A microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy and its application

Abstract

The present invention provides a microfluidic drug-sensitive plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy and its application, wherein the microfluidic drug-sensitive plate is provided with a bottom layer, a middle layer and a top layer from bottom to top; the middle layer is a cylindrical structure that runs through from top to bottom, and the lower bottom of the cylindrical structure is fixed to the bottom layer; the middle layer is provided with at least one detection unit; the detection unit is connected to a waste liquid pool, a sample addition hole, an antimicrobial drug treatment hole, a cleaning liquid hole and a SERS active substrate hole through a microfluidic pipeline; the top layer covers the upper bottom of the cylindrical structure of the middle layer. The microfluidic drug-sensitive plate of the present invention has low detection cost, high detection efficiency, accurate results, high sensitivity, easy operation, and can realize high-throughput detection of samples, and has important application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial drug sensitivity detection, and in particular relates to a microfluidic drug sensitivity plate for rapidly detecting microbial drug sensitivity based on Raman spectroscopy and an application thereof. Background Art

[0002] Since the discovery of penicillin by British scientist Alexander Fleming in 1928, antibiotics have become the first-line treatment for infectious diseases in clinical practice. However, with the widespread use of broad-spectrum antibiotics, the problem of antibiotic resistance has become increasingly serious in clinical practice. In 2019, the detection rates of carbapenem-resistant Acinetobacter baumannii, third-generation cephalosporin-resistant Escherichia coli, and quinolone-resistant Escherichia coli all exceeded 50%, which shows the severity of the problem of bacterial resistance. The problem of invasive fungal resistance should not be ignored either. With the continuous increase in antibiotic resistance, some "super bacteria" and "super fungi" with strong toxicity and resistance to most highly effective antibiotics have been continuously exposed in clinical practice. In 2019, the "super fungus" Candida auris broke out. Once infected with this fungus, the mortality rate exceeded 60%, and nearly 50% of the infected people died within 90 days. The continuous spread of drug-resistant strains has brought huge challenges to human health.

[0003] One of the important causes of antimicrobial resistance is the irrational use of antimicrobial drugs. Clinically, it mainly manifests itself in the selection of antimicrobial drugs based solely on habit and clinical experience, the use of broad-spectrum antimicrobial drugs in excessive doses and beyond the scope, and the incorrect use of ineffective or poorly effective antimicrobial drugs. Therefore, in order to effectively curb the spread of antimicrobial resistance, the most important measure is to achieve early and accurate diagnosis and treatment of infectious diseases, that is, on the basis of identification of pathogenic microorganisms, select antimicrobial drugs according to the results of the antibiotic susceptibility test (AST) to achieve the rational use of antimicrobial drugs.

[0004] At present, the widely recognized standardized methods for fungal drug sensitivity tests at home and abroad are mainly the disc diffusion method and dilution method (agar or broth dilution method) recommended by the Clinical and Laboratory Standards Institute (CLSI) of the United States. The disc diffusion method is to stick a disc containing a quantitative antimicrobial drug on an agar plate inoculated with the bacteria to be tested. The drug contained in the disc absorbs the water in the agar and dissolves, and then continuously diffuses to the area around the disc, forming a decreasing gradient concentration. The growth of the bacteria to be tested is inhibited within the antibacterial concentration range around the disc, thereby producing a transparent inhibition zone. The size of the inhibition zone reflects the sensitivity of the bacteria to be tested to the measured drug, and is negatively correlated with the minimum inhibitory concentration (MIC) of the drug to the bacteria to be tested, that is, the larger the inhibition zone, the smaller the MIC. The disc diffusion method is a semi-quantitative drug sensitivity test method, which is simple and easy to use, but cannot give an intuitive MIC value, and the test results are affected by many factors. The dilution method drug sensitivity test can be used to quantitatively test the in vitro activity of antimicrobial drugs against a certain bacterium, which is divided into agar dilution method and broth dilution method. During the experiment, the concentration of antimicrobial drugs is usually diluted by multiples (lg2). The lowest drug concentration that can inhibit the visible growth of the test bacteria is called the minimum inhibitory concentration (MIC). The test concentration range of a specific antimicrobial drug should include the concentration that can detect the interpretive breakpoints of bacteria (sensitive, intermediate and resistant), and should also include the MIC of the quality control reference strain. The dilution method is accurate and has a wide range of applications, but it is cumbersome, time-consuming and labor-intensive, and is not conducive to clinical use.

