Composite functional porous metal membrane and detection system for monitoring and identifying airborne microorganisms, and method for monitoring and identifying airborne microorganisms

Abstract

The present application relates to the technical field of biological sensing, and discloses a composite functional porous metal membrane and detection system for monitoring and identifying airborne microorganisms, and a method for monitoring and identifying airborne microorganisms. The monitoring and identification method comprises: applying an electric field to the composite functional porous metal membrane, causing the membrane to freely switch charge and capture airborne microorganisms to be tested; adding a fluorescent probe to the composite functional porous metal membrane, which binds with the microorganisms to be tested to obtain a fluorescent sample, and collecting imaging of light emitted after irradiation of excitation light, to achieve preliminary determination and addressing of the microorganisms to be tested; performing Raman spectroscopy scanning on individual bacteria, and comparing with a standard Raman spectrum library to achieve species identification of the individual bacteria. Relying on the "composite functional porous metal membrane," an integrated process of "selective enrichment-total bacteria monitoring-species identification" is realized; the present invention takes selective enrichment of the microorganisms to be tested as an entry point, lays the foundation for preliminary judgment accuracy and species identification accuracy, and improves the efficiency and precision of monitoring and identifying airborne microorganisms.

Description

A composite functional porous metal film and detection system for monitoring and identifying airborne planktonic microorganisms, and a method for monitoring and identifying airborne planktonic microorganisms

[0001] Cross-reference to related applications

[0002] This application claims priority to the Chinese patent application No. 202410831125.2, filed on June 25, 2024, and entitled "A composite functional porous metal film and detection system for monitoring and identifying airborne planktonic microorganisms, and a method for monitoring and identifying airborne planktonic microorganisms", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the field of biosensing technology, in particular to a composite functional porous metal film and detection system for monitoring and identifying airborne planktonic microorganisms, and a method for monitoring and identifying airborne planktonic microorganisms. BACKGROUND

[0004] Airborne planktonic microorganisms have been a concern for domestic and foreign research institutes. There are mainly two categories in the related field: one is clean environment monitoring, such as medical clean monitoring (operating room, emergency room, etc.), industrial clean monitoring (pharmaceutical factory, food factory, etc.), etc.; the other is infectious risk monitoring, such as hospital visit and nosocomial infection monitoring (fever clinic area, emergency area, etc.), port entry and exit inspection and quarantine, etc.

[0005] The airborne planktonic microorganisms have the significant characteristics of "extremely low concentration, but with reproductive ability". Most of the microorganisms in the air die quickly due to the influence of humidity, temperature, and ultraviolet light, and only a small part of the microorganisms with strong environmental tolerance (such as bacteria, especially pathogenic bacteria) can survive for a long time. Therefore, the concentration of airborne planktonic microorganisms is extremely low, but their reproductive ability makes their harm cannot be ignored even if the concentration is low. Therefore, it is particularly important to quickly identify the airborne planktonic microorganisms.

[0006] In addition, there are many interferences in the identification of airborne planktonic microorganisms. The main part of the planktonic microorganisms is bacteria, fungi, etc. with a size of about 2 μm, which has a very similar suspension characteristic in the air as PM2.5 (floating particles with a diameter of about 2.5 μm or less). These large amounts of PM2.5 particles with similar diameters, mostly inorganic soot or organic pollen, bring great interference to the monitoring and identification of planktonic microorganisms. However, there are significant differences between PM2.5 non-biological particles and planktonic microorganisms, and between planktonic non-pathogenic microorganisms and planktonic pathogenic microorganisms, which must be accurately identified.

[0007] The existing scheme is to combine the "preliminary judgment module", "sampling module" and "identification module" three instruments or modules, that is, the "preliminary judgment module" realizes high-sensitivity detection of "extremely low concentration" microorganisms by high-sensitivity fluorescence detection; the "detection module" solves the high-frequency false alarm caused by "various interference" by specific biological detection such as immunity or nucleic acid; and the "sampling module" realizes the connection between the preliminary judgment module and the detection module by converting the "airborne target" into the "liquid-phase analysis target".

[0008] However, the combination of the existing technology "preliminary judgment-sampling-detection" three instruments or modules has the following defects: (1) the preliminary judgment cannot accurately identify microorganisms and non-microbial particles such as pollen, and the subsequent manual repeated sampling, detection and investigation are required due to high false alarm frequency; (2) the sample difference is large, and the target types are various, which makes the sampling and detection screening time-consuming, and prolongs the disposal cycle.

[0009] The monitoring and identification based on "sampling-preliminary judgment-detection" cannot be integrated, automated and real-time, which is the key reason why it cannot be widely used in personnel-intensive areas (such as shopping malls, airport ports, subway stations, etc.) like dust particle counters and VOC measuring instruments (i.e. formaldehyde measuring instruments) to play the role of environmental monitoring. SUMMARY

[0010] Therefore, the present application provides an air floating microorganism monitoring and identification technology, specifically including providing a composite functional porous metal film for monitoring and identifying air floating microorganisms, a detection system for monitoring and identifying air floating microorganisms comprising the composite functional porous metal film, and a method for monitoring and identifying air floating microorganisms using the composite functional porous metal film, which enables real-time online total number monitoring and species identification of air floating microorganisms.

[0011] The present application provides a composite functional porous metal film and a detection system for monitoring and identifying air floating microorganisms, and an air floating microorganism monitoring and identification method, to solve the problems of low detection efficiency and difficulty in ensuring accuracy of the existing scheme.

[0012] In a first aspect, the present application provides an air floating microorganism monitoring and identification method, comprising:

[0013] adding the microorganism to be detected to the composite functional porous metal film; under the action of an electric field, the side of the composite functional porous metal film in contact with the microorganism to be detected exhibits flexible charging, the composite functional porous metal film comprises a substrate and a nanomaterial layer, the nanomaterial layer directly contacts and adsorbs the microorganism to be detected; the substrate has a plurality of pores, the nanomaterial layer covers one side surface of the substrate, and the side surface of the nanomaterial layer away from the substrate is used to form a Raman signal enhancement field;

[0014] adding a fluorescent probe to the side of the composite functional porous metal film with the microorganism to be detected, combining the fluorescent probe with the microorganism to be detected to obtain a fluorescent sample to be detected; emitting excitation light to the fluorescent sample to be detected on the composite functional porous metal film to make the fluorescent sample to be detected emit emission light; collecting the emission light and imaging to obtain quantity information and position information of the microorganism to be detected;

[0015] According to the obtained position information of the microorganism to be detected, focusing on the microorganism to be detected at one of the position information, emitting Raman excitation light to the microorganism to be detected, and capturing Raman scattering light emitted by the microorganism to be detected to obtain a Raman spectrum of a single bacterium; comparing the Raman spectrum of the single bacterium with standard Raman spectra in a Raman standard spectrum library to determine the species of the single bacterium.

