Microfluidic drug sensitive chip and application thereof

Abstract

The invention discloses a microfluidic drug sensitive chip and application thereof, and belongs to the technical field of microbial drug sensitive detection. The microfluidic drug sensitive chip comprises a drug preloading cover sheet layer, a bacterial culture cover sheet layer and a substrate, the drug preloading cover sheet layer and the bacterial culture cover sheet layer can be reversibly bonded with the substrate. The micro-fluidic drug sensitive chip provided by the invention can be applied to bacterial drug sensitivity detection or drug-resistant bacteria screening, can realize high-throughput and rapid bacterial drug sensitivity test or drug-resistant bacteria screening, and can provide more accurate response information of bacteria to various drugs while greatly shortening the test time.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial drug sensitivity detection, and in particular relates to a microfluidic drug sensitivity chip and applications thereof. Background Art

[0002] In vitro antibiotic susceptibility testing (AST) is a standardized testing technique for evaluating the in vitro antibacterial / bactericidal efficacy of antimicrobial agents. Its test results have important guiding value for clinical precision medication, drug resistance monitoring, and infection control. Currently, the following three AST technical systems are mainly used in the field of clinical microbiology: (1) Disk diffusion method (Kirby-Bauer method). This technique is based on the principle of concentration gradient diffusion of antimicrobial drugs in agar medium. The results are interpreted by measuring the diameter of the inhibition zone formed around the standardized drug-sensitive paper disc. In the specific implementation, the filter paper disc containing the quantitative antimicrobial agent is attached to the bacterial solution coated plate, and a characteristic inhibition zone is formed after incubation. The inhibition zone diameter-MIC value negative correlation model established according to the CLSI standard is used to achieve semi-quantitative analysis. Although this method has the technical advantages of convenient operation, low cost and visual results, its detection accuracy is easily affected by multiple factors: including but not limited to ① the degree of standardization of culture medium components; ② the control accuracy of bacterial suspension concentration; ③ the uniformity of agar layer thickness; ④ the batch-to-batch difference in drug loading on the disc. Existing studies have shown that this technical system has technical defects such as a high MIC prediction error rate (about 15%-20%), a long detection cycle (18-24 h) and false drug resistance phenotype misjudgment.

[0003] (2) Dilution method (gold standard for quantitative detection). This technology achieves accurate determination of the minimum inhibitory concentration (MIC) by constructing a series of antimicrobial drug concentration gradients (usually using the two-fold dilution method). According to the difference in carriers, it can be divided into two categories: broth microplate dilution method and agar plate dilution method. After the experimental system is inoculated with a standardized bacterial suspension and cultured at a constant temperature, the bacterial growth inhibition endpoint is judged by naked eye observation or optical instruments. Compared with the diffusion method, its MIC test results have a higher clinical compliance rate (>95%) and repeatability (CV<5%). However, due to the limitations of large reagent consumption (12-15 concentration gradients are required for a single test), cumbersome operation procedures (precise preparation of stock solution and gradient dilution is required), and long time consumption (24-48h), this technology is mainly used for scientific research verification and confirmation of special drug resistance phenotypes.

[0004] (3) E-test method (concentration gradient diffusion technology), as a composite detection system, combines the ease of operation of the diffusion method with the quantitative advantages of the dilution method. The core technology carrier is a prefabricated antibiotic concentration gradient strip (5 mm × 50 mm, covering a concentration range of 0.002-32 μg / mL), with a precisely calibrated MIC scale printed on its surface. During the test, the gradient strip is incubated with the test bacteria to form a characteristic elliptical inhibition zone, and the MIC is directly read by the corresponding concentration value at the intersection of the inhibition zone edge and the strip. Research data show that its results are consistent with the standard dilution method up to 92%-98%. However, this technology has bottlenecks in industrial application, such as the high cost of the test strip (the unit price is about 10-15 times that of conventional paper strips), limited drug susceptibility spectrum coverage (currently commercially available test strips only cover 70% of commonly used clinical antimicrobial drugs), and insufficient timeliness (requiring 16-20 hours of incubation). The above-mentioned technical systems generally have common technical difficulties in clinical applications, such as long detection cycle (>16 h), high requirements for operational standardization, and positive correlation between detection cost and accuracy, which restrict the application effectiveness of AST technology in rapid drug sensitivity testing scenarios.

