Microfluidic SERS (Surface Enhanced Raman Scattering) acquisition system and method for air fungal disease spores

Abstract

The invention provides a microfluidic SERS (Surface Enhanced Raman Scattering) collection system and method for air fungal disease spores, and the system comprises a microfluidic purification module which is used for separating and enriching extracted gas to obtain enriched spores; the SERS sensing module is used for mixing the enriched spores and the enhanced particles to obtain a detected solution; the spectrum acquisition module is used for acquiring the spectrum of the detected solution and outputting the spectrum; and the control module is electrically connected with the microfluidic purification module, the SERS sensing module and the spectrum acquisition module and is used for controlling the start-stop time sequence of the modules. The micro-fluidic purification module realizes purification of spores from a physical method, the SERS sensing unit generates a detected solution, the spectrometer acquires Raman spectrum data of the solution, and the modules are small in size, so that the system can be deployed on an unmanned aerial vehicle, data acquisition can be conveniently performed at any position, the acquisition process is full-automatic, personnel participation is not needed, and the cost is low. And the detection efficiency can be greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of spore separation and detection, and in particular to a microfluidic SERS collection system and method for airborne fungal disease spores. Background Art

[0002] Before the occurrence of airborne diseases of crops and the spread of diseases over a large area, the first thing that happens is that the spores of airborne diseases are spread along the airflow in the air. The spores adhere to the host through diffusion, and then germinate and infect to show the symptoms of the disease. At present, spore catchers are mainly divided into fixed spore catchers and movable spore catchers. The traditional fixed spore catcher is fixed to the ground with a fixed bracket. The spore catcher can only be fixed in one position to collect spores in the air. The collection efficiency is often low, and the collected data is not comprehensive. Although the movable spore catcher can be moved for collection, it cannot collect spores at different heights, and there is also the problem of incomplete collected data. Traditional spore detection methods such as morphological identification, separation and culture identification and polymerase chain reaction (PCR) methods, but there are many types of impurities and spores in the air, and there are disadvantages such as complex pre-processing, poor anti-interference ability, long detection time and poor accuracy, which make it difficult to detect online. Therefore, it is urgent to develop a system that can flexibly and accurately collect spore-related data. Summary of the invention

[0003] In view of the above defects of the prior art, the present invention provides a microfluidic SERS acquisition system and method for airborne fungal disease spores to solve the technical problems of inconvenient spore-related data acquisition and inaccurate data.

[0004] To achieve the above-mentioned purpose and other related purposes, the present invention provides a microfluidic SERS acquisition system for airborne fungal disease spores, comprising: a microfluidic purification module, used to separate and enrich the extracted gas to obtain enriched spores; a SERS sensing module, used to mix the enriched spores and enhanced particles to obtain a tested solution; a spectrum acquisition module, used to acquire the spectrum of the tested solution and then output it; and a control module, the control module is electrically connected to the microfluidic purification module, the SERS sensing module and the spectrum acquisition module, and is used to control the start and stop timing of these modules.

[0005] In one embodiment of the present invention, the microfluidic purification module includes a first channel, a second channel, a third channel, an enrichment zone, an impurity zone and a micro air pump; one end of the first channel is connected to the air, and the other end is separated into two second channels; the other end of the second channel is separated into two third channels; the other ends of the four third channels are respectively connected to three enrichment zones and one impurity zone; the enrichment zone is used to collect spores in the air, and the impurity zone is used to collect and discharge separated impurities; the micro air pump is connected to the first channel, and is used to generate negative pressure in the microfluidic purification module.

[0006] In one embodiment of the present invention, the SERS sensing module includes a secondary purification unit, a mixing unit, a pressurizing unit and a detection channel. The secondary purification unit is used to secondary purify the spores collected by the microfluidic purification module, the mixing unit is used to mix the secondary purified spores and the enhancing particles to obtain the solution to be tested, and the pressurizing unit is used to pump the solution to be tested into the detection channel.

[0007] In one embodiment of the present invention, the secondary purification unit includes a purification channel, a first injector and a magnetic ring. The enrichment zone in the microfluidic purification module and the first injector are connected to one end of the purification channel, and the magnetic ring is sleeved on the other end of the purification channel. The first injector is used to inject MNP@PNIPAMAA-CAS solution into the purification channel. The MNP@PNIPAMAA-CAS solution combines with the spores and is captured when passing through the magnetic ring to achieve secondary purification of the spores.