[0016] Beneficial effects: relying on the composite functional porous metal film as the only carrier to realize the true integration of the three functions of "sampling-judgment-detection", and upgrading the three-step process to "selective enrichment (sampling)-total bacteria monitoring (judgment)-species identification (detection)", that is, instead of taking the particle mixture in the air as the whole analysis object, the microorganism to be detected is first adsorbed and fixed by the flexible charging characteristics of the composite functional porous metal film, that is, the selective enrichment of the microorganism to be detected is taken as the starting point, thereby laying a foundation for the subsequent judgment accuracy and species identification accuracy, then the selective permeability of the composite functional porous metal film is used to retain the fluorescent probe of the fluorescent sample to be detected combined with the microorganism to be detected, while the uncombined fluorescent probe and other impurities such as solvents are removed to reduce the interference of impurity signals, realize the total bacteria judgment addressing of the microorganism to be detected, and then the Raman signal enhancement field of the nanomaterial layer on the composite functional porous metal film is used to obtain the Raman spectrum of a certain microorganism to be detected with a target characteristic peak, and through comparison with the standard Raman spectrum in the Raman standard spectrum library, the species of a certain bacterium is determined. The air floating microorganism monitoring and identification method of the present application provides a technical basis for realizing the integration, automation and real-time monitoring and identification of "sampling-judgment-detection".

[0017] In an optional embodiment, under the action of an electric field, the composite functional porous metal film has one of positive charge, negative charge and pulse charge;

[0018] The composite functional porous metal film has positive charges, and the composite functional porous metal film adsorbs air-borne microorganisms;

[0019] The composite functional porous metal film has negative charges, and the composite functional porous metal film releases air-borne microorganisms;

[0020] The composite functional porous metal film has pulse charges, and the composite functional porous metal film performs adsorption and release cycles on the air-borne microorganisms close to the composite functional porous metal film.

[0021] In an optional embodiment, a fluorescent probe is added to the side of the composite functional porous metal film with the microorganism to be detected, and the fluorescent probe is combined with the target sample to obtain a to-be-detected fluorescent sample, including:

[0022] The fluorescent probe solution is added dropwise to the side of the composite functional porous metal film with the microorganism to be detected;

[0023] The fluorescent probe is incubated with the microorganism to be detected for 1 min to 5 min, the fluorescent probe enters the microorganism to be detected, and the fluorescent probe is combined with the microorganism to be detected to obtain a to-be-detected fluorescent sample;

[0024] Excess to-be-detected fluorescent probe solution is washed away, and the fluorescent probe that is not combined with the microorganism to be detected passes through the nanomaterial layer and is discharged from the hole of the substrate;

[0025] The composite functional porous metal film obtains the quantity information and the position information of the microorganism to be detected, and is also used for detection of microorganisms in water bodies and microorganisms on solid surfaces.

[0026] In the composite functional porous metal film of the present application, the nanomaterial layer has pores for the passage of uncombined fluorescent probes, and the nanomaterial layer covers the substrate and forms small holes with a diameter smaller than the through holes of the substrate along with the through holes, that is, the composite functional porous metal film has porous permeability. First, the small fluorescent probe can pass through the pores of the nanomaterial layer itself, and then be discharged from the small holes. In this way, the excess fluorescent probe is washed away, and the composite functional porous metal film with such permeability reduces the interference of impurity fluorescent signals during addressing, thereby improving the accuracy of total bacteria addressing on the composite functional porous metal film of the present application.

[0027] In an optional embodiment, the size of the fluorescent probe is 2 nm to 10 nm; and the fluorescent probe is one or more of carbon quantum dots, fluorescent quantum dots, and fluorescent dyes.

[0028] In an optional embodiment, the standard Raman spectrum in the Raman standard spectrum library has an effective characteristic peak, the effective characteristic peak is obtained through independent Raman spectrum scanning of more than three bacteria of the same kind, and the generated Raman spectrum has a significant difference from the control spectrum, which is characterized as an effective characteristic peak.

[0029] The Raman spectrum of the single bacterium has target characteristic peaks for comparison with effective characteristic peaks;

[0030] The Raman spectrum of the single bacterium is also used for detection of microorganisms in water bodies and on solid surfaces.

[0031] In a second aspect, the application provides a composite functional porous metal film, comprising a substrate and a nanomaterial layer, the substrate being a porous metal plate or a metal mesh; the nanomaterial layer is coated on one side surface of the substrate, and the side surface of the nanomaterial layer away from the substrate is used to form a Raman signal enhancement field; the nanomaterial layer is suitable for adsorbing a to-be-detected fluorescent sample, the to-be-detected fluorescent sample comprising to-be-detected microorganisms and fluorescent probes combined together, the nanomaterial layer having pores for passage of uncombined fluorescent probes, the to-be-detected microorganisms being suitable for emitting Raman scattering light under irradiation of Raman excitation light, and the fluorescent probes being suitable for emitting fluorescent emission light under irradiation of fluorescent excitation light.

[0032] Beneficial effects: by setting a specific nanomaterial layer on the substrate, firstly, the nanomaterial layer has selective permeability, on one hand, the nanomaterial layer itself has small pores for passage of fluorescent probes, so that the fluorescent probes combined with the to-be-detected microorganisms form the to-be-detected fluorescent sample and remain on the composite functional porous metal film, on the other hand, other substances such as uncombined fluorescent probes pass through the composite functional porous metal film, reducing interference of background signals such as fluorescent probes during total bacteria addressing, and improving accuracy; secondly, when the fluorescent excitation light irradiates the nanomaterial layer where the to-be-detected fluorescent sample is located, the to-be-detected fluorescent sample emits emission light, facilitating acquisition of position information and total number information of the to-be-detected fluorescent sample, and realizing total number of to-be-detected microorganisms and initial judgment of addressing; thirdly, when a certain to-be-detected microorganism on the nanomaterial layer is irradiated with Raman excitation light, the to-be-detected microorganism absorbs the excitation light and emits Raman scattering light, and at the same time, the Raman excitation light causes oscillation of molecules of substances on the surface of the nanomaterial layer, forming a Raman signal enhancement field, the Raman scattering light signal of the single to-be-detected microorganism is enhanced, so that the single bacterium Raman spectrum of the to-be-detected microorganism can be obtained, and then the characteristic peaks are compared with standard Raman spectra in a Raman standard spectrum library, realizing species identification of the single bacterium of the to-be-detected microorganism. The composite functional porous metal film of the application has flexible charging property, porous permeability and Raman enhancement property, providing a support carrier for realizing integrated and automated precision capture of planktonic microorganisms, total bacteria addressing and species identification.

[0033] In an optional embodiment, the nanomaterial layer comprises a single metal element layer evaporated or a nanomaterial structure layer coated.

[0034] In an optional embodiment, the single metal element layer evaporated comprises one or more of a titanium nanoparticle film, a zinc nanoparticle film, a tin nanoparticle film, and a lead nanoparticle film.