[0005] Furthermore, many clinical situations often require the combined use of two or more antimicrobial agents. These include treating severe infections with unknown pathogens that are ineffective with a single antimicrobial agent, treating mixed infections caused by multiple bacteria, or treating infections caused by multidrug-resistant strains. In these situations, in vitro combined antimicrobial susceptibility testing is essential before treatment. Currently, commonly used combined antimicrobial susceptibility testing methods include the broth dilution checkerboard method, the agar dilution checkerboard method, the single-drug disc bridge method, the combined-drug disc method, and the paper strip method. While the first two methods offer relatively accurate results, they are time-consuming, material-intensive, and labor-intensive. While the paper disc and paper strip methods are simple and easy to perform, their results can sometimes be difficult to interpret, significantly limiting their practical application. Most antimicrobial susceptibility analysis methods require bacterial pre-culture, where conditions such as mixing level, pH, nutrient concentration, temperature, and space dimensions can affect bacterial metabolism and growth. Traditional in vitro culture experiments typically require 24 hours or longer to achieve a sufficiently high cell concentration. Therefore, a high-throughput, rapid microfluidic antimicrobial susceptibility chip for bacterial antimicrobial susceptibility testing is needed to address these challenges. Summary of the Invention

[0006] In response to the above-mentioned existing technologies, the present invention provides a microfluidic drug sensitivity chip and its application, which can achieve high-throughput and rapid bacterial drug sensitivity testing, greatly shorten the drug sensitivity testing time and provide more accurate drug sensitivity quantitative information.

[0007] In order to achieve the above-mentioned object, the technical solution adopted by the present invention is: a microfluidic drug sensitivity chip, comprising a drug pre-loaded cover sheet layer, a bacterial culture cover sheet layer and a substrate; the drug pre-loaded cover sheet layer comprises a drug inlet and a drug outlet connected through a drug main pipeline, the drug main pipeline is provided with a plurality of drug side pipelines connected to the drug main pipeline, the ends of all drug side pipelines are provided with micro chambers, and the diameters of the micro chambers increase or decrease successively from the drug inlet side to the drug outlet side, and exhaust holes are provided at the bottom of all micro chambers, and the exhaust holes pass through the bottom of the drug pre-loaded cover sheet layer; the bacterial culture cover sheet layer comprises a bacterial inlet and a bacterial outlet connected through the bacterial main pipeline, the bacterial main pipeline is provided with a plurality of bacterial side pipelines connected to the bacterial main pipeline, and the ends of all bacterial side pipelines are provided with culture chambers of the same size, and the culture chambers pass through the bottom of the bacterial culture cover sheet layer; both the drug pre-loaded cover sheet layer and the bacterial culture cover sheet layer can be reversibly bonded to the substrate, and the position where the exhaust holes are combined with the substrate corresponds to the position where the culture chambers are combined with the substrate.

[0008] On the basis of the above technical solution, the present invention can also be improved as follows.

[0009] Furthermore, the diameters of the drug inlet and the drug outlet are 0.8-1.2 mm, the width of the drug main pipeline is 300-400 μm, the width of the drug side pipeline is 200-300 μm, at least 8 microchambers are provided, the microchambers are cylindrical, and their diameters increase from 1 mm to 2.4 mm; the diameter of the exhaust hole is 300-400 μm.

[0010] Furthermore, the diameters of the bacterial inlet and the bacterial outlet are 0.8-1.2 mm, the width of the bacterial main channel is 300-400 μm, and the width of the bacterial side channel is 200-300 μm; the culture chamber is cylindrical and has a diameter of 500 μm.

[0011] Furthermore, the material of the drug pre-loaded cover layer and the bacterial culture cover layer is modified polydimethylsiloxane, and the raw materials for preparing the modified polydimethylsiloxane are polydimethylsiloxane, a crosslinker and a modifier, wherein the modifier is oleylamine or polyethyleneimine, and the crosslinker is sylgard 184 silicone elastomer curing agent.

[0012] Furthermore, the modifier is polyethyleneimine, and the amounts of polydimethylsiloxane, cross-linking agent, and modifier are 10 g:1 g:20-40 μL; and the material of the substrate is glass.

[0013] Furthermore, microfluidic drug sensitivity chips are used in drug sensitivity detection or drug-resistant bacteria screening.

