Micro-fluidic chip for quantitatively detecting drug sensitivity of bacteria and detection method of micro-fluidic chip

Abstract

The invention discloses a micro-fluidic chip for quantitatively detecting bacterial drug sensitivity and a detection method of the micro-fluidic chip, and relates to the technical field of micro-fluidic chip analys.The micro-fluidic chip is a Y-shaped channel and comprises a main pipeline, two sample feeding channels are formed in one end of the main pipeline, and a sample discharging channel is formed in the other end of the main pipeline; a plurality of culture chambers are arranged on the side of the main pipeline through side pipelines. The invention also discloses a method for quantitatively detecting the drug sensitivity of bacteria by using the micro-fluidic chip. According to the micro-fluidic chip, high-flux and rapid bacterial drug sensitivity test can be realized, the drug sensitivity test time is greatly shortened, and meanwhile, more accurate drug sensitivity quantitative information can be provided.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic chip analysis, and particularly relates to a microfluidic chip for quantitatively detecting bacterial drug susceptibility and a detection method thereof. Background Art

[0002] In vitro Antibiotic Susceptibility Test (AST) is a key technology for providing a scientific basis for rational clinical drug use by measuring the in vitro inhibitory effect of antibacterial drugs on pathogenic microorganisms. This detection method has important clinical value for accurately guiding the use of antibiotics, curbing the spread of drug-resistant strains, and improving the success rate of treating infectious diseases. Currently, the mainstream drug susceptibility detection methods can be divided into three categories according to the experimental principle: diffusion method, dilution method, and concentration gradient method, each with its own characteristics and applicable to different detection scenarios.

[0003] In the field of microfluidic chips for drug susceptibility detection, Y-shaped microfluidic chips are often used to generate drug concentration gradients to test the responses of microorganisms or cells at different doses due to their simple geometric structure and controllable hydrodynamic characteristics. However, this design has significant limitations in practical applications, especially when a long-term stable flow rate is required to maintain the concentration gradient. The core problems are mainly reflected in the following three aspects:

[0004] (1) Time-consuming: The bottleneck in establishing and maintaining the concentration gradient

[0005] The Y-shaped chip forms a concentration gradient through the laminar diffusion mixing of two fluid streams, and its gradient generation rate highly depends on the flow rate stability. Since the fluid in the microchannel is mainly laminar flow, the mixing mainly relies on molecular diffusion rather than turbulence, resulting in a slow gradient formation. For example, in bacterial drug susceptibility tests, it usually takes several hours or even longer to reach a stable linear concentration gradient, and the entire detection cycle (including gradient generation, drug exposure, and reaction observation) may thus be extended to more than 24 hours. In addition, to maintain the gradient stability, the fluid must be continuously driven by an external pump (such as an injection pump or a pressure pump), which not only increases the equipment dependence but also may cause micro-leakage or material fatigue at the chip interface due to long-term mechanical pressure, further affecting the repeatability of the experimental results.

[0006] (2) Reagent waste: Inefficient fluid utilization mechanism

[0007] Although microfluidic chips are advantageous in "consuming trace amounts of reagents", the design of the Y-shaped chip may have the opposite effect during long-term operation. The traditional Y-shaped chip needs to continuously inject the mother liquor to maintain the gradient, while only a small part of the total consumed drugs actually participate in the reaction (for example, only the middle laminar flow region participates in the diffusion), and the remaining reagents are finally discarded.

[0008] (3) Liquid evaporation: Chamber concentration change

[0009] The chamber volume of a microfluidic chip is usually in the microliter or even nanoliter range. Liquid evaporation can cause a reduction in the sample volume, resulting in a significant increase in the solute concentration, which directly affects the quantitative analysis results of the experiment. For example, in long-term cell culture, the evaporation of the culture medium may change the osmotic pressure and nutrient concentration, leading to cell death or abnormal function.

[0010] Therefore, it is necessary to provide a Y-shaped microfluidic chip for drug sensitivity testing with rapid detection, low reagent cost, and high moisture retention to solve the above problems. Summary of the Invention

[0011] To solve the above technical problems, the object of the present invention is to provide a microfluidic chip for quantitatively detecting bacterial drug sensitivity and its detection method. The microfluidic chip can achieve high-throughput and rapid bacterial drug sensitivity testing, greatly shortening the drug sensitivity testing time, and at the same time can provide more accurate drug sensitivity quantitative information.