[0035] In the present application, under the irradiation of Raman excitation light, the single metal element layer formed by evaporation has a lower background signal than other metal materials, which can effectively reduce the interference of single bacteria detection.

[0036] In an alternative embodiment, the nanomaterial of the coated nanomaterial structure layer includes one or more of gold, silver, and copper; the structure of the coated nanomaterial structure layer includes one or more of nanorods, nanoflowers, and nanospheres; and the nanomaterial in the nanomaterial layer is arranged in 1-6 layers.

[0037] In the present application, the nanomaterial layer formed by the coating growth method as a nanomaterial layer has pores through which the fluorescent probe passes and through which the bacteria cannot pass, and together with the substrate, it forms the selective transmission characteristics of the composite functional porous metal film of the present application. The nanomaterial layer is arranged in 1 to 6 layers, and the number of layers of nanomaterial within this range can help to increase the enhancement of the target signal. Beyond this range, there is no further improvement in signal enhancement, but the cost and process are increased. When the multiple layers of nanomaterial layers are superimposed on each other, it helps to block the voids of a single layer of material, improve the consistency of the overall nanomaterial layer, and thus improve the strength uniformity of the Raman enhancement field, enhancing the reliability and accuracy of single-bacterium Raman enhancement identification.

[0038] Due to the material composition of the composite functional porous metal film, under an electric field, the side of the composite functional porous metal film that captures the planktonic microorganisms can be flexibly charged positively, negatively, or pulsed. When the composite functional porous metal film is positively charged, it is adsorbed with negatively charged planktonic microorganisms, when it is negatively charged, it repels negatively charged planktonic microorganisms and is released, and when it has a pulsed charge, it performs a cycle of adsorption and release of negatively charged planktonic microorganisms that approach, providing a basis for the automated monitoring method of the present application.

[0039] In a third aspect, the present application also provides a detection system for monitoring and identifying air planktonic microorganisms, comprising: the composite functional metal film described above, a power supply device, a laser light source, a sample detection device, and a Raman spectrometer, the power supply device being connected to the nanomaterial layer of the composite functional metal film; the laser light source is arranged above the composite functional metal film, and the laser light source is adapted to emit fluorescent excitation light to the fluorescent sample to be tested, so that the fluorescent probe in the fluorescent sample to be tested emits fluorescent emission light; the sample detection device is arranged on one side of the laser light source, and the sample detection device is adapted to receive the fluorescent emission light to obtain the quantity information and position information of the fluorescent sample to be tested; the Raman spectrometer emits Raman excitation light to the fluorescent sample to be tested, and captures the Raman scattering light emitted by the microorganisms to be tested in the fluorescent sample to be tested.

[0040] Beneficial effects: the composite functional metal film receives the to-be-tested fluorescent sample, the composite functional metal film stably adsorbs the to-be-tested microorganism in suspension, and the fluorescent probe is labeled in situ to form the to-be-tested fluorescent sample; the laser light source vertically irradiates the upper surface of the entire composite functional metal film, and the to-be-tested fluorescent sample emits fluorescent emission light at multiple positions; the sample detection device arranged on the side above collects the fluorescent emission light to form an image, and the specific position of each single bacterium and the total number of bacteria in the to-be-tested fluorescent sample are obtained; the Raman spectrometer clearly focuses on a single bacterium to emit Raman excitation light, scans to obtain the Raman spectrum of the bacterium, compares the standard Raman spectrum in the Raman standard spectrum library, determines the species of the bacterium through the comparison of target characteristic peaks and effective characteristic peaks, and realizes selective enrichment sampling, accurate preliminary judgment addressing and accurate single-bacterium detection. BRIEF DESCRIPTION OF DRAWINGS

[0041] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0042] FIG. 1 is a structural schematic diagram of a composite functional porous metal film according to an embodiment of the present application;

[0043] FIG. 2 is an exploded view of the composite functional porous metal film according to an embodiment of the present application;

[0044] FIG. 3 is a schematic diagram of the composite functional porous metal film capturing air-suspended microorganisms according to an embodiment of the present application;

[0045] FIG. 4 is a schematic diagram of obtaining a to-be-tested fluorescent sample in air-suspended microorganism monitoring and identification according to an embodiment of the present application;

[0046] FIG. 5 is a schematic diagram of performing fluorescent addressing of to-be-tested microorganisms in air-suspended microorganism monitoring and identification according to an embodiment of the present application;

[0047] FIG. 6 is an image of the to-be-tested fluorescent sample under a microscope according to FIG. 5;

[0048] FIG. 7 is a schematic diagram of a single-bacterium Raman monitoring and identification method in air-suspended microorganism monitoring and identification according to an embodiment of the present application;

[0049] FIG. 8 is a comparison diagram of Raman spectra at a single-bacterium position and at a position without a single bacterium in the air-suspended microorganism monitoring and identification method according to an embodiment of the present application;

[0050] FIG. 9 is a comparison diagram of Raman spectra at an Escherichia coli position and at a position without an Escherichia coli in the air-suspended microorganism monitoring and identification method according to an embodiment of the present application;

[0051] Figure 10 is a comparison chart of Raman spectra of Staphylococcus epidermidis position and no Staphylococcus epidermidis position in the air floating microorganism monitoring and identification method according to the embodiments of the present application.

[0052] Legend: 10, composite functional porous metal film; 1, substrate; 11, hole; 2, nanomaterial layer; 21, nanomaterial structure; 20, laser light source; 30, sample detection device; 40, Raman spectrometer; 50, detection box; 51, inlet; 52, first outlet; 53, second outlet; 100, fluorescent sample to be detected; 101, microorganism to be detected; 101a, first microorganism; 102b, second microorganism; 102, fluorescent probe. DETAILED DESCRIPTION

[0053] The present application will be further described below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are merely illustrative of the present application and are not to be used to limit the present application. In addition, it is to be noted that only the parts related to the present application are shown in the accompanying drawings and not all the parts. In the following description, the description of well-known structures and techniques is omitted to avoid unnecessary confusion of the concept of the present application. In the accompanying drawings, various structural diagrams according to the embodiments of the present application are shown. These diagrams are not drawn to scale, in which some details are exaggerated for the purpose of clear expression, and some details can be omitted. The shapes of various regions, layers, and the relative size and position relationship therebetween shown in the diagrams are only exemplary, and in actuality, there can be deviations due to manufacturing tolerances or technical limitations, and a person skilled in the art can additionally design regions / layers with different shapes, sizes, and relative positions according to actual needs. In the context of the present application, when a layer / element is referred to as being located “on” another layer / element, the layer / element can be directly located on the other layer / element, or there can be an intermediate layer / element therebetween. In addition, if a layer / element is located “on” another layer / element in one orientation, the layer / element can be located “under” the other layer / element when the orientation is reversed.