[0014] Furthermore, drug sensitivity testing or drug-resistant bacteria screening includes the following steps: S1. Bond the substrate to the drug pre-loaded cover sheet, pass the drug solution into the microfluidic drug sensitivity chip to ensure that the microchamber is filled, then empty the channel to retain only the drug solution in the microchamber, and then freeze-dry the microfluidic drug sensitivity chip, and fix the drug powder on the substrate through the vent hole; S2. Separate the substrate from the drug pre-loaded cover sheet, then bond the substrate to the bacterial culture cover sheet, ensuring that the drug powder is fixed on the substrate in a one-to-one correspondence with the culture chamber on the substrate; introduce the test bacterial solution into the microfluidic drug sensitivity chip to ensure that the culture chamber is filled, and then empty the channel; moisturize and seal the microfluidic drug sensitivity chip; S3. The microfluidic drug sensitivity chip processed in step S2 is placed under constant temperature conditions for incubation, and the growth of the test bacteria in the chamber is observed under a phase contrast microscope to evaluate the sensitivity of the test bacteria to the tested drugs or screen out drug-resistant bacteria.

[0015] Furthermore, the drug solution and the test bacterial solution are introduced into the microfluidic drug sensitivity chip by negative pressure injection or microfluidic pump injection; The negative pressure injection steps are as follows: connecting the drug sampling port or bacterial sampling port of the microfluidic drug sensitivity chip to a syringe using a micro-channel, evacuating the air in the microfluidic drug sensitivity chip in advance with the syringe and sealing it; then dripping the drug solution or bacterial liquid into the drug sampling port or bacterial sampling port of the microfluidic drug sensitivity chip, until the drug solution or bacterial liquid fills the entire microfluidic drug sensitivity chip; The steps of microfluidic pump injection are as follows: load the drug solution or bacterial solution into the syringe, then fix the syringe on the microfluidic pump, and then connect the syringe outlet to the bacterial sampling port or drug sampling port through a pipe; set the microfluidic pump flow rate to 1-3μL / min, and wait for the bacterial solution to fill the entire microfluidic drug sensitivity chip.

[0016] Furthermore, after the pipeline is emptied in step S2, a sealing oil is introduced into the pipeline of the microfluidic drug sensitivity chip to separate the liquid in the culture chamber; the sealing oil is paraffin oil, edible oil or fluorocarbon oil.

[0017] Furthermore, in step S3, the microfluidic drug sensitivity chip is placed on the PTC constant temperature heating chip and cultured at 37° C. for 1-4 h.

[0018] The beneficial effects of the present invention are as follows: (1) The microfluidic drug sensitivity chip of the present invention realizes drug concentration gradient pre-loading by repeated bonding of the drug pre-loaded cover layer and the substrate, thereby avoiding the tedious operations such as plate preparation and drug dilution during the detection process, which can greatly reduce the labor intensity of the detection personnel. (2) The culture microcavity based on the bacterial culture cover layer is used to culture bacteria, which can better control the growth environment of bacteria and avoid the traditional drug sensitivity test being easily affected by external environmental interference. (3) By directly counting and observing the bacteria in the culture chamber under the phase contrast mode of the microscope, the drug sensitivity test time can be greatly shortened, and more accurate drug sensitivity quantitative information can be provided compared with the traditional plate bacterial culture mode. (4) For traditional methods, before performing bacterial drug sensitivity testing, it is often necessary to first separate the single colony by plating the plate, and then pick the separated single colony, amplify and culture it, and then use the amplified and cultured bacterial sample for drug sensitivity testing analysis. Generally, it takes 12 hours to separate the single colony and 12 hours to amplify and culture it. The culture process of drug sensitivity testing usually lasts 16-24 hours. The present invention is based on the direct observation mode under the microscope of microcavity growth. Even when the concentration of the bacterial solution sample is relatively low, the bacteria distributed in each microcavity are in a state of mutual isolation. No amplification and culture process is required, and the drug sensitivity test experiment can be directly carried out. At the same time, due to the small area and shallow depth of the microcavity, single bacteria can be directly observed in the microcavity, and accurate test results can be obtained in only 1-4 hours, which is much shorter than the drug sensitivity test time of the traditional method. (5) During the use of the microfluidic drug sensitivity chip of the present invention, different concentration gradients of drug freeze-dried powder are set in the microcavities of different bacterial culture cover sheets. Multiple microfluidic drug sensitivity chips can be used to integrate several drugs at the same time, and each drug is set with several concentration gradients. It can save time and effort to complete the drug sensitivity MIC test of multiple drugs required for clinical use at one time. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the structure of the drug pre-loaded cover layer; 1, drug inlet; 2, microchamber; 3, exhaust hole; 4, drug main pipeline; 5, drug side pipeline; 6, drug outlet; Figure 2 Schematic diagram of the structure of the bacterial culture cover layer; 7, bacterial inlet; 8, bacterial main pipeline; 9, bacterial side pipeline; 10, culture chamber; 11, bacterial outlet; Figure 3 This is a schematic diagram of the process of applying the microfluidic drug sensitivity chip to drug sensitivity detection or drug-resistant bacteria screening; Figure 4 The bacterial growth in the microfluidic drug sensitivity chip was observed using phase contrast microscopy. Figure A shows the culture chamber results corresponding to the minimum inhibitory concentration (MIC) at which growth was inhibited, and Figure B shows the culture chamber results where growth was not inhibited. DETAILED DESCRIPTION