[0008] In one embodiment of the present invention, the mixing unit includes a mixing channel and a second injector, the second injector is connected to one end of the purification channel away from the magnetic ring, one end of the mixing channel is connected to one end of the purification channel where the magnetic ring is set, the second injector is used to inject the silver nano-enhanced particle solution, and the mixing channel has several turns along its length to achieve mixing of the silver nano-enhanced particle solution and the spores after secondary purification.

[0009] In one embodiment of the present invention, the mixing channel and the detection channel are connected through a cache channel, and the mixing channel and the detection channel are both vertically connected to the cache channel; the pressurizing unit is connected to one end of the cache channel close to the mixing channel, and the tested solution obtained by mixing in the mixing channel is cached in the cache channel, and the pressurizing unit introduces the cached solution into the detection channel through high-pressure nitrogen.

[0010] In one embodiment of the present invention, a cleaning module and a waste liquid collection module are also included; the purification channel, the mixing channel, the cache channel and the detection channel are an integrated structure, the purification channel is connected to the cleaning module at one end away from the mixing channel, the cleaning module is used to pump deionized water into the purification channel, and the deionized water flows along each channel of the integrated structure to clean each channel, and the detection channel is connected to the waste liquid collection module at one end away from the cache channel for collecting waste liquid during the detection and cleaning process.

[0011] In one embodiment of the present invention, the spectrum acquisition module includes a laser and a spectrometer. The laser emitted by the laser reacts with the solution to be tested in the detection channel. The Raman signal generated by the reaction is received by the spectrometer and converted into spectral data for output.

[0012] In one embodiment of the present invention, the MNP@PNIPAMAA-CAS solution is prepared according to the following steps: acrylic acid with a concentration of 100 mM and magnetic nanoparticles are reacted at a volume ratio of 5:1, and activated by EDC / NHS for 12 hours to obtain a MNP-AA solution; N-isopropylacrylamide with a concentration of 100 mM, acrylic acid with a concentration of 100 mM, bis(2-methylpropylene)ethoxy disulfide with a concentration of 50 mM, sodium dodecyl sulfate with a concentration of 100 mM, and the MNP-AA solution are mixed in a container at a volume ratio of 60:3.4:12:1:20. ; After blowing nitrogen into the mixed solution for 20 minutes, ammonium persulfate with a concentration of 10 mM and a volume ratio of 10 was quickly added; after heating for 4 hours under an ultrasonic atmosphere, centrifugation and purification were performed to obtain a MNP@PNIPAMAA solution; the MNP@PNIPAMAA solution was activated by EDC / NHS for 12 hours, and a caspofungin solution with a concentration of 10 g / mL was added, and the volume ratio of the MNP@PNIPAMAA solution to the caspofungin solution was 9:1; after stirring for 12 hours, centrifugation and purification were performed to obtain the MNP@PNIPAMAA-CAS solution.

[0013] To achieve the above-mentioned purpose and other related purposes, the present invention also provides a microfluidic SERS collection method for airborne fungal disease spores, comprising: turning on a micro air pump in a microfluidic purification module to enrich the spores in an enrichment area; turning on a first injector to inject an MNP@PNIPAMAA-CAS solution into a purification channel, and closing the first injector after a preset first time; turning on a second injector to inject an enhanced particle solution into the purification channel, and closing the second injector after a preset second time; turning on a pressurizing unit, and closing it after a preset third time; turning on a laser and a spectrometer to collect spectral data, and closing it after a preset fourth time; turning on a micro water pump in a cleaning module, and closing it after a preset fifth time; turning on the first injector again to perform the next detection, and after the detection is completed, closing the micro air pump in the microfluidic purification module.

[0014] Beneficial effects of the present invention: The present invention proposes a microfluidic SERS acquisition system and method for airborne fungal disease spores. The device uses a microfluidic purification module to physically purify the spores, and then automatically produces a detectable solution through a SERS sensing unit. Finally, a spectrometer is used to collect Raman spectrum data of the solution. These modules are relatively small in size, so that the system can be deployed on a drone, which is convenient for data acquisition at any location. At the same time, the acquisition process is fully automatic and does not require human participation, which can greatly improve the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 A functional block diagram of a collection system provided by an embodiment of the present invention; Figure 2 A schematic diagram of the structure of a microfluidic purification module provided in one embodiment of the present invention; Figure 3 This is a schematic structural diagram of a SERS sensor module provided in one embodiment of the present invention. Explanation of the reference numerals: 100, microfluidic purification module; 101, first channel; 102, second channel; 103, third channel; 104, enrichment zone; 105, impurity zone; 106, micro gas pump; 200, SERS sensing module; 210, secondary purification unit; 211, purification channel; 212, first injector; 213, magnetic ring; 220, mixing unit; 221, mixing channel; 222, second injector; 230, pressurizing unit; 240, cache channel; 250, detection channel; 300, spectrum acquisition module; 301, laser; 302, spectrometer; 400, control module; 500, cleaning module; 600, waste liquid collection module. DETAILED DESCRIPTION

[0017] The following describes the embodiments of the present invention through specific specific embodiments, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. It should be noted that the following embodiments and the features in the embodiments can be combined with each other without conflict. In addition to the specific methods, equipment, and materials used in the embodiments, according to the technical personnel in this technical field's grasp of the prior art and the records of the present invention, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to implement the present invention.