[0054] In the related art, an aerosol alarm is usually used for preliminary judgment of microorganism detection, and harmful microorganisms are timely alarmed. However, the aerosol alarm cannot effectively distinguish microorganisms from pollen, tail gas particles, dust and the like, resulting in false alarms of many non-microorganism substances. After the alarm, monitoring personnel need to collect and detect these samples for investigation. It is proved through many subsequent manual investigation practices that most of the aerosol alarm results are not microorganism threats. Therefore, the existing preliminary judgment has poor accuracy. On the other hand, common detection methods for microorganism detection include immunochromatography and nucleic acid detection. The number of one-time multi-target detection types is generally 10-20. If the target to be detected is not covered, multiple changes are required. The single detection time combined with multiple detection screening times is long, and the detection efficiency is low.

[0055] To solve the problems of poor preliminary judgment accuracy and low detection efficiency in the related art, as shown in FIGS. 1 and 2, the embodiment provides a composite functional porous metal film 10 for monitoring and identifying air plankton microorganisms, which includes a substrate 1 and a nanomaterial layer 2. The substrate 1 is a porous metal plate or metal mesh. The nanomaterial layer 2 is coated on one side surface of the substrate 1. The side surface of the nanomaterial layer 2 away from the substrate 1 is used to form a Raman signal enhancement field. The nanomaterial layer 2 is suitable for adsorbing a to-be-detected fluorescent sample 100. The to-be-detected fluorescent sample 100 includes a to-be-detected microorganism 101 and a fluorescent probe 102 combined together. The nanomaterial layer 2 has pores for the fluorescent probe not combined with the to-be-detected microorganism to pass through. The to-be-detected microorganism 101 is suitable for emitting Raman scattering light under irradiation of Raman excitation light. The fluorescent probe 102 is suitable for emitting fluorescent emission light under irradiation of fluorescent excitation light.

[0056] Specifically, the substrate 1 is provided as a thin metal sheet or metal mesh with an array of holes 11, which can be one of a circular, square, diamond or other shape, and the diameter of the holes 11 on the substrate 1 is 1-10 μm; as shown in FIG. 1 and FIG. 2, the nanomaterial layer 2 covers the upper surface of the substrate 1, and covers the holes 11. In order to effectively adsorb, the substrate 1 and the coated thin film-shaped nanomaterial layer 2 or the grown nanomaterial layer 2 with a certain morphology will usually choose a metal material. Metal is an atomic structure, and Raman spectrum is a molecular spectrum, so under normal circumstances, metal will not form a Raman spectrum. However, in the actual experiment process, it is found that metal materials have different degrees of noise signals under the irradiation of Raman excitation light, which will interfere with the detection of single bacteria. Therefore, in the embodiment, the nanomaterial layer 2 with low background signal (i.e. low noise signal) is used as the metal film layer covering the substrate 1 and realizing the energized adsorption medium. This is because, on the one hand, the selective permeability of the nanomaterial layer 2 can filter out impurity fluorescent probes that are not combined with the microorganisms to be detected, reducing the interference of the fluorescent signal; on the other hand, the nanomaterial layer 2 has the performance of blocking external impurity signals and itself as a metal layer to produce low noise. The background refers to the interference signal produced by other factors in the detection device in addition to the target factor in the sample to be tested, so the low background signal refers to the low interference signal. The nanomaterial layer 2 has the functions of stable adsorption of bacteria and Raman enhancement.

[0057] The composite functional porous metal film 10 of the embodiment, by setting the specific nanomaterial layer 2 on the substrate 1, firstly, the nanomaterial layer 2 has porous permeability, on the one hand, the nanomaterial layer 2 itself has small pores for the unbound fluorescent probes to pass through, so that the fluorescent probes 102 combined with the microorganisms 101 to be detected are retained on the nanomaterial layer 2, and on the other hand, other impurities such as unbound fluorescent probes pass through the small pores of the nanomaterial layer 2 and are discharged from the small holes formed by the holes 11 of the conformal substrate 1, reducing the interference of background signals such as impurity fluorescent probes and improving the sampling accuracy; secondly, the nanomaterial layer 2 accurately and stably adsorbs the fluorescent sample 100 to be detected when powered on, when the fluorescent excitation light irradiates the nanomaterial layer 2 where the fluorescent sample 100 to be detected is located, the fluorescent sample 100 to be detected emits fluorescent emission light, which is convenient for the detector to obtain the position information and total number information of the fluorescent sample 100 to be detected, and realizes the total number and addressing preliminary judgment of the microorganisms 101 to be detected, and then when the nanomaterial layer 2 is irradiated with Raman excitation light, the microorganisms 101 to be detected in the fluorescent sample 100 to be detected absorb the Raman excitation light and emit Raman scattering light, and at the same time, the Raman excitation light makes the molecules of the substances on the surface of the nanomaterial layer 2 vibrate, forming a Raman signal enhancement field, and the Raman scattering light signal is enhanced, so that the detector can obtain a higher intensity and accurate single-bacterium Raman spectrum of the microorganisms 101 to be detected, and then compare the characteristic peaks with the standard Raman spectrum in the Raman standard spectrum library to determine the species of the single bacterium of the microorganisms to be detected.

[0058] The composite functional metal film of the present application has flexible charging property, porous permeability and Raman enhancement characteristics, effectively reduces the interference signals in detection, realizes accurate capture and recognition of microorganisms, and can realize in-situ identification of single bacteria without changing and converting the target, and has high detection efficiency.

[0059] In one embodiment, the nanomaterial layer 2 can be formed by evaporating or magnetron sputtering spraying metal nanoparticles on the upper surface side of the substrate 1, and the nanomaterial layer 2 is a film layer formed by dense arrangement of metal particles in a microcosm, and the nanomaterial layer 2 can be one or more of a titanium nanoparticle film, a zinc nanoparticle film, a tin nanoparticle film, and a lead nanoparticle film; under the irradiation of Raman excitation light, the nanomaterial layer 2 formed by these metal nanoparticles is suitable for generating a low background signal, effectively reducing the interference with single-bacterium detection.

[0060] In one embodiment, the nanomaterial layer 2 can also be a coated nanomaterial structure layer, and the nanomaterial of the nanomaterial structure layer can be one or more of gold, silver, and copper; the nanomaterial structure 21 grown in the nanomaterial layer 2 includes one or more of nanorods, nanoflowers, and nanospheres, and the coated nanomaterial structure layer is provided in 1-6 layers.

[0061] That is, the nanomaterial layer 2 can be one or more of silver nanorods, silver nanoflowers, silver nanospheres, gold nanorods, gold nanoflowers, gold nanospheres, copper nanorods, copper nanoflowers, and copper nanospheres. That is, the nanomaterial layer 2 is a layer formed by arranging nanorods, nanospheres, and nanoflowers in a nanomaterial structure 21 in a microscopic view, as shown in the dashed box in FIG. 2.