[0020] The specific implementation methods of the present invention are described in detail below with reference to the embodiments.

[0021] Example 1 A microfluidic drug sensitivity chip 1. Structure of microfluidic drug sensitivity chip: Microfluidic drug sensitivity chip includes drug pre-loaded cover layer, bacterial culture cover layer and substrate.

[0022] The schematic diagram of the structure of the drug pre-loaded cover layer is as follows Figure 1 As shown, the drug preloaded cover sheet includes a drug inlet 1 and a drug outlet 6 connected by a main drug conduit 4. Several drug-side conduits 5 are provided on the main drug conduit 4, connected to the main drug conduit 4. Each of the drug-side conduits 5 is provided with a microchamber 2 at its end. The diameter of the microchambers 2 increases or decreases from the drug inlet 1 to the drug outlet 6. All of the microchambers 2 have vent holes 3 at their bottoms, extending through the bottom of the drug preloaded cover sheet. The diameters of the drug inlet 1 and drug outlet 6 are 0.8-1.2 mm, the width of the main drug conduit 4 is 300-400 μm, and the width of the drug-side conduits 5 is 200-300 μm. There are at least eight cylindrical microchambers 2, each with a diameter increasing from 1 mm to 2.4 mm. The diameter of the vent holes 3 is 300-400 μm.

[0023] The schematic diagram of the structure of the bacterial culture cover layer is as follows Figure 2 As shown, the bacterial culture cover sheet includes a bacterial inlet 7 and a bacterial outlet 11 connected by a bacterial main pipe 8. The bacterial main pipe 8 is provided with several bacterial side pipes 9 connected to the bacterial main pipe 8. The ends of all bacterial side pipes 9 are provided with culture chambers 10 of the same size. The diameters of the bacterial inlet 7 and the bacterial outlet 11 are 0.8-1.2 mm, the width of the bacterial main pipe 8 is 300-400 μm, and the width of the bacterial side pipes 9 is 200-300 μm. The culture chambers 10 are cylindrical with a diameter of 500 μm.

[0024] Both the drug pre-loaded cover layer and the bacterial culture cover layer can be reversibly bonded to the substrate. The bonding occurs at the substrate attached to the underside of the drug pre-loaded cover layer, or at the underside of the bacterial culture cover layer. The locations where the vents 3 bond to the substrate correspond to the locations where the culture chambers 10 bond to the substrate. A preferred method for reversible bonding in this embodiment is to modify the surfaces of the substrate, drug pre-loaded cover layer, and bacterial culture cover layer to impart adhesiveness, allowing for repeated attachment and removal.

[0025] 2. Material of microfluidic drug sensitivity chip: (1) The surface of the drug pre-loaded cover layer exhibits hydrophilic properties, and the pre-loaded drug is made of antibiotic powder and ultrapure water. The material of the drug pre-loaded cover layer is modified polydimethylsiloxane, and the raw materials for preparing the modified polydimethylsiloxane are polydimethylsiloxane, a cross-linking agent, and a modifier. Among them, the modifier is one of oleylamine and polyethyleneimine (PEIE). In this embodiment, the modifier is preferably PEIE, and the cross-linking agent is sylgard 184 silicone elastomer curing agent (sylgard 184 organic elastic curing agent). The amount ratio of polydimethylsiloxane, cross-linking agent, and modifier is 10 g:1 g:20 μL.