[0018] It should be understood that the terms used in the embodiments of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art.

[0019] The structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions under which the present invention can be implemented, so they have no substantive technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. In addition, the drawings only show the components related to the present invention rather than the number, shape, and size of the components in actual implementation. The type, quantity, and proportion of each component in actual implementation can be changed arbitrarily, and the component layout type may also be more complicated.

[0020] In the following description, numerous details are discussed to provide a more thorough explanation of the embodiments of the present invention. However, it is obvious to those skilled in the art that the embodiments of the present invention can be implemented without these specific details. In some of the embodiments, well-known structures and devices are shown in the form of block diagrams rather than in detail to avoid making the embodiments of the present invention difficult to understand.

[0021] The flowchart and block diagram in the accompanying drawings illustrate the system architecture, functions and operations that may be implemented according to the methods and computer program products of various embodiments disclosed in the present invention. In this regard, each box in the flowchart or block diagram can represent a module, a program segment, or a part of a code, and the module, the program segment, or a part of the code contains one or more executable instructions for implementing the specified logical function. It should also be noted that in some alternative implementations, the functions marked in the box can also occur in a different order from the order marked in the accompanying drawings. For example, two boxes represented in succession can actually be executed substantially in parallel, and they can sometimes be executed in the opposite order, depending on the functions involved. It should also be noted that each box in the block diagram and / or the flowchart, and the combination of the boxes in the block diagram and / or the flowchart can be implemented with a dedicated hardware-based system that performs the specified function or operation, or can be implemented with a combination of dedicated hardware and computer instructions.

[0022] Before introducing the solution of the present invention, it should be noted that the channel size of the present invention is very small, so the overall size is also relatively small. Figure 2 and Figure 3 In order to clearly illustrate the connection relationship between the various components, the accompanying drawings are not drawn according to the actual scale, and these drawings are only for illustration.

[0023] See also Figure 1 , Figure 1 A microfluidic SERS acquisition system for airborne fungal disease spores provided in one embodiment of the present invention includes a microfluidic purification module 100, a SERS sensor module 200, a spectrum acquisition module 300, and a control module 400. The microfluidic purification module 100 is used to separate and enrich the extracted gas to obtain enriched spores; the SERS sensor module 200 is used to mix the enriched spores with enhanced particles to obtain a tested solution; the spectrum acquisition module 300 is used to collect the spectrum of the tested solution and then output it; the control module 400 is electrically connected to the microfluidic purification module 100, the SERS sensor module 200, and the spectrum acquisition module 300, and is used to control the start and stop timing of these modules.

[0024] The device uses a microfluidic purification module 100 to physically purify the spores, then uses a SERS sensing unit to automatically produce a detectable solution, and finally uses a spectrometer 302 to collect Raman spectrum data of the solution. These modules are relatively small in size, so that the system can be deployed on a drone, which is convenient for data collection at any location. At the same time, the collection process is fully automatic and does not require human participation, which can greatly improve the detection efficiency.

[0025] See also Figure 2 In a specific embodiment of the present invention, the microfluidic purification module 100 includes a first channel 101, a second channel 102, a third channel 103, an enrichment area 104, an impurity area 105 and a micro air pump 106; one end of the first channel 101 is connected to the air, and the other end is separated into two second channels 102; the other end of the second channel 102 is separated into two third channels 103; the other ends of the four third channels 103 are respectively connected to three enrichment areas 104 and one impurity area 105; the enrichment area 104 is used to collect spores in the air, and the impurity area 105 is used to collect and discharge the separated impurities; the micro air pump 106 is connected to the first channel 101, and is used to generate negative pressure in the microfluidic purification module 100.