[0005] At present, commercial automated systems such as VITEK 2, Microscan Panel, BD Phoenix and Sensititre are widely used in routine clinical testing, shortening the AST time from several days to about 6 to 16 hours. However, the above conventional methods all require 24 to 48 hours to isolate pathogenic microorganisms from patient samples through pure culture, and 6 to 72 hours to complete drug sensitivity testing, with a total time of 30 hours to 7 days, and it is even more difficult to detect pathogenic microorganisms that are difficult to culture or grow slowly. In clinical practice, the earlier the targeted treatment of infectious diseases, the better the effect, and the golden window period for rescue of some acute infections is only 12 hours. Therefore, the development of rapid antibiotic susceptibility test (RAST) technology is of great significance to clinical medicine.

[0006] Invention patent CN110537087A discloses a drug sensitivity test kit. Although it can produce an antibiotic gradient, it relies on the resazurin colorimetric method, which cannot complete drug sensitivity testing in a short time, and resazurin is not metabolized by non-fermenting Gram-negative bacteria. Therefore, this method cannot be used for drug sensitivity testing of all pathogenic microorganisms. Invention patent CN110542757A discloses a method for quantitative detection of bacteria and drug sensitivity testing using a microfluidic chip. Antigen-antibody technology is used to quantitatively detect bacteria, and drug sensitivity testing is performed simultaneously. However, the drug sensitivity test results are based on color development reactions and are difficult to complete in a short time. Invention patent CN106238112A discloses a microfluidic chip and its application in pathogen identification and drug sensitivity testing, but the results are also judged based on color development reactions, which takes a long time.

[0007] Therefore, how to provide a product and method that can quickly detect the sensitivity of microbial drugs has become an urgent problem to be solved. Summary of the invention

[0008] In view of the deficiencies in the prior art and actual needs, the present invention provides a microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy and its application, which can realize the detection of a series of drug concentrations on a drug sensitivity plate, and is used in conjunction with a fully automatic Raman spectroscopy drug sensitivity detection system to realize the automation of drug sensitivity detection, greatly reduce the workload, and shorten the detection time.

[0009] To achieve this object, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy, wherein the microfluidic drug sensitivity plate is provided with a bottom layer, a middle layer and a top layer from bottom to top;

[0011] The middle layer is a cylindrical structure that penetrates from top to bottom, and the bottom of the cylindrical structure is fixed to the bottom layer;

[0012] The middle layer is provided with at least one detection unit;

[0013] The detection unit is connected to the waste liquid pool, the sample addition hole, the antimicrobial drug treatment hole, the cleaning liquid hole and the SERS active substrate hole through a microfluidic pipeline;

[0014] The top layer covers the upper bottom of the tubular structure of the middle layer.

[0015] In the present invention, Raman spectroscopy is an analytical method based on the Raman scattering effect, which analyzes the scattered spectrum with different frequencies from the incident light to obtain information on molecular vibration and rotation, and is applied to molecular structure research. After the action of antibiotics, microorganisms will produce certain biochemical reactions, such as changes in their own composition, metabolites, assimilation rate, etc. These changes are detected by Raman spectroscopy to determine whether the antibiotics are effective against microorganisms. The outstanding advantage of this method is that it can achieve real-time detection.

[0016] In the present invention, the microfluidic drug sensitivity plate is used to detect the drug sensitivity of microorganisms, and only a very small amount of sample is needed to simultaneously detect multiple gradient concentration drugs, thereby reducing the screening cost, improving the detection efficiency, and realizing automated detection. The detection process is easy to operate, the steps are simple, the time is short, and quantitative detection can be realized, which has practical application value.

[0017] In the present invention, the bottom layer provides a substrate with low background interference for Raman spectrum detection.

[0018] Preferably, the number of the waste liquid pool, the sample addition hole, the cleaning liquid hole and the SERS active substrate hole is one.

[0019] Preferably, the number of the antimicrobial drug treatment holes is at least 2, for example, it can be 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0020] Preferably, exhaust holes are arranged above the waste liquid pool, the cleaning liquid hole and the SERS active substrate hole.