[0062] The nanomaterial layer 2 described above is provided in 1-6 layers. Within this range, the number of layers of nanomaterials can assist in increasing the enhancement of the target signal. Beyond this range, there is no further improvement in signal enhancement, but the cost and process are increased. Alternatively, the nanomaterial layer 2 is provided in 2-6 layers, such as 2 layers, 4 layers, or 6 layers. The multiple layers of the nanomaterial layer 2 are stacked on each other, which helps to form gaps that shield a single layer of material by being misaligned, improves the consistency of the overall nanomaterial layer 2, and further improves the strength uniformity of the Raman enhancement field, thereby enhancing the reliability and accuracy of the single-bacterium Raman enhancement identification.

[0063] The enhancement effect related to the rough surface of gold, silver, copper, etc. is called surface-enhanced Raman scattering. The enhancement mechanism is produced by the synergistic effect of physical and chemical enhancement mechanisms, which facilitates the capture of the Raman scattering spectrum of the surface of a single bacterium.

[0064] Specifically, the surface of a bacterium is composed of many proteins and different modified proteins. Each bacterium surface has its unique protein composition and higher structure. Raman is a non-elastic collision between incident photons and molecules, which produces the vibration or rotation energy level of the molecule, and the frequency of the photon can change. The original incident light frequency and the new frequency component exist in the scattering spectrum, and the structure of the molecule can be understood through the new frequency component.

[0065] In the Raman surface enhancement effect, the Raman excitation light excites the vibration of the substance molecules. The bacterium is located on the nanomaterial layer 2 formed by silver, gold, and other nanorods, nanoflowers, etc. A large number of small protrusions or recesses are formed on the nanomaterial layer 2. This structure supports the collective oscillation of localized surface plasmons, which produces a locally enhanced electromagnetic field, thereby greatly enhancing the Raman scattering signal of the surface molecules of the bacterium adsorbed on the nanomaterial layer 2. This enhancement effect belongs to the physical enhancement caused by the electromagnetic field. In addition, it also includes chemical enhancement induced by charge transfer between the surface protein molecules of the bacterium and the metal surface. The two work together to significantly improve the efficiency of the Raman scattering of a single bacterium, thereby realizing the Raman spectrum detection of a single bacterium.

[0066] In one embodiment, the nanomaterial layer 2 can be positively charged, or negatively charged, or pulsed charged. The nanomaterial layer 2 is coated on the upper surface of the substrate 1 and the inner wall of the hole 11, so that the substrate 1 is electrically the same as the nanomaterial layer 2. The nanomaterial layer 2 is controlled by an external power supply device, and the type and intensity of the charge can be flexibly controlled. Generally, the isoelectric point of bacteria is 3-4, at which point the bacteria are negatively charged. The composite functional porous metal film 10 is externally powered through the nanomaterial layer 2, and the surface potential is positively charged. The electrical properties of bacteria are determined by the environment. When the pH of the environment in which the bacteria live is greater than or equal to the isoelectric point, the bacteria are negatively charged. When the pH is equal to the isoelectric point, the bacteria are not charged. When the pH is less than the isoelectric point, the bacteria are positively charged. In most cases, the pH of the environment in which the bacteria live is greater than the isoelectric point, so the surface of the bacteria is usually negatively charged. In this embodiment, the composite functional porous metal film 10 is negatively charged, and the negatively charged airborne planktonic microorganisms are stably adsorbed on the positively charged side of the composite functional porous metal film 10, i.e. the nanomaterial layer 2, reducing the interference of other airborne impurities.

[0067] Referring to FIGS. 3-7, the present embodiment further provides a detection system for monitoring and identifying airborne planktonic microorganisms, comprising: the composite functional porous metal film 10 described above, a power supply device, a laser light source 20, a sample detection device 30, and a Raman spectrometer 40, wherein the power supply device is connected to the nanomaterial layer 2 of the composite functional porous metal film 10; the laser light source 20 is arranged above the composite functional porous metal film 10, and is adapted to emit fluorescent excitation light to a to-be-tested fluorescent sample 100, so that a fluorescent probe 102 in the to-be-tested fluorescent sample 100 emits fluorescent emission light; the sample detection device 30 is arranged on one side of the laser light source 20, and is adapted to receive the fluorescent emission light to obtain quantity information and position information of the to-be-tested fluorescent sample 100; and the Raman spectrometer 40 emits Raman excitation light to the to-be-tested fluorescent sample 100, and captures Raman scattering light emitted by a to-be-tested microorganism 101 in the to-be-tested fluorescent sample 100.

[0068] The overall structure of the detection system is not shown, and each functional part is described separately. Different functional modules collect and process data of the to-be-tested fluorescent sample 100.

[0069] First, the composite functional porous metal film 10 receives the to-be-tested fluorescent sample 100. The to-be-tested microorganism 101 in the to-be-tested fluorescent sample 100 can be an airborne planktonic microorganism. As shown in FIG. 3, the composite functional porous metal film 10 is positively charged by being powered, and then the to-be-tested microorganism 101 in the air, including different forms of first microorganisms 101a and second microorganisms 101b, such as different bacteria with negative charges, is stably fixed on the nanomaterial layer 2 of the composite functional porous metal film 10. Then, the fluorescent probe 102 is labeled in situ to form the to-be-tested fluorescent sample 100, as shown in FIG. 4.

[0070] Then the composite functional porous metal film 10 with the fluorescent sample 100 to be tested is placed under the laser light source 20, the laser light source 20 vertically irradiates the upper surface of the entire composite functional porous metal film 10, and the fluorescent emission light is emitted from multiple fluorescent samples 100 to be tested. The fluorescent emission light is collected and imaged by the sample detection device 30 arranged on the side, as shown in FIG. 5, to obtain the specific positions of the microorganisms 101 to be tested, such as each single bacterium, and the total number of bacteria in the fluorescent sample 100 to be tested. FIG. 6 shows images of the fluorescent samples 100 to be tested at different positions on the composite functional porous metal film 10. The sample detection device 30 can be an image sensor or other image acquisition and analysis device. Of course, the sample detection device 30 can be arranged at other positions according to actual conditions, as long as it can meet the collection of fluorescent emission light.

[0071] Then the composite functional porous metal film 10 is placed under the Raman spectrometer 40, and the Raman spectrometer 40 clearly focuses on a single bacterium to emit Raman excitation light. The wavelength of the Raman excitation light depends on the characteristics of the microorganism 101 to be tested. Common Raman excitation light wavelengths include 325 nm, 405 nm, 488 nm, 532 nm, etc. In this embodiment, the bacterium is irradiated with Raman excitation light with a wavelength of 532 nm, and the Raman scattering spectrum of the bacterium is scanned. By comparing the standard Raman spectrum in the Raman standard spectrum library with the target characteristic peak obtained, the species of the bacterium is determined. The Raman spectrum of a single bacterium has a target characteristic peak, a Raman standard spectrum library, and at least three independent tests of each bacterium when establishing. The effective characteristic peak has a significant difference with the control and is repeated in independent cell tests, and is used as an effective characteristic peak.