[0026] In a microfluidic system, the design of the enrichment zone 104 is usually based on the principle of microfluidics, and the efficient separation and enrichment of target molecules or cells is achieved by precisely controlling the flow of fluids and the transmission of substances. The microfluidic purification module 100 in the present invention is used to collect spores. During the design, the length and radius of each channel are determined according to the spore size, microchannel force analysis and the magnitude of the dielectrophoretic force, and the parameters such as the radius of the spore enrichment zone, the spacing of the triangular gradient inner rib structure, the inlet flow rate of the microfluidic chip, the width and length of the pretreatment channel, the width of the airflow outlet channel, the separation angle and the electric field voltage are adjusted to maximize the spore enrichment efficiency by adjusting these parameters.

[0027] During the design, simulation can be performed using Comsol Multiphysics software. In this embodiment, preferably three levels of channels (i.e., the first channel 101, the second channel 102, and the third channel 103) are designed, and three enriched regions 104 and one impurity region 105 are designed, and the following parameters are used for construction: the length of the first channel 101 is 11000 μm, the width is 2000 μm, and the separation angle from the first channel 101 to the second channel 102 is 30°; the length of the second channel 102 is 9000 μm, the width is 1500 μm, and the separation angle from the second channel 102 to the third channel 103 is 30°; the length of the third channel 103 is 2300 μm, the width is 1500 μm, and the third channel 103 directly connects three enriched regions 104 and one impurity region 105, the radius of the enriched region 104 is 1000 μm, and the radius of the impurity region 105 is 1000 μm. The microfluidic purification module 100 of this size can be used to collect spores well, with an enrichment efficiency as high as 98%.

[0028] When the micro air pump 106 is turned on, the micro air pump 106 is in the suction mode. Under the action of the micro air pump 106, each channel presents a negative pressure, which will attract the external air containing spores from the first channel 101 and divert it to other channels. After passing through the microfluidic channel, it is enriched in the three enrichment areas 104 respectively. The spores collected in the three enrichment areas 104 will enter the subsequent SERS sensor module 200. The pressure of the micro air pump 106 can be adjusted to control the flow rate of the incoming air. Impurities in the air will be collected in the impurity area 105 and discharged.

[0029] See also Figure 3 In a specific embodiment of the present invention, the SERS sensing module 200 includes a secondary purification unit 210, a mixing unit 220, a pressurizing unit 230 and a detection channel 250. The secondary purification unit 210 is used for secondary purification of spores collected by the microfluidic purification module 100, the mixing unit 220 is used for mixing the secondary purified spores and the enhanced particles to obtain a solution to be tested, and the pressurizing unit 230 is used for pumping the solution to be tested into the detection channel 250.

[0030] In order to achieve efficient and synchronous separation and enrichment of wheat airborne fungal disease spores in different regions, solve the problem of high impurities and inaccurate detection in conventional detection of air, and enable faster and more accurate capture and detection of spores, we designed a SERS sensor module 200. Because the impurities cannot be completely removed by the physical model when the air containing spores passes through the microfluidic purification module 100, the fungal spores are further captured in the SERS sensor module 200. The ultrafast nano-capturing agent MNP@PNIPAMAA-CAS is used. When it is mixed with the fungal spores in the air and passes through the channel containing the strong magnetic ring 213, the fungal spores will adhere to the magnetic ring 213, and the impurity solution will be discharged through the waste liquid outlet. At the same time, the introduction of enhanced silver nanoparticles improves the detection sensitivity and specificity of the SERS technology, so that the combination of the two further improves the speed and accuracy of detection.

[0031] In a specific embodiment of the present invention, the secondary purification unit 210 includes a purification channel 211, a first injector 212 and a magnetic ring 213. The enrichment area 104 in the microfluidic purification module 100 and the first injector 212 are connected to one end of the purification channel 211, and the magnetic ring 213 is sleeved on the other end of the purification channel 211. The first injector 212 is used to inject the MNP@PNIPAMAA-CAS solution into the purification channel 211. The MNP@PNIPAMAA-CAS solution combines with the spores and is captured when passing through the magnetic ring 213 to achieve secondary purification of the spores. The secondary purification unit 210 is set because the microfluidic purification module 100 is purified by physical means, and its purification effect is relatively not particularly good. In order to enhance the subsequent spectrum collection, the fewer impurities contained in the spores, the more accurate the spectrum data obtained by the subsequent collection, so the secondary purification unit 210 is added in this embodiment.