[0012] The technical solution of the present invention to solve the above technical problems is as follows: Provide a microfluidic chip for quantitatively detecting bacterial drug sensitivity. The microfluidic chip has a Y-shaped channel. The microfluidic chip includes a main pipeline. One end of the main pipeline is provided with two sample injection channels, and the other end of the main pipeline is provided with a sample output channel. A plurality of culture chambers are provided on the side of the main pipeline through side pipelines.

[0013] Setting of the culture chamber: The main pipeline is provided with a plurality of side pipelines, and each side pipeline is connected to a culture chamber.

[0014] Further, a culture chamber is provided on the side of the sample injection channel through a side pipeline. The culture chamber is located inside or outside the sample injection channel.

[0015] The beneficial effect of adopting the further technical solution is: It can be used for negative and positive controls.

[0016] Further, the width of the sample injection channel and the sample output channel is 150 - 250 μm; the inclination angle between the sample injection channel and the main pipeline is 45 - 50°.

[0017] Further, the width of the main pipeline is 800 - 1000 μm; the width of the side pipeline is 80 - 100 μm, and the length is 150 - 250 μm; the interval between the side pipelines is 400 - 500 μm; the diameter of the culture chamber is 250 - 350 μm.

[0018] Further, the thickness of the microfluidic chip is 40 - 60 μm.

[0019] Further, separation layers are provided at both ends of the main pipeline.

[0020] The beneficial effect of adopting the further technical solution is: to avoid the premature mixing of the two liquids and interfere with the control group.

[0021] Furthermore, the upper surface of the microfluidic chip is bonded to a PDMS (polydimethylsiloxane) film.

[0022] Still further, the thickness of the PDMS film is 1 - 1.5 mm.

[0023] The present invention also provides a method for quantitatively detecting the drug sensitivity of bacteria, which uses the above-mentioned microfluidic chip for detection, and includes the following steps:

[0024] Bacterial suspensions containing and not containing drugs are respectively introduced into the two injection channels. The bacterial suspensions diffuse in the main pipeline and form a drug concentration gradient in the culture chamber. After cultivation, the drug sensitivity of the bacterial strain is judged according to the growth condition of the bacteria.

[0025] Furthermore, a microfluidic injection pump is used for injection.

[0026] Furthermore, the flow rates of the two injection channels are 1 - 3 μL / min, which can be the same or different.

[0027] Furthermore, the drug is an antibiotic.

[0028] Furthermore, the concentration of the bacterial suspension is 0.8 - 1.5×10 5 CFU / mL.

[0029] Furthermore, after the injection of the bacterial suspension is completed, the liquid in the main pipeline is emptied and an oil phase is added for sealing.

[0030] The beneficial effect of adopting the further technical solution is: to ensure the independence of each chamber, and at the same time, there is no need to continuously introduce the bacterial suspension and the drug, reducing the consumption of reagents.

[0031] Still further, the oil phase is paraffin oil.

[0032] Furthermore, the microfluidic chip is placed on top of a liquid storage pool filled with water for bacterial cultivation.

[0033] The beneficial effect of adopting the further technical solution is: the chamber volume of the microfluidic chip is usually in the microliter or even nanoliter level. The evaporation of the liquid will cause the reduction of the sample volume, resulting in a significant increase in the solute concentration, which directly affects the quantitative analysis results of the experiment. The PDMS film allows water vapor molecules to pass through, thereby maintaining the liquid volume in the culture chamber and ensuring the accuracy of the analysis experiment results.

[0034] Furthermore, a bacterial metabolism indicator is added to the bacterial suspension, and the drug sensitivity of the bacteria is judged according to the fluorescence intensity in the culture chamber.

[0035] The present invention has the following beneficial effects:

[0036] 1. The Y-shaped microfluidic chip of the present invention forms a concentration gradient based on the diffusion effect and isolates and cultures bacteria at different concentrations, which can better control the growth environment of bacteria, avoid the influence of traditional drug sensitivity tests being easily interfered by the external environment, and improve the accuracy of detection.