[0026] The preparation of the drug pre-loaded cover sheet and the bacterial culture cover sheet includes the following steps: designing a mold using 3D graphics design software, then directly fabricating the mold using a method selected from CNC engraving, laser engraving, and 3D printing. The material is prepared according to the aforementioned modified polydimethylsiloxane ratio, poured into the designed mold, placed in a vacuum desiccator to remove air bubbles, and baked in a 95°C oven for approximately 2 hours. The dried mold is then removed and perforated to obtain the drug pre-loaded cover sheet.

[0027] The bacterial culture cover sheet was prepared by soft photolithography, which included the following steps: using Solidworks software to draw a model and process a mask, performing photolithography to obtain a mold, and then pouring the material prepared according to the ratio of the modified polydimethylsiloxane into the mold, baking it in a 95°C oven for about 2 hours, and drying it to obtain the bacterial culture cover sheet.

[0028] (2) The substrate is preferably made of glass.

[0029] Example 2 Method for using the microfluidic drug sensitivity chip: The schematic diagram of the process of applying the microfluidic drug sensitivity chip provided in this embodiment to drug sensitivity testing is as follows: Figure 3 The specific process is as follows: S1. Prepare a drug solution of the required concentration; bond the substrate to the drug pre-loaded cover layer, and use negative pressure injection to pass the drug solution from the drug injection port 1 into the microfluidic drug sensitivity chip to ensure that the microchamber 2 is completely filled. Then empty the pipeline to retain only the drug solution in the microchamber, and then freeze-dry the microfluidic drug sensitivity chip at -40°C. During the freeze-drying process, the drug powder is fixed on the substrate through the exhaust hole 3.

[0030] The drug solution is introduced into the chip from the drug injection port 1 by negative pressure injection. The specific steps are as follows: the drug outlet 6 of the microfluidic drug sensitivity chip is connected to the syringe using a microchannel, the air in the microfluidic drug sensitivity chip is pre-evacuated by the syringe and sealed with tape to achieve a negative pressure state of the microfluidic drug sensitivity chip; then the drug solution is dropped into the drug injection port 1 of the microfluidic drug sensitivity chip, the drug injection port 1 is opened, and the drug solution will quickly fill the entire microfluidic drug sensitivity chip.

[0031] S2. Separate the substrate from the drug pre-loaded cover layer, and then bond the substrate to the bacterial culture cover layer. The position where the drug powder is fixed on the substrate corresponds one-to-one to the position where the substrate is combined on the culture chamber 10; then, the test bacterial liquid is introduced into the microfluidic drug sensitivity chip from the bacterial liquid inlet 7 by negative pressure injection, ensuring that the culture chamber is completely filled with bacterial liquid, empty the pipeline, and then pass paraffin oil into the pipeline of the microfluidic drug sensitivity chip to seal and separate the microcavities, ensuring that the liquids in the microcavities are separated and do not affect each other; the bacterial liquid dissolves the antibiotic powder in the microcavity, and then the entire microfluidic drug sensitivity chip is placed in a high moisture-retaining environment, and then paraffin oil, edible oil or fluorocarbon oil is added to the bacterial inlet 7 and the bacterial outlet 11 to ensure sealing.

[0032] The test bacterial solution is introduced into the microfluidic drug sensitivity chip through the bacterial solution inlet 7 by negative pressure injection. The specific operation is as follows: the bacterial outlet 11 of the microfluidic drug sensitivity chip is connected to a syringe using a microchannel, the bacterial inlet 7 of the bacterial culture cover layer is first sealed with tape, and then the bacterial solution is dripped onto the tape to form a closed microcavity / microchannel system in the microfluidic drug sensitivity chip. The microfluidic drug sensitivity chip is then degassed using a syringe, and the bacterial solution is then dripped onto the bacterial inlet 7 of the microfluidic drug sensitivity chip, and the bacterial inlet 7 is opened. Because the dissolved air in the PDMS cover layer is extracted during the degassing process, a certain vacuum is established in the PDMS block. When the bacterial inlet 7 of the degassed microfluidic drug sensitivity chip is opened, the degassed PDMS cover layer absorbs the air in the microcavity / microchannel, generating a negative pressure in the microcavity / microchannel, driving the bacterial solution in the bacterial inlet 7 into and filling the entire microcavity / microchannel system of the chip. After the bacterial solution injection is completed, ensure that the chambers are completely filled with bacterial solution, and remove the residual drug / bacteria solution at the bacterial solution injection port 7. Since the syringe at the end of the bacterial sampling port 11 of the microfluidic drug sensitivity chip is still in a negative pressure state, the liquid in the pipeline will be sucked away at this time, while the liquid in the microcavity is retained due to the effect of resistance, thereby achieving discretization of the bacterial solution. The entire process is completed in a very short time, ensuring that the antibiotic powder is just dissolved by the liquid in the microcavity, and a liquid containing a mixture of antibiotics and bacterial solution of different concentrations is obtained in the microcavity.