[0072] To ensure the reliability of the detection system, the detection system is injected with the to-be-detected microorganism 101, including the air carried during sampling, when the to-be-detected microorganism 101 is air-borne microorganism. Therefore, in an embodiment, the detection system further comprises a detection box 50 and a power device. The detection box 50 has an inlet 51, a first outlet 52 and a second outlet 53. The composite functional porous metal film 10 is arranged in the detection box 50 and located below the inlet 51. The first outlet 52 is located on one side of the detection box 50 and is suitable for discharging gaseous medium, such as air medium in air-borne microorganism. The second outlet 53 is located at the lower end of the detection box 50 and is suitable for discharging liquid and solid medium, such as solvent in fluorescent probe solution and fluorescent probe not combined with the to-be-detected microorganism. The power device is suitable for passing the to-be-detected sample from the inlet 51 of the detection box 50. The to-be-detected sample includes air and air-borne microorganism. The to-be-detected microorganism 101 is air-borne microorganism. The to-be-detected microorganism 101 is fixed to the nanomaterial layer 2 of the composite functional porous metal film 10. The air is discharged from the first outlet 52. The second outlet 53 is suitable for discharging waste liquid, such as waste liquid of fluorescent probe solution when the fluorescent probe 102 is combined with the to-be-detected microorganism 101.

[0073] The embodiment also includes an air-borne microorganism monitoring and identification method applied to the above-mentioned detection system for monitoring and identifying air-borne microorganism. The monitoring and identification method comprises the following steps:

[0074] Step S100, adding the to-be-detected microorganism 101 to the composite functional porous metal film 10. Under the action of the electric field, the side of the composite functional porous metal film 10 in contact with the to-be-detected microorganism 101 presents flexible charging. The composite functional porous metal film 10 comprises a substrate 1 and a nanomaterial layer 2. The nanomaterial layer 2 directly contacts and adsorbs the to-be-detected microorganism 101. The substrate 1 has a plurality of holes 11. The nanomaterial layer 2 covers one side surface of the substrate 1. The side surface of the nanomaterial layer 2 away from the substrate 1 is used to form a Raman signal enhancement field.

[0075] The composite functional porous metal film 10 is positively charged through the electrified nanomaterial layer 2. The to-be-detected microorganism 101 in the air, such as bacteria, is negatively charged and stably adsorbed on the nanomaterial layer 2 of the composite functional porous metal film 10. The nanomaterial layer 2 may, for example, be formed of titanium nanoparticles. The nanomaterial layer 2 may also be one, three or five layers of silver nanorods. Of course, other types can also be selected. See the above-mentioned embodiments, which will not be described in detail here.

[0076] Step S200, adding the fluorescent probe 102 to the side of the composite functional porous metal film 10 with the to-be-detected microorganism 101 to combine the fluorescent probe 102 with the to-be-detected microorganism 101 to obtain the to-be-detected fluorescent sample 100.

[0077] That is, the fluorescent probe 102 is added to the upper surface of the nanomaterial layer 2, and is combined with the microorganism 101 to be detected. The fluorescent probe 102 in the embodiment is a kind of nanobio probe, and the planktonic microorganism adsorbed on the composite functional porous metal film 10 has structural gaps on the cell membrane and cell wall, so that the nanobio probe enters and accumulates. The nanobio probe has the characteristics of non-quenching of aggregate fluorescence, and has the characteristics of universality, effectiveness, specificity and stability of microbial staining. The nanobio probe also has the characteristics of increasing the permeability of the bacterial cell membrane. The nanobio probe has the characteristics of photoluminescence, and emits fluorescent emission light under the irradiation of excitation light, which is convenient for the detection and addressing of the planktonic microorganism.

[0078] In step S300, the excitation light is emitted to the composite functional porous metal film 10 to make the fluorescent sample 100 emit emission light.

[0079] For example, the laser light source 20 is used to emit excitation light, and the wavelength of the excitation light of the laser light source 20 is related to the selected material of the fluorescent probe 102. The nanobio probe enters and accumulates, and generates the detected fluorescent emission light under the irradiation of the excitation light.

[0080] In step S400, the emission light is collected and imaged to obtain the quantity information and position information of the microorganism 101 to be detected.

[0081] The nanobio probe enters and accumulates in the planktonic microorganism, and generates the detected fluorescent emission light under the irradiation of the excitation light. The sample detection device 30 collects the emission light to obtain the preliminary judgment result of the planktonic microorganism in the form of imaging counting, that is, the total number of bacteria and addressing.

[0082] The above S200-S400 realizes the preliminary judgment and addressing of the total number of bacteria of the microorganism to be detected, which is convenient for the subsequent identification of the species of the single bacterium in the microorganism to be detected.

[0083] In step S500, according to the obtained position information of all the microorganisms 101 to be detected, one position information is focused, the Raman excitation light is emitted to the selected one of the microorganisms 101 to be detected, and the Raman scattering light emitted by the microorganism is captured to obtain the Raman spectrum of the single bacterium. The Raman spectrum of the single bacterium is compared with the standard Raman spectrum in the Raman standard spectrum library to identify the species of the single bacterium.

[0084] Referring to Figure 8, the broken line b1, b2 and b3 are the Raman spectra obtained by using the Raman spectrometer 40 to perform multiple Raman experiments on the same position of a single bacterium, and the broken line a is the Raman spectrum obtained by performing a Raman experiment on a position without a single bacterium. It can be clearly seen that the Raman spectrum of the single bacterium has obvious characteristic peaks, while the Raman spectrum of the region without the single bacterium presents a relatively flat pattern. Therefore, the obtained Raman spectrum of the single bacterium with obvious characteristic peaks is compared with the Raman standard spectrum library, and the species category of the single bacterium is obtained by comparing the target characteristic peaks and the effective characteristic peaks.

[0085] Compared with the existing air floating microorganism alarm, the application has higher accuracy in detection principle and detection mode. The application physically splices the three modules of "sampling-primary judgment-detection" relying on the "composite functional porous metal film 10" as the only carrier to realize true integration, and improves the three-step process to "selective enrichment (sampling)-total bacteria monitoring (primary judgment)-species identification (detection)", that is, instead of taking the particle mixture in the air as the whole analysis object, the nanomaterial layer 2 of the composite functional porous metal film 10 is used to adsorb and fix the microorganism to be detected 101, that is, the selective enrichment of the microorganism to be detected 101 is taken as the starting point, thereby laying a foundation for the subsequent primary judgment accuracy and species identification accuracy. Then, the selective permeability of the nanomaterial layer 2 is used to retain the fluorescent probe 102 combined with the microorganism to be detected 101, while the uncombined fluorescent probe and other impurities such as solvents can be removed, thereby reducing the interference of impurity signals and realizing the total bacteria identification of the microorganism to be detected 101. Then, the nanomaterial layer 2 has a Raman signal enhancement field, and the enhanced Raman signal of the microorganism to be detected 101 is obtained, the typical Raman spectrum and characteristic peaks are obtained, the single microorganism, that is, the species category of a certain bacterium, is obtained by comparing with the standard Raman spectrum in the Raman standard spectrum library, and the detection efficiency and accuracy of the air floating microorganism are effectively improved.