[0037] 2. After the Y-shaped microfluidic chip of the present invention quickly forms a concentration gradient, the liquid in the main pipeline is emptied. The liquid in the side pipeline will not flow out due to capillary action, and when an oil phase is introduced for sealing, it can better save reagents and avoid waste.

[0038] 3. The Y-shaped microfluidic chip of the present invention is bonded to the PDMS film, and water vapor can penetrate through the film into the microcavity array. By providing a high-humidity environment, the volatilization of the liquid in the chamber is reduced. Even if part of the chamber liquid volatilizes, the water vapor penetrating through the film can refill the chamber again, avoiding the interference of concentration changes during the cultivation process on the analysis results. Description of the Drawings

[0039] Figure 1 It is a schematic top view structure diagram of the cross-section of the microfluidic chip in Example 1;

[0040] Figure 2 It is a schematic diagram of the simulated concentration gradient of the microfluidic chip in Example 1 based on COMSOL simulation software;

[0041] Figure 3 It is a fluorescence image of the growth of bacteria in the culture chamber;

[0042] Figure 4 It is a comparison diagram of the liquids in the culture chambers of the microfluidic chips in Example 1 and Comparative Example 1;

[0043] Among them, 1. Sampling channel; 2. Culture chamber; 3. Side pipeline; 4. Main pipeline; 5. Sampling outlet channel. Detailed Embodiments

[0044] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0045] Example 1

[0046] A microfluidic chip for quantitatively detecting the drug sensitivity of bacteria, the structural schematic diagram is as Figure 1 shown. The microfluidic chip has a Y-shaped channel, including a main pipeline 4. One end of the main pipeline 4 is provided with two sampling channels 1, the other end of the main pipeline 4 is provided with a sampling outlet channel 5, and several culture chambers 2 are arranged on the side of the main pipeline 4 through side pipelines 3;

[0047] A culture chamber 2 is provided on the side of the sample injection channel 1 through a side channel 3; the widths of the sample injection channel 1 and the sample outlet channel 5 are 200 μm; the inclination angle between the sample injection channel 1 and the main channel 4 is 45°; the width of the main channel 4 is 900 μm; the width of the side channel 3 is 90 μm, the length is 200 μm, and the interval is 450 μm; the diameter of the culture chamber is 200 μm; the upper surface of the microfluidic chip is bonded to the PDMS film.

[0048] Example 2

[0049] A method for quantitatively detecting bacterial drug susceptibility, which uses the microfluidic chip of Example 1 for detection, includes the following steps:

[0050] Bacterial suspensions containing and not containing drugs are respectively introduced into the two sample injection channels 1. The bacterial suspensions diffuse in the main channel 4 to form a drug concentration gradient in the culture chamber 2. After the sample injection of the suspension is completed, the liquid in the main channel 4 is emptied and an oil phase is added for sealing. The microfluidic chip is placed on top of a liquid storage pool filled with water for bacterial culture. After the culture, the drug susceptibility of the strain is judged according to the growth status of the bacteria.

[0051] Comparative Example 1

[0052] A microfluidic chip for drug susceptibility detection, which is different from Example 1 in that the PDMS film is not bonded.

[0053] Test Example 1

[0054] The generation of the concentration gradient of the microfluidic chip of Example 1 was simulated based on COMSOL simulation software. The results are as Figure 2 shown. It can be seen that a concentration gradient was successfully formed in each culture chamber of the microfluidic chip, which can be used for drug susceptibility detection.

[0055] Test Example 2

[0056] Drug susceptibility tests were respectively carried out using the microfluidic chips of Example 1 and Comparative Example 1. The detection steps using the microfluidic chip of Example 1 are as follows:

[0057] (1) Prepare the antibiotic solution: Take out the refrigerated vancomycin powder and dilute it to 100 μg / mL with LB broth;

[0058] (2) Dilute the bacterial suspension: The cultured Staphylococcus aureus was diluted to 10 5 CFU / mL with LB broth and the antibiotic solution prepared in step (1) respectively, and a cell proliferation and toxicity detection reagent (AlamarBlue) was added thereto at a volume ratio of 9:1;

[0059] (3) Injecting bacterial solution: Use a microfluidic pumping device to inject bacterial solutions with and without vancomycin into the two injection channels 1 respectively, and maintain a stable flow rate for a period of time to ensure that the culture chamber 2 is completely filled with liquid;