[0033] S3. Place the bacterial culture cover sheet and substrate processed in step S2 on a PTC constant temperature heating chip and culture at 37°C for 1-4 hours. Take out the coverslip and substrate every hour to observe and record the growth of bacteria in the microcavity using a phase contrast microscope. Compare the growth of bacteria in each microcavity and find the minimum concentration corresponding to the chamber where bacterial growth is inhibited, which is the minimum inhibitory concentration (MIC). This can be used to evaluate the sensitivity of the test bacteria to the tested drug and the minimum inhibitory concentration of a specific drug for the test bacteria.

[0034] Example 3 Method for using the microfluidic drug sensitivity chip: The schematic diagram of the process of applying the microfluidic drug sensitivity chip provided in this embodiment to drug sensitivity testing is as follows: Figure 3 The specific process is as follows: S1. Prepare a drug solution of the required concentration; bond the substrate to the drug pre-loaded cover layer, and use a microfluidic pump to pass the drug solution from the drug inlet 1 into the microfluidic drug sensitivity chip to ensure that the microchamber 2 is completely filled. Then empty the pipeline to retain only the drug solution in the microchamber, and then freeze-dry the microfluidic drug sensitivity chip at -40°C. During the freeze-drying process, the drug powder is fixed on the substrate through the exhaust hole 3.

[0035] The drug solution was introduced into the chip from drug inlet 1 by microfluidic pump injection. The specific steps were as follows: the drug solution was loaded into a 1 mL syringe, the syringe was fixed on the microfluidic pump, and the syringe outlet was connected to the drug inlet 1 through a PVC hose; the flow rate of the microfluidic pump was set to 1-3 μL / min, preferably 3 μL / min, and the bacterial solution was allowed to fill the entire microfluidic drug sensitivity chip.

[0036] S2. Separate the substrate from the drug pre-loaded cover layer, and then bond the substrate to the bacterial culture cover layer. The position where the drug powder is fixed on the substrate corresponds one-to-one to the position where the substrate is combined on the culture chamber 10; then, the test bacterial liquid is injected into the microfluidic drug sensitivity chip from the bacterial liquid inlet 7 by a microfluidic pump to ensure that the culture chamber is completely filled with the bacterial liquid, empty the pipeline, and then pass paraffin oil into the pipeline of the microfluidic drug sensitivity chip to seal and separate the microcavities, ensuring that the liquids in the microcavities are separated and do not affect each other; the bacterial liquid dissolves the antibiotic powder in the microcavity, and then the entire microfluidic drug sensitivity chip is placed in a high moisture-retaining environment, and then paraffin oil, edible oil or fluorocarbon oil is added to the bacterial inlet 7 and the bacterial outlet 11 to ensure sealing.

[0037] The test bacterial solution was introduced into the microfluidic drug sensitivity chip from the bacterial solution inlet 7 by microfluidic pump injection. The specific steps were as follows: the test bacterial solution was placed in a 1 mL syringe, the syringe was then fixed on the microfluidic pump, and the syringe outlet was connected to the drug inlet 1 through a PVC hose; the flow rate of the microfluidic pump was set to 1-3 μL / min, preferably 3 μL / min, and the entire microfluidic drug sensitivity chip was filled with the bacterial solution.

[0038] S3. Place the bacterial culture cover sheet and substrate processed in step S2 on a PTC constant temperature heating chip and culture at 37°C for 1-4 hours. Take out the coverslip and substrate every hour to observe and record the growth of bacteria in the microcavity using a phase contrast microscope. Compare the growth of bacteria in each microcavity and find the minimum concentration corresponding to the chamber where bacterial growth is inhibited, which is the minimum inhibitory concentration (MIC). This can be used to evaluate the sensitivity of the test bacteria to the tested drug and the minimum inhibitory concentration of a specific drug for the test bacteria.