[0086] In one embodiment, the standard Raman spectrum in the Raman standard spectrum library has an effective characteristic peak, the effective characteristic peak is obtained by scanning the independent Raman spectrum of more than three same bacteria, the effective characteristic peak has a significant difference from the control, and the effective characteristic peak repeatedly appears in the spectrum of more than three independent Raman spectra; the Raman spectrum of a single bacterium has a target characteristic peak, and the target characteristic peak is used for comparison with the effective characteristic peak.

[0087] In one embodiment, under the action of an electric field, the composite functional porous metal film 10 has one of positive charge, negative charge and pulse charge.

[0088] The composite functional porous metal film 10 has positive charge, and the composite functional porous metal film 10 adsorbs the air floating microorganism.

[0089] The composite functional porous metal film 10 carries a negative charge, and the composite functional porous metal film 10 releases air-borne microorganisms;

[0090] The composite functional porous metal film 10 carries a pulse charge, and the composite functional porous metal film 10 performs the cycle of adsorption and release on the approaching air-borne microorganisms.

[0091] In one embodiment, step S200, a fluorescent probe 102 is added to the side of the composite functional porous metal film 10 with the microorganisms to be detected 101, the fluorescent probe 102 binds to the microorganisms to be detected 101, and a fluorescent sample to be detected 100 is obtained, including the following steps:

[0092] Step S201, a fluorescent probe 102 solution is added to the side of the composite functional porous metal film 10 with the microorganisms to be detected 101.

[0093] Step S202, the fluorescent probe 102 and the microorganisms to be detected 101 are incubated for 1-5 minutes to allow the fluorescent probe 102 to enter the microorganisms to be detected 101, and the fluorescent probe 102 binds to the microorganisms to be detected 101 to obtain the fluorescent sample to be detected 100.

[0094] Step S203, the excess fluorescent probe solution is washed away; the fluorescent probe in the fluorescent probe solution that is not combined with the microorganisms to be detected 101 passes through the nano material layer 2 and is discharged from the hole 11 of the substrate 1. The waste liquid in the fluorescent probe 102 solution is discharged from the second outlet 53 of the detection box 50, and a large number of microorganisms to be detected 101 labeled by the fluorescent probe 102, i.e. the fluorescent sample to be detected 100, is obtained.

[0095] In this application, the nano material layer 2 in the composite functional porous metal film 10 has pores for the uncombined fluorescent probe to pass through, and the nano material layer covers the substrate 1 and forms small holes with the shaped hole 11, i.e. the composite functional porous metal film 10 has porous permeability, first the small fluorescent probe 102 can pass through the pores of the nano material layer 2 itself, and then be discharged from the small holes, so that the excess fluorescent probe is washed away, and only the fluorescent probe 102 combined with the microorganisms to be detected 101 remains on the composite functional porous metal film 10. The nano material layer 2 arranged in this way reduces the interference of impurity fluorescent signals on detection, so that the microbial detection on the composite functional porous metal film 10 in this application has the characteristics of low background, and improves the accuracy of detection.

[0096] In one embodiment, the size of the above-mentioned fluorescent probe 102 is 2-10 nm; the fluorescent probe 102 can be one or more of carbon quantum dots, fluorescent quantum dots, and fluorescent dyes.

[0097] In this embodiment, the Raman spectra of E. coli and S. epidermidis in the Raman standard spectrum library are exemplarily shown. Five single bacteria Raman spectrum independent detections are performed on E. coli and S. epidermidis respectively, and the E. coli Raman spectrum shown in FIG. 9 and the S. epidermidis Raman spectrum shown in FIG. 10 are obtained.

[0098] In FIG. 9, the broken lines d1, d2, d3, and d4 are Raman spectra obtained by performing multiple Raman experiments on the same position of E. coli, and the broken line c is a Raman spectrum obtained by performing a Raman experiment on a position without E. coli. In FIG. 10, the broken lines f1, f2, f3, and f4 are Raman spectra obtained by performing multiple Raman experiments on the same position of S. epidermidis, and the broken line e is a Raman spectrum obtained by performing a Raman experiment on a position without S. epidermidis. It can be clearly seen that the Raman spectrum characteristic peaks of E. coli and S. epidermidis have good repeatability, the characteristic peaks have significant differences with the baseline of the control and are repeated in each independent cell test as effective characteristic peaks, and E. coli and S. epidermidis have unique Raman spectrum effective characteristic peaks.

[0099] In one embodiment, the monitoring identification method can also be used to detect planktonic microorganisms in water bodies. Exemplarily, the substrate 1 of the composite functional porous metal film 10 adopts a metal mesh, and tin metal elements are attached to the metal mesh in the form of nanoparticles by an evaporation method to form a nano material layer 2 attached to the substrate 1 with reduced pore size, so that the substrate 1 has a basis for enrichment of planktonic microorganisms. Alternatively, the nano material layer 2 is a metal nano material layer formed by tiling silver nanorods synthesized from silver nano materials, so that the Raman spectrum signal of microorganisms on the composite functional porous metal film 10 is enhanced. Three layers of silver nanorods are coated on the substrate 1 as the signal-enhancing nano material layer 2 to complete the fabrication of the composite functional porous metal film 10.

[0100] When sampling the sample of planktonic microorganisms in water bodies, the water body containing microorganisms is dropped on one side of the composite functional porous metal film 10 having the nano material layer 2, and incubated for 5 min to allow the negatively charged microorganisms to be more stably adsorbed to the composite functional porous metal film 10. The water sample is removed and naturally dried.

[0101] In another embodiment, the monitoring identification method can also be used to monitor and identify microorganisms on the surface of an object. Exemplarily, the nano material layer 2 of the composite functional porous metal film 10 is a metal nano material layer formed by tiling nanorods synthesized from gold nano materials, so that the Raman spectrum signal of microorganisms on the composite functional porous metal film 10 is enhanced. One layer of gold nanorods is directly synthesized on the substrate 1 as the signal-enhancing nano material layer 2 to complete the fabrication of the composite functional porous metal film 10.

[0102] In the sampling of microorganism sample on solid surface, the sampling swab is used to wipe the solid surface, the sampling swab is eluted in sterile water to prepare a sample containing microorganism, the sample is dropped on one side of the composite functional porous metal film 10 with nanomaterial layer 2, and incubated for 5 min to make the negatively charged microorganism more firmly adsorbed on the composite functional porous metal film 10, and then the water sample is removed and naturally dried.