[0032] The structure of the first injector 212 is similar to that of the second injector 222, which includes a micro pump, a micro liquid storage tank and a catheter. The liquid can be exported by turning on or off the micro pump, and the pressure during export can be adjusted by adjusting the micro pump. The micro pump can be a micro air pump 106, which drives the liquid to flow by applying air pressure to avoid excessive or low pressure. A pressure sensor can be installed at the end of the channel of each pump, and the micro pump can be controlled when the liquid reaches a certain set pressure. The control unit can adjust the output power of different pumps to control the pressure of different channels. In the present invention, the pump pressure with enhanced particles is the highest, which is used to promote the mixing of enhanced particles and spores, and the pump pressure of purified spores is the lowest to prevent enhanced particles or MNP@PNIPAMAA-CAS solution from flowing back into this channel. For the sake of insurance, the installation positions of the two injectors and the microfluidic purification module 100 on the purification channel 211 are different, and when they are connected to the channel, the injection port is arranged obliquely as much as possible to move each solution toward the side of the magnetic ring 213.

[0033] The micro air pump 106 in the present invention may be, for example, a micro pneumatic diaphragm pump, which has stable airflow output, good pressure control capability, corrosion resistance and long life.

[0034] In a specific embodiment of the present invention, the above-mentioned MNP@PNIPAMAA-CAS solution is prepared according to the following steps.

[0035] (1) Acrylic acid (AA) with a concentration of 100 mM and magnetic nanoparticles (MNPs) were reacted in a volume ratio of 5:1 and activated with EDC / NHS for 12 hours to obtain an MNP-AA solution. During this time, AA combined with MNPs (-NH2). The reaction temperature was 4°C to ensure that AA was successfully attached to the surface of the magnetic particles.

[0036] (2) N-isopropylacrylamide (NIPAM) with a concentration of 100 mM, acrylic acid (AA) with a concentration of 100 mM, bis(2-methylpropylene)ethoxy disulfide (BMEDS) with a concentration of 50 mM, sodium dodecyl sulfate (SDS) with a concentration of 100 mM, and MNP-AA solution are mixed in a container (e.g., a flask) at a volume ratio of 60:3.4:12:1:20.

[0037] (2) After nitrogen was blown into the mixed solution for 20 minutes, ammonium persulfate (APS) with a concentration of 10 mM and a volume ratio of 10 was quickly added. The volume ratio here is obtained by comparing the volumes of each solution in step 1.

[0038] (3) After heating for 4 hours under ultrasonic atmosphere, centrifugation and purification are performed to obtain MNP@PNIPAMAA solution; the frequency of the ultrasonic wave can be, for example, 80 MHz, the rotation speed during centrifugation can be, for example, 14000 rpm, and the centrifugation time can be 10 minutes. During the purification process, ultrapure water can be used for purification, and purification can be performed multiple times. The purpose of purification is to remove exposed MNPs and small molecules.

[0039] (4) After the MNP@PNIPAMAA solution was activated by EDC / NHS for 12 hours, a 10 g / mL caspofungin solution was added, and the volume ratio of the MNP@PNIPAMAA solution to the caspofungin solution was 9:1. In this step, EDC / NHS activation refers to the use of two chemical reagents, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide), in the bioconjugation technology to chemically modify the target molecule for subsequent coupling reactions. This process is a common technique in biochemistry and molecular biology experiments, which is used to activate the carboxyl group (-COOH) and react with the amine group (-NH2) to form a stable amide bond (-CO-NH-).

[0040] (5) After stirring for 12 hours, centrifugation and purification are performed to obtain MNP@PNIPAMAA-CAS solution. The centrifugation and purification steps can be the same as those in step 3.

[0041] MNP@PNIPAMAA-CAS is mainly used as a nanoparticle trap in the present invention. The first pass through the microfluidic purification module 100 cannot completely screen out impurities in the spores. The MNP@PNIPAMAA-CAS solution will specifically bind to the fungal spores after the initial screening, causing the cell walls to carry magnetic nanoparticles. When passing through the subsequent magnetic ring 213, the fungal spores will be bound in the channel where the magnetic ring 213 is located under the magnetic force, and other impurities will flow out along the subsequent channels with the solution.

[0042] In a specific embodiment of the present invention, the mixing unit 220 includes a mixing channel 221 and a second injector 222. The second injector 222 is connected to the end of the purification channel 211 away from the magnetic ring 213. One end of the mixing channel 221 is connected to the end of the purification channel 211 where the magnetic ring 213 is set. The second injector 222 is used to inject the silver nano-enhanced particle solution. The mixing channel 221 has several turns along its length to achieve mixing of the silver nano-enhanced particle solution and the spores after secondary purification. In this embodiment, silver nanoparticles are used as enhancement particles. The enhancement particles can amplify the Raman signal, increase the signal intensity, and further improve the signal quality. The mixing efficiency is also increased by setting several turns in the mixing channel 221 to ensure that the surfaces of the spores and the silver nanoparticles are fully in contact.