[0021] Preferably, a valve is provided between the waste liquid pool and the sample addition hole.

[0022] Preferably, the valve comprises a mechanical valve or a pneumatic valve.

[0023] In the present invention, the valve can open or close the channel according to demand.

[0024] Preferably, one-way valves are arranged behind the antimicrobial drug treatment hole, at the outlet of the cleaning liquid hole and at the outlet of the SERS active substrate hole.

[0025] In the present invention, a one-way valve is arranged behind the antimicrobial drug treatment hole. When bacterial solution is added, the bacterial solution fills the antimicrobial drug treatment hole without overflowing, thereby preventing the antimicrobial drug from dissolving in the bacterial solution and overflowing to affect the drug concentration; a one-way valve is arranged at the outlet of the cleaning liquid hole and the outlet of the SERS active substrate hole to ensure the one-way flow of the liquid and prevent the contamination that may be caused by the backflow of the liquid.

[0026] Preferably, a bubble cap with deformation ability is arranged at the bottom of the cleaning liquid hole and the SERS active substrate hole to squeeze the liquid into the channel.

[0027] Preferably, the bottom layer corresponding to the antibiotic treatment hole is coated with 4-MPBA and antibiotics.

[0028] In the present invention, the boronic acid group contained in the 4-MPBA molecule can reversibly bind to the peptidoglycan of the bacterial / fungal cell wall, thereby specifically capturing various bacteria / fungi.

[0029] Preferably, the antibiotics coated on the bottom layer corresponding to the different antibiotic treatment holes have gradient concentrations.

[0030] Preferably, the bubble cap corresponding to the cleaning liquid hole is filled with cleaning liquid.

[0031] Preferably, the bubble corresponding to the SERS active substrate hole contains a SERS active substrate.

[0032] Preferably, the SERS active substrate comprises any one of gold colloid, silver colloid or gold-silver composite nanoparticles, or a combination of at least two thereof.

[0033] Preferably, the material of the bottom layer includes quartz glass.

[0034] In the present invention, the bottom layer is made of quartz glass as a preparation material, which can provide a substrate with low background interference for Raman spectrum detection.

[0035] Preferably, the material of the top layer comprises transparent plastic film polypropylene.

[0036] In a second aspect, the present invention provides a method for using the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to the first aspect, the method comprising:

[0037] Add bacterial solution to the sample well, enter the antimicrobial drug treatment well, and culture;

[0038] Use cleaning solution to clean, and then add SERS active substrate;

[0039] Detect and determine the drug sensitivity of microorganisms.

[0040] Preferably, the bacterial solution contains heavy water with a mass fraction of 30% to 100%. The mass fraction can be, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, and other specific point values ​​within the numerical range can be selected, which will not be described one by one here.

[0041] Preferably, the culture time is 0.5 to 4 h, for example, it can be 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h or 4 h, and other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0042] Preferably, the cleaning further includes a step of opening a valve.

[0043] Preferably, the cleaning further includes a step of discharging waste liquid.

[0044] Preferably, the step of closing the valve is further included before adding the SERS active substrate.

[0045] Preferably, the detecting comprises detecting using a micro-Raman spectrometer.

[0046] Preferably, the judgment standard is the presence and size of the deuterium peak.

[0047] As a preferred technical solution, the method for using the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy of the present invention comprises the following steps:

[0048] (1) Using a sampling needle, a bacterial solution containing a culture medium prepared with 30% to 100% heavy water is added to the sample well, and then enters the well containing the gradient concentration of antibiotics through a microfluidic channel for culturing for 0.5 to 4 hours;

[0049] (2) opening the valve, squeezing out the cleaning liquid pre-sealed in the cleaning liquid hole, cleaning the antimicrobial drug treatment hole, and discharging the waste liquid into the waste liquid pool; closing the valve, squeezing the SERS active substrate in the SERS active substrate hole into the antimicrobial drug treatment hole;

[0050] (3) Use a micro-Raman spectrometer to detect and determine the drug sensitivity of the microorganism based on the presence and size of the deuterium peak.