[0039] Example 4 A method for detecting the minimum inhibitory concentration of clindamycin against Staphylococcus aureus using a microfluidic drug sensitivity chip 1. Structure of microfluidic drug sensitivity chip: The structure of the microfluidic drug sensitivity chip is the same as that of Example 1. The materials of the drug pre-loaded cover layer and the bacterial culture cover layer are both modified polydimethylsiloxane, which is made by mixing polydimethylsiloxane, a cross-linking agent, and polyethyleneimine in a ratio of 10 g:1 g:30 μL; the substrate is made of glass.

[0040] On the drug pre-loading cover layer, the diameters of the drug inlet 1 and the drug outlet 6 are 1.0 mm, the width of the drug main pipeline 4 is 300 μm, and the width of the drug side pipeline 5 is 200 μm. There are 8 cylindrical microchambers 2, and their diameters increase from the side of the drug inlet 1 to the side of the drug outlet 6, namely 1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2 and 2.4 mm respectively; the diameter of the exhaust hole 3 is 300 μm, and the exhaust hole 3 runs through the bottom of the drug pre-loading cover layer, and its depth is 1 mm.

[0041] On the bacterial culture cover layer, the diameters of the bacterial inlet 7 and the bacterial outlet 11 are 1.0 mm, the width of the bacterial main channel 8 is 300 μm, and the width of the bacterial side channel 9 is 200 μm; the culture chamber 10 is cylindrical with a diameter of 500 μm. The culture chamber 10 runs through the bottom of the bacterial culture cover layer and has a depth of 1 mm.

[0042] 2. The steps for detecting the minimum inhibitory concentration are the same as those in Example 2: (1) Preparing a substrate loaded with clindamycin in a concentration gradient by using a drug pre-loaded layer substrate; (2) Bond the substrate pre-loaded with clindamycin to the bacterial culture cover sheet, and introduce bacteria at a concentration of about 10 6 cfu / mL of Staphylococcus aureus liquid was sealed and moisturized and placed on a PTC constant temperature heating chip at 37°C for in-situ constant temperature culture; (3) The growth of bacteria in the microcavities with different clindamycin concentrations was observed and recorded using the microscope phase mode at 0, 1, and 2 h of culture. The lowest drug concentration at which bacterial growth was inhibited was the minimum inhibitory concentration of clindamycin.

[0043] Use phase contrast mode to observe bacterial growth in the culture chamber. Figure 4 As shown, Figure 4 A is a picture of the chamber corresponding to the minimum antibiotic concentration that inhibits the growth of Staphylococcus aureus. The concentration of clindamycin in the chamber is 0.6 μg / mL, which means that the minimum inhibitory concentration of clindamycin against Staphylococcus aureus is 0.6 μg / mL.

[0044] The steps of drug-resistant bacteria screening are the same as those of drug sensitivity testing in Example 4. According to the test results, bacteria that are insensitive to antibiotics are drug-resistant bacteria.

[0045] Although the specific embodiments of the present invention have been described in detail in conjunction with the embodiments, this should not be construed as limiting the scope of protection of this patent. Within the scope described by the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A microfluidic drug sensitivity chip, characterized by: The invention comprises a drug pre-loaded cover sheet layer, a bacterial culture cover sheet layer and a base sheet; the drug pre-loaded cover sheet layer comprises a drug inlet (1) and a drug outlet (6) connected via a drug main pipeline (4); the drug main pipeline (4) is provided with a plurality of drug side pipelines (5) connected to the drug main pipeline (4); the ends of all drug side pipelines (5) are provided with micro chambers (2); and the diameters of the micro chambers (2) increase or decrease in sequence from the drug inlet (1) side to the drug outlet (6) side; and the bottoms of all micro chambers (2) are provided with exhaust holes (3), and the exhaust holes (3) penetrate the drug pre-loaded cover sheet layer. The bottom of the bacterial culture cover sheet comprises a bacterial sample inlet (7) and a bacterial sample outlet (11) connected through a bacterial main pipeline (8); the bacterial main pipeline (8) is provided with a plurality of bacterial side pipelines (9) connected to the bacterial main pipeline (8); the ends of all bacterial side pipelines (9) are provided with culture chambers (10) of the same size, and the culture chambers (10) pass through the bottom of the bacterial culture cover sheet; the drug pre-loaded cover sheet and the bacterial culture cover sheet can be reversibly bonded to the substrate, and the position where the exhaust hole (3) is combined with the substrate corresponds to the position where the culture chamber (10) is combined with the substrate.