[0043] In a specific embodiment of the present invention, the mixing channel 221 and the detection channel 250 are connected through the buffer channel 240, and the mixing channel 221 and the detection channel 250 are vertically connected to the buffer channel 240; the pressurizing unit 230 is connected to the end of the buffer channel 240 close to the mixing channel 221, and the solution to be tested obtained by mixing in the mixing channel 221 is buffered in the buffer channel 240, and the pressurizing unit 230 introduces the buffered solution into the detection channel 250 through high-pressure nitrogen. The buffer channel 240 mainly plays the role of buffering and is conducive to the installation of the pressurizing unit 230, and the detection channel 250 is specially set for spectral detection.

[0044] In a specific embodiment of the present invention, it also includes a cleaning module 500 and a waste liquid collection module 600; the purification channel 211, the mixing channel 221, the cache channel 240 and the detection channel 250 are an integrated structure, and the end of the purification channel 211 away from the mixing channel 221 is connected to the cleaning module 500, and the cleaning module 500 is used to pump deionized water into the purification channel 211, and the deionized water flows along the channels of the integrated structure to clean the channels, and the end of the detection channel 250 away from the cache channel 240 is connected to the waste liquid collection module 600 for collecting waste liquid during the detection and cleaning process.

[0045] The cleaning module 500 includes a deionized water storage unit and a pumping unit. The control unit controls the pumping unit to work and inject deionized water into each channel of the SERS sensor module 200 to clean it. The cleaning process is fully automatic. After the cleaning module 500 is set, the residue in the channel of the SERS sensor module 200 is prevented from affecting the next measurement, further improving the accuracy of the detection.

[0046] The waste liquid collection module 600 is mainly used to collect waste liquid generated during the collection process and the cleaning process, such as waste liquid during secondary purification, waste liquid during detection, and waste liquid during cleaning. The waste liquid collection module 600 can be a waste liquid collection bottle, and the collected waste liquid can ensure its safe discharge or reprocessing.

[0047] See also Figure 2 In a specific embodiment of the present invention, the spectrum acquisition module 300 includes a laser 301 and a spectrometer 302 arranged on both sides of the detection channel 250. The laser emitted by the laser 301 reacts with the solution to be tested in the detection channel 250. The Raman signal generated by the reaction is received by the spectrometer 302 and converted into spectral data for output.

[0048] The laser 301 and the spectrometer 302 can both be selected according to the needs. In the present invention, for example, a 633nm laser 301 can be used, whose maximum output power is 80mW and the power adjustment tuning range is 1~100%, for stimulating Raman spectral response. The spectrometer 302 can be, for example, a S3000-UV-NIR model fiber spectrometer 302, which has an ultra-wide spectrum measurement range (180~1102nm), a wavelength resolution as small as 0.2nm, a signal-to-noise ratio as low as 300:1, and contains an SMA905 fiber optic interface and a USB interface for easy connection to the control unit.

[0049] The mixed liquid is transmitted to the detection channel 250, and the laser 301 emits light of a specific wavelength to excite the analyte molecules. The excited signal is transmitted to the spectrometer 302, and the spectrometer 302 converts the Raman signal into a spectrum, through which the chemical composition or concentration of the target substance can be analyzed. These spectral data can be transmitted wirelessly, or directly analyzed and processed by the control unit.

[0050] The control unit controls the start and stop of each pump through digital output, ensuring the automation and continuous operation of the entire collection process and reducing manual intervention.

[0051] In a specific embodiment of the present invention, a microfluidic SERS collection method for airborne fungal disease spores is also provided, comprising the following steps.

[0052] S1. Turn on the micro air pump 106 in the microfluidic purification module 100 so that the spores are enriched in the enrichment area 104 and the impurities flow out through the outlet of the impurity area 105 .

[0053] S2, open the first injector 212, inject the MNP@PNIPAMAA-CAS solution into the purification channel 211, and close the first injector 212 after a preset first time. When the spores are mixed with the MNP@PNIPAMAA-CAS solution and pass through the magnetic ring 213 with strong magnetic force, the fungal spores adhere to the inner side of the channel, and the impurity solution is discharged through the waste liquid outlet.

[0054] S3, open the second injector 222, inject the enhanced particle solution into the purification channel 211, and close the second injector 222 after a preset second time; through this step, the enhanced particles can enter the mixing area through the injection port and be fully mixed in the microfluidic mixing area for 1 to 2 seconds to promote their combination with fungal spores.