[0103] In the above description, the technical details of the patterning, etching and other techniques for each layer are not described in detail. However, those skilled in the art should understand that the layers, regions and the like with desired shapes can be formed by various technical means. In addition, those skilled in the art can also design methods that are not exactly the same as the methods described above in order to form the same structure. In addition, although each embodiment is described above separately, this does not mean that the measures in each embodiment cannot be used advantageously in combination.

[0104] Although the embodiments of the present application are described in conjunction with the drawings, various modifications and changes can be made by those skilled in the art without departing from the spirit and scope of the present application, and such modifications and changes fall within the scope defined by the appended claims.

Claims

1. An air-borne microorganism monitoring discrimination method characterized by, The application relates to a composite functional porous metal film and a method for detecting microorganisms. The composite functional porous metal film is added with the microorganisms to be detected. Under the action of an electric field, one side of the composite functional porous metal film in contact with the microorganisms to be detected is provided with flexible charging, the composite functional porous metal film comprises a substrate and a nanomaterial layer, the nanomaterial layer is directly contacted with the microorganisms to be detected through electrification and adsorption fixation, and selective enrichment is carried out. The substrate has a plurality of holes, the nanomaterial layer is arranged on one side surface of the substrate, and the side surface of the nanomaterial layer away from the substrate is used for forming a Raman signal enhancement field. A fluorescent probe is added to the side of the composite functional porous metal film with the microorganisms to be detected, the fluorescent probe is combined with the microorganisms to be detected to obtain a fluorescent sample to be detected. The selective permeability of the composite functional porous metal film is used to retain the fluorescent probe of the fluorescent sample to be detected combined with the microorganisms to be detected, excitation light is emitted to the fluorescent sample to be detected at a plurality of positions on the upper surface of the composite functional porous metal film, and the fluorescent sample to be detected emits emission light. The emission light is collected and imaged to obtain quantity information and position information of the microorganisms to be detected, total bacteria discrimination addressing of the microorganisms to be detected is realized. According to the obtained position information of the microorganisms to be detected, the microorganisms to be detected at one position information are focused, Raman excitation light is emitted to the microorganisms to be detected, and Raman scattering light emitted by the microorganisms to be detected is captured to obtain a Raman spectrum of a single bacterium; the Raman spectrum of the single bacterium is compared with standard Raman spectra in a Raman standard spectrum library, and the species of the single bacterium is judged.

2. The method according to claim 1, wherein Under the action of an electric field, the composite functional porous metal film has one of positive charge, negative charge and pulse charge. The composite functional porous metal film has positive charge, and the composite functional porous metal film adsorbs air-borne microorganisms. The composite functional porous metal film has negative charge, and the composite functional porous metal film releases air-borne microorganisms. The composite functional porous metal film has pulse charge, and the composite functional porous metal film carries out adsorption and release circulation of the air-borne microorganisms close to the composite functional porous metal film.

3. The method according to claim 1, wherein The fluorescent probe is added to the side of the composite functional porous metal film with the microorganisms to be detected, the fluorescent probe is combined with the microorganisms to be detected to obtain a fluorescent sample to be detected. The fluorescent probe solution is added dropwise to the side of the composite functional porous metal film with the microorganisms to be detected. The fluorescent probe and the microorganisms to be detected are incubated for 1 min to 5 min, the fluorescent probe enters the microorganisms to be detected, and the fluorescent probe is combined with the microorganisms to be detected to obtain a fluorescent sample to be detected. Excess fluorescent probe solution is washed away, and the fluorescent probe not combined with the microorganisms to be detected in the fluorescent probe solution passes through the nanomaterial layer and is discharged from the holes of the substrate. The composite functional porous metal film is also used for detection of microorganisms in water bodies and microorganisms on solid surfaces.

4. The method according to claim 3, wherein The size of the fluorescent probe is 2 nm to 10 nm, and the fluorescent probe is one or more of carbon quantum dots, fluorescent quantum dots and fluorescent dyes.

5. The method of claim 1, wherein the method is used for monitoring airborne microorganisms. The standard Raman spectrum in the Raman standard spectrum library has effective characteristic peaks, which are obtained from independent Raman spectrum scanning of more than three same bacteria, and the effective characteristic peak spectrum has a significant difference from the baseline spectrum; The Raman spectrum of the single bacterium has target characteristic peaks, which are used for comparison with the effective characteristic peaks; The Raman spectrum of the single bacterium is obtained and the species determination is also used for detection of microorganisms in water and microorganisms on solid surfaces.

6. A composite functional porous metal film for monitoring and identifying airborne microorganisms, which is used in the method for monitoring and identifying airborne microorganisms according to any one of claims 1 to 5, characterized by It comprises: a substrate, which is a porous metal plate or a metal mesh; a nanomaterial layer covering one side surface of the substrate, the nanomaterial layer away from the one side surface of the substrate is used to form a Raman signal enhancement field; The nanomaterial layer is suitable for adsorbing a to-be-tested fluorescent sample, the to-be-tested fluorescent sample includes a to-be-tested microorganism combined with a fluorescent probe, the nanomaterial layer has pores for uncombined fluorescent probes to pass through, the to-be-tested microorganism is suitable for emitting Raman scattering light under irradiation of Raman excitation light, and the fluorescent probe is suitable for emitting fluorescent emission light under irradiation of fluorescent excitation light.

7. The composite functional porous metal film for monitoring and discriminating airborne microorganisms according to claim 6, wherein, The nanomaterial layer includes an evaporated single metal element layer or a coated nanomaterial structure layer.

8. The composite functional porous metal film for monitoring and discriminating airborne microorganisms according to claim 7, wherein, The evaporated single metal element layer includes one or more of a titanium nanoparticle film, a zinc nanoparticle film, a tin nanoparticle film, and a lead nanoparticle film.

9. The composite functional porous metal film for monitoring and discriminating airborne microorganisms according to claim 7, wherein, The nanomaterial of the coated nanomaterial structure layer includes one or more of gold, silver, and copper; the nanomaterial structure of the coated nanomaterial structure layer includes one or more of nanorods, nanoflowers, and nanospheres; and the coated nanomaterial structure layer is provided in 1-6 layers.

10. A detection system for monitoring and discriminating airborne microorganisms, characterized by, It comprises: The composite functional porous metal film for monitoring and identifying air planktonic microorganisms according to any one of claims 6-9; A power supply device connected to the nanomaterial layer of the composite functional porous metal film; A laser light source arranged above the composite functional porous metal film, the laser light source is suitable for emitting fluorescent excitation light to the to-be-tested fluorescent sample to make the fluorescent probe in the to-be-tested fluorescent sample emit fluorescent emission light; A sample detection device arranged on one side of the laser light source, the sample detection device is suitable for receiving the fluorescent emission light to obtain quantity information and position information of the to-be-tested fluorescent sample; A Raman spectrometer emitting Raman excitation light to the to-be-tested fluorescent sample and capturing Raman scattering light emitted by the to-be-tested microorganism in the to-be-tested fluorescent sample.

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