[0051] In the third aspect, the present invention provides the application of the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy as described in the first aspect and / or the method of using the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy as described in the second aspect in microbial drug sensitivity detection.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The microfluidic drug sensitivity plate of the present invention can perform drug sensitivity determination using a microliter volume, which greatly reduces the reagent consumption and reduces the cost of drug screening; the required amount of bacteria is small, and it can be directly used for the enrichment detection of pathogenic microorganisms in clinical samples; multiple steps such as gradient drug preparation, bacterial liquid distribution, cultivation, and drug effect detection are integrated into a drug sensitivity plate, realizing the automation of drug sensitivity activity determination; the microfluidic drug sensitivity plate can integrate multiple microarrays to realize the determination of multiple pathogenic microorganisms and multiple antibacterial drugs, realize high-throughput drug screening, and can perform quantitative detection, with accurate results and sensitive reactions, and has outstanding advantages such as direct application to clinical sample detection, simple operation, and short time consumption;

[0054] (2) The microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy of the present invention adopts a closed microfluidic design, and the sample containing the bacterial or fungal suspension is loaded into the microfluidic chip. All further sample processing is performed in an automatic manner without any personal contact with the sample, thereby saving hands-on time and avoiding further contact between the operator and the infected sample, making the detection process safer;

[0055] (3) The present invention can be used not only for drug sensitivity testing of pure cultured bacteria or fungi, but also for drug sensitivity testing of bacteria or fungi enriched in clinical samples, including but not limited to blood, urine, bronchoalveolar lavage fluid, cerebrospinal fluid, joint effusion, etc. Clinical samples are selectively lysed before filtration, and the pathogenic microorganisms retained on the filter membrane are enriched by reverse elution and can be used for subsequent drug sensitivity tests. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is a schematic diagram of the structure of a microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy in Example 1 of the present invention;

[0057] In the figure, 1-waste liquid pool, 2-sample loading hole, 3-antimicrobial drug treatment hole, 4-cleaning liquid hole, 5-SERS active substrate hole, 6-valve, 7-check valve. DETAILED DESCRIPTION

[0058] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0059] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0060] Example 1

[0061] This embodiment provides a series of microfluidic drug sensitivity plates based on Raman spectroscopy for rapid detection of microbial drug sensitivity, and its structural schematic diagram is shown in FIG. Figure 1 shown.

[0062] The microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy is provided with a bottom layer, a middle layer and a top layer from bottom to top;

[0063] The middle layer is a cylindrical structure that penetrates from top to bottom, and the bottom of the cylindrical structure is fixed to the bottom layer;

[0064] The middle layer is provided with a detection unit;

[0065] The detection unit is connected to the waste liquid pool 1, the sample addition hole 2, the antimicrobial drug treatment hole 3, the cleaning liquid hole 4 and the SERS active substrate hole 5 through a microfluidic pipeline;

[0066] The top layer covers the upper bottom of the cylindrical structure of the middle layer;

[0067] The number of the waste liquid pool 1, the sample addition hole 2, the cleaning liquid hole 4 and the SERS active substrate hole 5 is 1, and the number of the antimicrobial drug treatment holes 3 is 8;

[0068] An exhaust hole is arranged above the waste liquid pool 1, the cleaning liquid hole 4 and the SERS active substrate hole 5;

[0069] A valve 6 is arranged between the waste liquid pool 1 and the sample adding hole 2, and the valve 6 is a mechanical valve;

[0070] One-way valves 7 are arranged behind the antimicrobial drug treatment hole 3, at the outlet of the cleaning liquid hole 4 and at the outlet of the SERS active substrate hole 5;

[0071] A bubble cap with deformation capability is arranged at the bottom of the cleaning liquid hole 4 and the SERS active substrate hole 5;

[0072] The bottom layer corresponding to the antimicrobial drug treatment hole 3 is coated with 4-MPBA (Sigma) and any one of the antimicrobial drugs fluconazole, flucytosine, amphotericin B, itraconazole, ketoconazole, isavuconazole, posaconazole, ravuconazole, voriconazole, anidulafungin, caspofungin, micafungin, penicillin, ampicillin, cefotaxime, ceftriaxone, cefepime, imipenem, meropenem, erythromycin, clindamycin, ciprofloxacin, levofloxacin, chloramphenicol or vancomycin (all of the above drugs are purchased from Sigma);

[0073] The concentrations of the antimicrobial drugs coated on the bottom layer corresponding to the different antimicrobial drug treatment holes 3 are shown in Table 1 and Table 2, respectively.