2. The microfluidic drug sensitivity chip according to claim 1, characterized in that: The diameters of the drug inlet (1) and the drug outlet (6) are 0.8-1.2 mm, the width of the drug main pipeline (4) is 300-400 μm, the width of the drug side pipeline (5) is 200-300 μm, at least 8 micro-chambers (2) are provided, and the micro-chambers (2) are cylindrical, with diameters increasing from 1 mm to 2.4 mm; the diameter of the exhaust hole (3) is 300-400 μm.

3. The microfluidic drug sensitivity chip according to claim 1, characterized in that: The diameters of the bacterial inlet (7) and the bacterial outlet (11) are 0.8-1.2 mm, the width of the bacterial main pipeline (8) is 300-400 μm, and the width of the bacterial side pipeline (9) is 200-300 μm; the culture chamber (10) is cylindrical and has a diameter of 500 μm.

4. The microfluidic drug sensitivity chip according to claim 1, characterized in that: The material of the drug pre-loaded cover layer and the bacterial culture cover layer is modified polydimethylsiloxane, and the raw materials for preparing the modified polydimethylsiloxane are polydimethylsiloxane, a crosslinking agent and a modifier, wherein the modifier is oleylamine or polyethyleneimine, and the crosslinking agent is sylgard 184 silicone elastomer curing agent.

5. The microfluidic drug sensitivity chip according to claim 4, characterized in that: The modifier is polyethyleneimine, and the amounts of polydimethylsiloxane, crosslinker, and modifier are 10 g:1 g:20-40 μL; the substrate is made of glass.

6. Use of the microfluidic drug sensitivity chip according to any one of claims 1 to 5 in drug sensitivity detection or drug-resistant bacteria screening.

7. Use according to claim 6, characterized in that Drug susceptibility testing or drug-resistant bacterial screening involves the following steps: S1. Bond the substrate to the drug pre-loaded cover layer, pass the drug solution into the microfluidic drug sensitivity chip, ensure that the microchamber (2) is filled, then empty the pipeline to retain only the drug solution in the microchamber (2), and then freeze-dry the microfluidic drug sensitivity chip, and fix the drug powder on the substrate through the exhaust hole (3); S2, separating the substrate from the drug pre-loaded cover layer, and then bonding the substrate to the bacterial culture cover layer, so that the position where the drug powder is fixed on the substrate corresponds to the position of the culture chamber (10) on the substrate; passing the test bacterial solution into the microfluidic drug sensitivity chip, ensuring that the culture chamber (10) is filled, and then emptying the pipeline; moisturizing and sealing the microfluidic drug sensitivity chip; S3. The microfluidic drug sensitivity chip processed in step S2 is placed under constant temperature conditions for cultivation, and the growth of the test bacteria in the chamber is observed under a phase contrast microscope to evaluate the sensitivity of the test bacteria to the tested drugs or screen out drug-resistant bacteria.

8. The use according to claim 7, characterized in that: The drug solution and the test bacterial solution are introduced into the microfluidic drug sensitivity chip by negative pressure injection or microfluidic pump injection; The steps of negative pressure sampling are as follows: connecting the drug outlet (6) or bacterial outlet (11) of the microfluidic drug sensitivity chip to a syringe using a micro-channel, and pre-evacuating the air in the microfluidic drug sensitivity chip through the syringe and sealing it; then dripping the drug solution or bacterial liquid into the drug inlet (1) or bacterial inlet (7) of the microfluidic drug sensitivity chip, and the drug solution or bacterial liquid fills the entire microfluidic drug sensitivity chip; The steps of microfluidic pump injection are as follows: put the drug solution or bacterial solution into the syringe, then fix the syringe on the microfluidic pump, and then connect the syringe outlet to the bacterial inlet (7) or drug inlet (1) through a pipe; set the microfluidic pump flow rate to 1-3 μL / min, and wait for the bacterial solution to fill the entire microfluidic drug sensitivity chip.

9. The use according to claim 7, characterized in that: After the pipeline is emptied in step S2, a sealing oil is introduced into the pipeline of the microfluidic drug sensitivity chip to separate the liquid in the culture chamber (10); the sealing oil is paraffin oil, edible oil or fluorocarbon oil.

10. The use according to claim 7, characterized in that: In step S3, the microfluidic drug sensitivity chip is placed on the PTC constant temperature heating chip and cultured at 37° C. for 1-4 hours.

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