nanoimprint technology is as follows: an imprinting polymer is coated on a substrate, and then the imprinting polymer is pressed with an imprinting template having a funnel-shaped cylinder on its lower surface. After demolding, the imprinting template is removed, and the residual adhesive on the imprinting polymer corresponding to the funnel-shaped cylinder is removed to form funnel-shaped through-holes. Finally, after demolding, the polymer material layer 101 containing a plurality of funnel-shaped through-holes 102 is obtained. Here, it should be noted that:

[0065] The imprint template can be a homemade PDMS template or a commercially available custom product made of nickel film or other materials that can achieve similar functions. The imprint polymer and substrate are also commercial products. The imprint polymer material can be PMMA, PS, PC, PE, PDMS, PEGMA, or other types of nanoimprint adhesives. The substrate material can be PET, PP, PTFE, or other materials. Preferably, PEGMA, which has good biocompatibility, is used as the imprint polymer material, and PET is used as the substrate material.

[0066] b. Nanoimprinting using hot pressing or UV embossing. Nanoimprinting can be performed using plane-to-plane, roll-to-plane, or roll-to-roll methods. Preferably, a roll-to-roll nanoimprinter is used for imprint replication, and a nickel template is used as the imprint template.

[0067] c. In order to ensure the stability of continuous nanoimprinting, the surface of the imprint template needs to be chemically modified first, that is, the imprint template is first cleaned in a mixture of concentrated sulfuric acid and hydrogen peroxide at a mass ratio of 2:1 at 120°C for 20 minutes.

[0068] d. Then, the imprint template treated in step c is placed in an ammonia solution (ammonia: hydrogen peroxide: water = 1:1:5 by mass) and treated for 20 minutes.

[0069] e. The imprint template treated in step d was then placed in an isooctane solution containing 1 mM perfluorodecyltrichlorosilane for 30 minutes for hydrophobic treatment.

[0070] f. Fix the chemically modified imprint template on the surface of the imprint roll.

[0071] g. Mix 1 g of 2-hydroxy-2-methylpropiophenone with 100 g of PEGMA to obtain a UV photosensitive adhesive. Select a UV curing light source with a wavelength of 365 nm and an imprinting speed of 1 to 10 mm / s.

[0072] h. Due to the residual glue left by nanoimprinting, ion etching and plasma etching are needed to remove the residual glue at the bottom of the funnel, and finally form an open shape.

[0073] i. Peel off the three-dimensional micron funnel membrane (i.e., the polymer material layer 101 containing a plurality of funnel-shaped through holes 102) after removing the residual glue from the base material to obtain a large-area three-dimensional micron funnel membrane, i.e., the polymer material layer 101 containing a plurality of funnel-shaped through holes 102.

[0074] In step (2), the preparation method of the support structure is specifically as follows: Figure 6 As shown, the following steps are included:

[0075] 1) Select a single-sided oxidized silicon wafer with a silicon oxide layer thickness of 2-10 μm (preferably 6 μm), and evaporate a layer of 50 to 200 nm (preferably 100 nm) of metal Cr on the non-oxidized surface;

[0076] 2) Spin-coat 2 to 10 μm thick photoresist AZ4620;

[0077] 3) performing UV photolithography and developing with 2.5 wt % TMAH (tetramethylammonium hydroxide);

[0078] 4) Place the developed pattern into a Cr etching solution (mainly composed of cerium ammonium nitrate and nitric acid, such as the chromium etching solution CE-8001-N from Transene, USA) to transfer the developed pattern to the Cr layer;

[0079] 5) Place in acetone to remove the photoresist layer;

[0080] 6) placing the substrate in a 25 wt % TMAH (tetramethylammonium hydroxide) solution and etching at 50-70° C. (preferably 60° C.) for 1-3 hours (preferably 2 hours) until the silicon oxide layer is etched, and then placing the substrate in a Cr etching solution to remove the Cr layer (i.e., the protective layer) to obtain a support structure 5;

[0081] 7) Alternatively, the substrate obtained after step 5) is subjected to deep silicon etching in a mixed gas of sulfur hexafluoride and oxygen until the silicon oxide layer is etched, and then placed in a Cr etching solution to remove the Cr layer (ie, the protective layer) to obtain a support structure.

[0082] As a preferred embodiment of the present invention, in step (5), the mass concentration of HF in the HF solution is preferably 10%, and the immersion time is preferably 3 minutes.