[0074] Table 1

[0075]

[0076]

[0077] Table 2

[0078] Antibacterial drugs Detection range (μg / mL) penicillin 0.06，0.125，0.25，0.5，1，2，4，8 Ampicillin 0.125，0.25，0.5，1，2，4，8，16 Ceftriaxone 0.125，0.25，0.5，1，2，4，8，16 Ceftriaxone 0.125，0.25，0.5，1，2，4，8，16 Cefepime 0.125，0.25，0.5，1，2，4，8，16 Imipenem 0.125，0.25，0.5，1，2，4，8，16 Meropenem 0.125，0.25，0.5，1，2，4，8，16 Erythromycin 0.125，0.25，0.5，1，2，4，8，16 Clindamycin 0.125，0.25，0.5，1，2，4，8，16 Ciprofloxacin 0.125，0.25，0.5，1，2，4，8，16 Levofloxacin 0.125，0.25，0.5，1，2，4，8，16 Chloramphenicol 0.125，0.25，0.5，1，2，4，8，16 Vancomycin 0.125，0.25，0.5，1，2，4，8，16

[0079] The bubble cap corresponding to the cleaning liquid hole 4 is filled with cleaning liquid, and the cleaning liquid is PBS buffer and Tris buffer (purchased from HyClone);

[0080] The bubble corresponding to the SERS active substrate hole 5 contains a SERS active substrate;

[0081] The SERS active substrate is gold colloid, silver colloid or gold-silver composite nanoparticles, and the preparation method is as follows:

[0082] Gold colloid: Dissolve 50 mg of HAuCl4 in 500 mL of deionized water and heat to boiling. Add 6 mL of 1% (W / W) trisodium citrate aqueous solution dropwise and stir vigorously. Finally, a wine-red colloid is formed.

[0083] Silver colloid: Take 500mL 1.0×10 -3 mol / L AgNO3 aqueous solution was heated to boiling, 12 mL of 1% (W / W) trisodium citrate solution was added dropwise to the above solution, and the solution was kept boiling while being vigorously stirred for 60 min, and the stirring was continued until the solution cooled.

[0084] Gold-silver composite nanoparticles: Dilute 50 mL of the prepared gold colloid with sodium citrate to 200 mL and heat to boiling. -3 mol / L AgNO3 aqueous solution is added dropwise, and stirring is required during the process. Then, a sodium citrate solution corresponding to the amount of AgNO3 added is added to reduce the silver and deposit it on the gold particles. The mixed solution is kept boiling for 0.5h and stirred and cooled at room temperature.

[0085] Example 2

[0086] This example uses the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy prepared in Example 1 to detect the quality control bacteria Candida parapsilosis ATCC22019, and the steps are as follows:

[0087] (1) Resuspend the bacteria in RPMI1640 medium prepared with 100% heavy water, add the bacterial solution into the sample well 2 using a sampling needle, and then enter the well 3 containing gradient concentrations of antibiotics through the microfluidic channel for culturing for 2 h;

[0088] (2) opening valve 6, squeezing out the cleaning liquid pre-sealed in the cleaning liquid hole 4, cleaning the antimicrobial drug treatment hole 3, and discharging the waste liquid into the waste liquid pool 1; closing valve 6, squeezing the SERS active substrate in the SERS active substrate hole 5 into the antimicrobial drug treatment hole 3;

[0089] (3) Use a micro-Raman spectrometer to detect and determine the drug sensitivity of the microorganism based on the presence and size of the deuterium peak.

[0090] The test results were compared with the quality control range of the MIC value, and the results are shown in Table 3. It can be seen that the MIC values ​​are within the quality control range when the microfluidic drug sensitivity plate in the present application is used for detection, indicating that the drug sensitivity test results in the present application meet the requirements of drug sensitivity test quality control. In addition, the MIC value of the present invention is low within the quality control range, indicating that compared with conventional detection methods, the present application has higher sensitivity, and the applicable scope and the sample types detected are richer.