[0060] (4) Sealing: Drain the liquid in the main pipeline 4, inject paraffin oil for sealing to ensure the independence of each culture chamber 2, and place the entire chip in a moisturizing environment (on top of a liquid reservoir filled with water);

[0061] (5) Culturing and observing: Place the microfluidic chip in a constant temperature incubator for 4 hours, then take out the microfluidic chip to observe the fluorescence intensity of the culture chamber array, and record the experimental data;

[0062] (6) Analyzing data: Compare the fluorescence intensities of each chamber and combine with the blank control. The concentration corresponding to the chamber with the fluorescence intensity closest to the negative control group is the minimum inhibitory concentration of the detected bacteria against the antibiotic.

[0063] The difference in using the microfluidic chip of Comparative Example 1 for detection is that it is not placed in a moisturizing environment and is directly placed in a constant temperature incubator for culturing.

[0064] The fluorescence image of the growth of bacteria in one culture chamber of the microfluidic chip used in this test example, as shown in Figure 3 shows that this method can judge the growth of bacteria through fluorescence intensity, and further analyze the antibacterial concentration of the drug.

[0065] After culturing using the microfluidic chips of Example 1 and Comparative Example 1, the liquid conditions in the culture chambers are as shown in Figure 4 shows that there is water evaporation in the microfluidic chip of Comparative Example 1 during the culturing process, while the culture chambers of the microfluidic chip of Example 1 with the PDMS film bonded and placed in a moisturizing environment are still filled with liquid. This indicates that this method can effectively avoid the concentration change caused by liquid volatilization during the drug sensitivity detection process, which is beneficial to improving the accuracy of the analysis results.

[0066] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A microfluidic chip for quantitatively detecting bacterial drug sensitivity, characterized in that, The microfluidic chip has a Y-shaped channel. The microfluidic chip includes a main pipeline. One end of the main pipeline is provided with two sample injection channels, and the other end of the main pipeline is provided with a sample output channel. A number of culture chambers are arranged on the side of the main pipeline through side pipelines.

2. The microfluidic chip for quantitatively detecting bacterial drug sensitivity according to claim 1, wherein Culture chambers are arranged on the side of the sample injection channel through side pipelines.

3. The microfluidic chip for quantitatively detecting bacterial drug susceptibility according to claim 1, characterized in that, The widths of the sample injection channel and the sample output channel are 150 - 250 μm; the inclination angle between the sample injection channel and the main pipeline is 45 - 50°.

4. The microfluidic chip for quantitatively detecting bacterial drug sensitivity according to claim 1, characterized in that, The width of the main pipeline is 800 - 1000 μm; the width of the side pipeline is 80 - 100 μm, and the length is 150 - 250 μm; the interval between the side pipelines is 400 - 500 μm; the diameter of the culture chamber is 250 - 350 μm.

5. The microfluidic chip for quantitatively detecting bacterial drug sensitivity according to claim 1, characterized in that Partition layers are arranged at both ends of the main pipeline.

6. The microfluidic chip for quantitatively detecting bacterial drug sensitivity according to claim 1, characterized in that, The upper surface of the microfluidic chip is bonded to a PDMS film.

7. A method for quantitatively detecting the drug sensitivity of bacteria, characterized in that, Using the microfluidic chip according to any one of claims 1 - 6 for detection, comprising the following steps: Bacterial suspensions containing and not containing drugs are respectively introduced into the two sample injection channels. The bacterial suspensions diffuse in the main pipeline, and a drug concentration gradient is formed in the culture chambers. After cultivation, the drug sensitivity of the bacterial strain is judged according to the growth status of the bacteria.

8. The method for quantitatively detecting bacterial drug sensitivity according to claim 7, wherein After the injection of the bacterial suspension is completed, the liquid in the main pipeline is emptied and an oil phase is added for sealing.

9. The method for quantitatively detecting bacterial drug susceptibility according to claim 7, wherein The microfluidic chip is placed on top of a liquid storage pool filled with water for bacterial culture.

10. The method for quantitatively detecting bacterial drug susceptibility according to claim 7, wherein A bacterial metabolism indicator is added to the bacterial suspension, and the drug sensitivity of the bacteria is judged according to the fluorescence intensity in the culture chambers.

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