[0083] As another preferred embodiment of the present invention, in step (6), the structure treated in step (5) is treated with oxygen plasma for 5 minutes, immersed in a 5wt% APTES ethanol solution for reaction for 20 minutes, and then immersed in a solution of precious metal nanoparticles such as 20 to 100 nm (preferably 60 nm) nanogold and nanosilver for reaction for 5 hours, and then rinsed with clean water to obtain a three-dimensional Raman enhanced film.

[0084] The funnel-shaped cavity (i.e., the funnel-shaped through-hole) of the 3D Raman-enhanced membrane for rapid bacterial identification described in this invention significantly increases the contact area between pathogens trapped within the cavity and the surrounding area, thereby obtaining more Raman information about the pathogens and improving the accuracy of pathogen identification. Furthermore, the use of a production-grade nanoimprinting method allows for large-scale production of 3D Raman-enhanced membranes, making this method highly marketable.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An analytical detection system comprising a three-dimensional Raman enhanced membrane for rapid identification of bacteria, characterized in that: The three-dimensional Raman enhancement film comprises a polymer material layer (101); a plurality of funnel-shaped through holes (102) are provided on the polymer material layer (101); and a Raman enhancement material layer (103) is provided on the surface of the polymer material layer (101) and the plurality of funnel-shaped through holes (102); The system comprises a sample pool (4), a support structure (5) and a waste liquid pool (6) which are arranged in sequence from top to bottom; the three-dimensional Raman enhancement membrane (1) is arranged between the sample pool (4) and the support structure (5), and the three-dimensional Raman enhancement membrane is connected to the sample pool (4) and the waste liquid pool (6) through a plurality of funnel-shaped through holes (102); the top of the sample pool (4) is open, and a Raman probe (7) is arranged above the sample pool; a liquid outlet is arranged on the waste liquid pool (6), and the liquid outlet can be connected to a negative pressure device; The lower surface of the sample pool (4) and the upper surface of the three-dimensional Raman enhancement film (1), the three-dimensional Raman enhancement film (1) and the upper surface of the support structure (5), and the lower surface of the support structure (5) and the upper surface of the waste liquid pool (6) are all tightly fitted and fixedly connected; The support structure (5) is a single-sided oxidized silicon sheet arranged at the edge of the lower surface of the three-dimensional Raman enhancement film (1); The inner side of the support structure (5) is in the shape of a truncated cone or a prism that is smaller at the top and larger at the bottom, or in the shape of a cylinder or a prism, and the inner side of the support structure (5) and the waste liquid pool (6) together form a waste liquid cavity; the negative pressure device is a small negative pressure pump or a simple syringe.

2. The analysis and detection system according to claim 1, characterized in that: The polymer material layer (101) is made of PMMA, PS, PC, PE, PDMS or PEGMA.

3. The analysis and detection system according to claim 1, characterized in that: The material of the Raman enhancement material layer (103) is precious metal nanoparticles.

4. The analysis and detection system according to claim 1, characterized in that: The thickness of the polymer material layer (101) is 5-30 μm; the upper opening size of the funnel-shaped through hole (102) is 3-30 μm, and the lower opening size is 0.2-3 μm.

5. The analysis and detection system according to claim 1, characterized in that: A plurality of funnel-shaped through holes (102) are distributed in an array on the polymer material layer (101); the material of the polymer material layer (101) is PEGMA.

6. The analysis and detection system according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) using nanoimprint technology to prepare a polymer material layer (101) containing a plurality of funnel-shaped through holes (102); (2) Selecting a single-sided oxidized silicon wafer, and sequentially subjecting it to a process of spinning, photolithography, etching, debonding, dry etching or wet etching, and removing a protective layer, to prepare a support structure (5); (3) Spin coating or spray coating a layer of 1-20 μm alcohol-soluble polymer on the surface of the support structure (5), wherein the polymer is one or more of alcohol-soluble resin, polyacrylate, polyvinyl butyral, and polyethylphenol; (4) immersing the polymer material layer (101) containing a plurality of funnel-shaped through holes (102) into ethanol, taking it out and spreading it flat on the surface of the support structure (5), and drying it at 20-80°C; (5) immersing the overall structure obtained in step (4) in an HF solution for 1-5 min to remove the support film at the support window, and then immersing it in ethanol for 1-10 min to remove the alcohol-soluble polymer at the support window; (6) The structure treated in step (5) is treated with oxygen plasma for 2-10 min, immersed in a 5 wt% APTES ethanol solution for reaction for 15-30 min, then immersed in a Raman enhancement material solution for reaction for 2-8 h, and rinsed with clean water to obtain a three-dimensional Raman enhancement film.

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