[0091] Table 3

[0092] MIC value quality control range (μg / mL) MIC of the present invention (μg / mL) Amphotericin B 0.12～1.0 0.5 Flucytosine 0.12～0.5 0.25 Fluconazole 0.5～2.0 1.0 Itraconazole 0.03～0.12 0.06 Voriconazole 0.015～0.06 0.0313 Posaconazole 0.015～0.06 0.0313

[0093] Example 3

[0094] In this example, the accuracy of the detection results of the microfluidic drug sensitivity plate prepared in Example 1 for rapid detection of microbial drug sensitivity based on Raman spectroscopy was tested.

[0095] (1) Resuscitation of bacterial strains: Collect sensitive and resistant Candida albicans isolated from clinical sites. Before use, take 10 μL of bacterial solution and streak it on Sabouraud solid agar (SDA) and culture it in a 35°C incubator for 24 h. Pick a single clone and streak it again;

[0096] (2) The MIC value of each strain to fluconazole was determined according to the microbroth dilution method recommended by CLSI, and the MIC value test was performed using the drug sensitivity card of the present invention. According to the sensitivity breakpoints specified by CLSI, when the MIC of Candida albicans to fluconazole is ≤2 μg / mL, it is judged as sensitive bacteria (S); when the MIC of Candida albicans to fluconazole is ≥8 μg / mL, it is judged as resistant bacteria (R).

[0097] The results are shown in Table 4. It can be seen that the MIC values ​​of fluconazole for each Candida albicans strain detected by the microfluidic drug sensitivity plate of the present invention are consistent with the detection results of the standard microbroth dilution method, indicating that the microfluidic drug sensitivity plate of the present invention has a very high accuracy. In addition, the microfluidic drug sensitivity plate in the present application also has the advantages of simple operation and short detection time, which realizes rapid and accurate detection of samples.

[0098] Table 4

[0099]

[0100] In Table 4, S represents sensitive and R represents resistant.

[0101] Example 4

[0102] This example uses the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy prepared in Example 1 to detect urine samples infected with Candida albicans, and the steps are as follows:

[0103] (1) Filter urine through a 0.22 μm filter membrane, reversely elute the pathogenic microorganisms retained on the filter membrane with PBS buffer, and centrifuge at 3000 g for 5 min to obtain bacterial precipitates;

[0104] (2) Resuspend the bacterial pellet in RPMI1640 medium prepared with 100% heavy water, add the bacterial solution into the sample well 2 using a sampling needle, and then enter the well 3 containing gradient concentrations of antibiotics through the microfluidic channel for culturing for 2 h;

[0105] (3) Open valve 6, squeeze out the cleaning liquid pre-sealed in the cleaning liquid hole 4, clean the antimicrobial drug treatment hole 3, and discharge the waste liquid into the waste liquid pool 1; close valve 6, squeeze the SERS active substrate in the SERS active substrate hole 5 into the antimicrobial drug treatment hole 3;

[0106] (4) Micro-Raman spectrometer was used to detect the drug sensitivity of the microorganism based on the presence and size of the deuterium peak, and the results were compared with the MIC value determined by the microbroth dilution method. The results are shown in Table 5.

[0107] The results showed that the samples were tested using the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy provided by the present invention, and the test results were consistent with the test results of the microbroth dilution method, indicating that the results of testing using the microfluidic drug sensitivity plate in the present application have similar detection precision and accuracy as the traditional microbroth dilution method, but are simpler to operate and take less time, which promotes the promotion and use of related products.

[0108] Table 5

[0109] Broth microdilution method (μg / mL) Raman MIC (μg / mL) Fluconazole 1.0 1.0 Voriconazole 0.06 0.06 Caspofungin 0.25 0.25 Micafungin 0.125 0.125

[0110] In summary, the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy described in the present invention only requires a very small amount of sample to perform drug sensitivity detection, reducing the actual dosage and reducing the cost of drug screening; the test results are accurate, parallelism is good, quantitative detection can be performed, and it is easy to operate and has high detection efficiency. It can be applied to high-throughput detection of clinical samples and has important application value.