[0055] S4, opening the pressurizing unit 230, and closing it after a preset third time. Through this step, the solution to be tested can be pumped into the detection channel 250.

[0056] S5, turning on the laser 301 and the spectrometer 302 to collect spectrum data, and then turning them off after a preset fourth time to ensure that complete spectrum data is received.

[0057] S6. Turn on the micro water pump in the cleaning module 500, and turn it off after a preset fifth time. This step is mainly to clean the entire acquisition system to avoid being affected when collecting data next time.

[0058] S7, opening the first injector 212 again to perform the next detection, and after the detection is completed, closing the micro air pump 106 in the microfluidic purification module 100.

[0059] In general, the present invention combines microfluidics with SERS technology to improve the detection sensitivity and speed. The microfluidic purification module 100 used can separate and enrich fungal spores in the inhaled air to improve the detection accuracy. The nano-capturing device MNP@PNIPAMAA-CAS is also formed by magnetic nanoparticles (MNP) and polymer nanoshells, and a new water-soluble chemical reagent caspofungin (CAS) is used, which is attached to the cell wall of specific fungi. Acrylic acid (AA) and N-isopropylacrylamide (NIPAM) are integrated into the compact shell layer by ultrasonic induced polymerization. NIPAM and AA are strongly cross-linked with magnetic nanoparticles during the polymerization process, rather than being adsorbed on the surface by electrostatic action, which improves the structural stability of MNP@PNIPAMAA. Due to the abundant COOH on the surface of AA, MNP@PNIPAMAA can easily combine with CAS through an amidation reaction to form an ultrafast nano-capturing agent MNP@PNIPAMAA-CAS, which can attach fungal spores near the magnetic ring 213. Because the thick wall structure of spores and the extracellular matrix together form a protective barrier, the spores are protected from external environmental stresses such as dryness, extreme temperatures and harmful chemicals. The new water-soluble chemical reagent caspofungin (CAS) can be adsorbed on the cell wall to capture fungal spores. At the same time, silver nano-enhanced particles can enhance the signal strength and further improve the signal quality.

[0060] At the same time, the microfluidic purification module 100, the SERS sensor module 200 and the portable Raman spectrometer 302 are small in size and can be carried on a drone. The automatic detection and analysis methods can reduce the number of workers required for detection and lower the professional technical operation requirements of the detection personnel. The detection range is wider and the detection efficiency can be greatly improved.

[0061] It should be noted that the main protection in the present invention is the collection structure. As for how to calculate the classification, identification and early warning of fungal pathogenic spores in the air based on the Raman spectrum curve, it is not the focus of this case. We can calculate according to the common scheme in the prior art: first, the collected SERS signal is processed and data analyzed, and the signal is analyzed using a few-sample algorithm and an adaptive peak detection algorithm to mine hidden features and filter key information. At the same time, the spores are quantitatively analyzed through a residual network with an embedded attention mechanism, and a hybrid model for long-term and short-term time series prediction is adopted. Finally, a large number of network model parameters and correlation parameters of time series results are trained and adjusted to obtain the optimal early disease prediction and epidemic dynamic prediction model with stable time series, so as to predict the possibility and time range of disease occurrence and provide timely early warning.

[0062] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A microfluidic SERS acquisition system for airborne fungal disease spores, characterized in that: include: A microfluidic purification module is used to separate and enrich the extracted gas to obtain enriched spores; A SERS sensing module is used to mix the enriched spores and the enhanced particles to obtain a test solution; A spectrum acquisition module, used for acquiring the spectrum of the solution to be tested and then outputting it; as well as A control module is electrically connected to the microfluidic purification module, the SERS sensing module and the spectrum acquisition module, and is used to control the start and stop timing of these modules.

2. The microfluidic SERS acquisition system for airborne fungal disease spores according to claim 1, characterized in that: The microfluidic purification module comprises a first channel, a second channel, a third channel, an enrichment area, an impurity area and a micro air pump; one end of the first channel is connected to the air, and the other end is separated into two second channels; the other end of the second channel is separated into two third channels; The other ends of the four third channels are respectively connected to the three enrichment areas and one impurity area; the enrichment area is used to collect spores in the air, and the impurity area is used to collect and discharge the separated impurities; The micro air pump is connected to the first channel and is used to generate negative pressure in the microfluidic purification module.

3. The microfluidic SERS acquisition system for airborne fungal disease spores according to claim 1, characterized in that: The SERS sensing module includes a secondary purification unit, a mixing unit, a pressurizing unit and a detection channel. The secondary purification unit is used for secondary purification of spores collected by the microfluidic purification module. The mixing unit is used for mixing the secondary purified spores and enhanced particles to obtain the solution to be tested. The pressurizing unit is used for pumping the solution to be tested into the detection channel.