[0111] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy, characterized in that: The microfluidic drug-sensitive plate is provided with a bottom layer, a middle layer and a top layer from bottom to top; The middle layer is a cylindrical structure that penetrates from top to bottom, and the bottom of the cylindrical structure is fixed to the bottom layer; The middle layer is provided with at least one detection unit; The detection unit is connected to the waste liquid pool, the sample addition hole and the antibiotic treatment hole in sequence through a microfluidic pipeline, and the cleaning liquid hole and the SERS active substrate hole are respectively connected to the antibiotic treatment hole through a microfluidic pipeline. An exhaust hole is arranged above the waste liquid pool, the cleaning liquid hole and the SERS active substrate hole; One-way valves are arranged behind the antimicrobial drug treatment hole, at the outlet of the cleaning liquid hole and at the outlet of the SERS active substrate hole; The bottom layer corresponding to the antimicrobial drug treatment hole is coated with 4-MPBA and antimicrobial drugs; the number of the antimicrobial drug treatment holes is at least 2; The antimicrobial drugs coated on the bottom layer corresponding to the different antimicrobial drug treatment holes are in gradient concentrations; The top layer covers the upper bottom of the tubular structure of the middle layer.

2. The microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 1, characterized in that: The number of the waste liquid pool, the sample addition hole, the cleaning liquid hole and the SERS active substrate hole is one.

3. The microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 1, characterized in that: A valve is arranged between the waste liquid pool and the sample adding hole.

4. The microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 3, characterized in that: The valve comprises a mechanical valve or a pneumatic valve.

5. The microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 1, characterized in that: A bubble cap with deformation capability is arranged at the bottom of the cleaning liquid hole and the SERS active substrate hole.

6. The microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 5, characterized in that: The bubble cap corresponding to the cleaning liquid hole is filled with cleaning liquid.

7. The microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 5, characterized in that: The bubbles corresponding to the SERS active substrate holes are filled with SERS active substrates.

8. The microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 7, characterized in that: The SERS active substrate includes any one of gold colloid, silver colloid or gold-silver composite nanoparticles or a combination of at least two of them.

9. The microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 1, characterized in that: The material of the bottom layer includes quartz glass.

10. The microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 1, characterized in that: The material of the top layer comprises a transparent plastic film polypropylene.

11. A method for using the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to any one of claims 1 to 10, characterized in that: The method of use includes: Add bacterial solution to the sample well, enter the antimicrobial drug treatment well, and culture; Use cleaning solution to clean, and then add SERS active substrate; Detect and determine the drug sensitivity of microorganisms.

12. The method for using the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 11, characterized in that: The bacterial solution contains heavy water with a mass fraction of 30% to 100%.

13. The method for using the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 11, characterized in that: The culture time is 0.5 to 4 h.

14. The method for using the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 11, characterized in that: The step of opening the valve is also included before the cleaning.

15. The method for using the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 11, characterized in that: The step of removing waste liquid is also included after the cleaning.

16. The method for using the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 11, characterized in that: The method further comprises the step of closing the valve before adding the SERS active substrate.

17. The method for using the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 11, characterized in that: The detection includes detection using a micro-Raman spectrometer.

18. The method for using the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 11, characterized in that: The criteria for the judgment are the presence and size of the deuterium peak.

19. The method for using the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy according to claim 11, characterized in that: The method of use includes: (1) Use a syringe to add a bacterial solution containing a culture medium prepared with 30% to 100% heavy water into the sample well, and then pass it through the microfluidic channel into the antibiotic treatment well containing gradient concentrations of antibiotics for 0.5 to 4 hours. (2) Open the valve, squeeze out the cleaning liquid pre-sealed in the cleaning liquid hole, clean the antimicrobial drug treatment hole, and discharge the waste liquid into the waste liquid pool; close the valve, squeeze the SERS active substrate in the SERS active substrate hole into the antimicrobial drug treatment hole; (3) Use a micro-Raman spectrometer to detect and determine the drug sensitivity of the microorganism based on the presence and size of the deuterium peak.

20. Application of the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy as described in any one of claims 1 to 10 and / or the method for using the microfluidic drug sensitivity plate for rapid detection of microbial drug sensitivity based on Raman spectroscopy as described in any one of claims 11 to 19 in microbial drug sensitivity detection.

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