4. The microfluidic SERS acquisition system for airborne fungal disease spores according to claim 3, characterized in that: The secondary purification unit includes a purification channel, a first injector and a magnetic ring. The enrichment area in the microfluidic purification module and the first injector are connected to one end of the purification channel, and the magnetic ring is sleeved on the other end of the purification channel. The first injector is used to inject MNP@PNIPAMAA-CAS solution into the purification channel. The MNP@PNIPAMAA-CAS solution combines with spores and is captured when passing through the magnetic ring to achieve secondary purification of spores.

5. The microfluidic SERS acquisition system for airborne fungal disease spores according to claim 4, characterized in that: The mixing unit includes a mixing channel and a second injector, wherein the second injector is connected to an end of the purification channel away from the magnetic ring, and one end of the mixing channel is connected to an end of the purification channel where the magnetic ring is set. The second injector is used to inject the silver nano-enhanced particle solution. The mixing channel has several turns along its length to achieve mixing of the silver nano-enhanced particle solution and the spores after secondary purification.

6. The microfluidic SERS acquisition system for airborne fungal disease spores according to claim 5, characterized in that: The mixing channel and the detection channel are connected through a cache channel, and the mixing channel and the detection channel are both vertically connected to the cache channel; the pressurizing unit is connected to one end of the cache channel close to the mixing channel, and the tested solution obtained by mixing in the mixing channel is cached in the cache channel, and the pressurizing unit introduces the cached solution into the detection channel through high-pressure nitrogen.

7. The microfluidic SERS acquisition system for airborne fungal disease spores according to claim 6, characterized in that: It also includes a cleaning module and a waste liquid collection module; the purification channel, the mixing channel, the cache channel and the detection channel are an integrated structure, the purification channel is connected to the cleaning module at one end away from the mixing channel, the cleaning module is used to pump deionized water into the purification channel, the deionized water flows along each channel of the integrated structure to clean each channel, and the detection channel is connected to the waste liquid collection module at one end away from the cache channel for collecting waste liquid during the detection and cleaning process.

8. The microfluidic SERS acquisition system for airborne fungal disease spores according to claim 3, characterized in that: The spectrum acquisition module includes a laser and a spectrometer. The laser emitted by the laser reacts with the solution to be tested in the detection channel. The Raman signal generated by the reaction is received by the spectrometer and converted into spectrum data for output.

9. The microfluidic SERS acquisition system for airborne fungal disease spores according to claim 4, characterized in that: The MNP@PNIPAMAA-CAS solution is prepared according to the following steps: Acrylic acid with a concentration of 100 mM and magnetic nanoparticles were reacted at a volume ratio of 5:1, and after activation with EDC / NHS for 12 hours, an MNP-AA solution was obtained; Mixing 100 mM N-isopropylacrylamide, 100 mM acrylic acid, 50 mM bis(2-methylpropylene)ethoxydisulfide, 100 mM sodium dodecyl sulfate and the MNP-AA solution in a container at a volume ratio of 60:3.4:12:1:20; After nitrogen was blown into the mixed solution for 20 minutes, ammonium persulfate with a concentration of 10 mM and a volume ratio of 10 was quickly added; After heating for 4 hours under ultrasonic atmosphere, the solution was centrifuged and purified to obtain MNP@PNIPAMAA solution. After the MNP@PNIPAMAA solution was activated by EDC / NHS for 12 hours, a 10 g / mL caspofungin solution was added, and the volume ratio of the MNP@PNIPAMAA solution to the caspofungin solution was 9:1; After stirring for 12 hours, centrifugation and purification were performed to obtain the MNP@PNIPAMAA-CAS solution.

10. A microfluidic SERS method for collecting airborne fungal disease spores, characterized in that: include: Turn on the micro air pump in the microfluidic purification module to allow the spores to be enriched in the enrichment area; Open the first injector, inject the MNP@PNIPAMAA-CAS solution into the purification channel, and close the first injector after a preset first time; Opening the second injector to inject the enhanced particle solution into the purification channel, and closing the second injector after a preset second time has passed; The pressurizing unit is turned on and then turned off after a preset third time; Turn on the laser and the spectrometer to collect spectral data, and turn off after a preset fourth time; Turn on the micro water pump in the cleaning module and turn it off after a preset fifth time; The first injector is opened again for the next detection, and after the detection is completed, the micro air pump in the microfluidic purification module is closed.

Images