阅读说明：本技术 一种利用微流控芯片定量检测细菌及药敏实验的方法 (method for quantitatively detecting bacteria and drug sensitivity experiment by using micro-fluidic chip ) 是由 张晓杰 宋波 于 2019-09-24 设计创作，主要内容包括：本发明提供了一种具有新型结构的微流控芯片,以及利用微流控芯片玻璃微珠定量检测细菌的方法。本发明的微流控芯片包括顶层、中层和底层结构,其中中层结构包括四个区域：入口区、检测区、浓度梯度发生器以及废液孔,所述入口区位于检测区和药敏反应池之间,同时,本发明将玻璃微珠填充到微流控芯片检测区中,采用抗原抗体技术定量检测细菌,并同步进行药敏检测。采用本发明的微流控芯片,能够实现细菌的快速准确检测,且灵敏度高,特异性高,同时药敏试剂用量小。(The invention provides a micro-fluidic chip with a novel structure and a method for quantitatively detecting bacteria by utilizing micro-fluidic chip glass beads. The micro-fluidic chip comprises a top layer, a middle layer and a bottom layer structure, wherein the middle layer structure comprises four areas: the invention discloses a micro-fluidic chip for detecting drug sensitivity, which comprises an inlet area, a detection area, a concentration gradient generator and a waste liquid hole, wherein the inlet area is positioned between the detection area and a drug sensitivity reaction tank, and meanwhile, glass beads are filled in the detection area of the micro-fluidic chip, and the antigen-antibody technology is adopted to quantitatively detect bacteria and synchronously carry out drug sensitivity detection. The micro-fluidic chip can realize the rapid and accurate detection of bacteria, and has high sensitivity, high specificity and small dosage of drug sensitive reagents.)

1. The microfluidic chip is characterized by comprising a top layer, a middle layer and a bottom layer, wherein the middle layer is of a PDMS structure, the bottom layer is a glass slide, and the middle layer comprises four regions: the device comprises an inlet area, a detection area, a concentration gradient generator and a waste liquid hole, wherein the inlet area is positioned between the detection area and the concentration gradient generator, the concentration gradient generator is of a Christmas tree type and comprises a drug sensitive culture pool, wherein n is equal to 1, 2, 3 and 4 … …;

The preparation method of the microfluidic chip comprises the following steps: sealing the middle layer of the microfluidic chip with the bottom layer of the microfluidic chip, respectively placing sterile filter paper sheets in the drug sensitive culture tank, sealing the middle layer of the microfluidic chip with the top layer of the microfluidic chip after plasma treatment, baking the layers at 90 ℃ for 2h before sealing the chips, and irradiating overnight by an ultraviolet lamp.

2. The microfluidic chip according to claim 1, wherein the inlet region comprises a sample well to be tested, a diluent/buffer solution well and a drug well, the sample well to be tested is provided with a detachable PVDF film with a thickness of 90-110 μm on one side of the concentration gradient generator, and the bottom layer of the inlet region is 2-4 μm higher than the bottom layers of the other regions.

3. The microfluidic chip according to claim 1, wherein the diameter of the drug sensitive culture pool is 2-5mm, and the diameter of the waste liquid hole is 0.5-1 mm.

4. The microfluidic chip according to claim 1, wherein the detection region comprises microtots and microbeads; the micro support is V-shaped or semicircular, the diameter of the micro bead is 60-90 μm, and the micro support is slightly wider than the micro bead to support the micro bead.

5. The microfluidic chip according to claim 1, wherein the waste liquid wells comprise a detection zone waste liquid well (1) and a concentration gradient generator waste liquid well (9), the waste liquid well (1) is communicated with the detection zone through a liquid channel, and the waste liquid well (9) is communicated with the downstream of the drug sensitive culture tank through a liquid channel.

6. the microfluidic chip according to claim 5, wherein a curved channel (10) is provided between the inlet region and the concentration gradient generator and between each drug sensitive culture cell, and the channel width between the detection region and the waste liquid hole (1) is 40 μm, and the width of all other channels is 100 μm.

7. the microfluidic chip according to claim 1, wherein the layers are baked at 90 ℃ for 2h and irradiated with an ultraviolet lamp overnight before the chip is sealed.

8. Use of the microfluidic chip according to any one of claims 1 to 7 for pathogen identification and drug susceptibility testing, comprising the steps of:

step S1: sterilizing each layer of the chip under high pressure, sealing the bottom layer and the middle layer, adding a culture medium into a drug sensitive culture tank, adding a sterile filter paper sheet into the drug sensitive culture tank, sealing the upper layer, storing the sealed whole chip in a refrigerator at 4 ℃, and irradiating the surface of the chip for 30min by using an ultraviolet lamp before use;

Step S2: preparing antibody-coated microbeads, pumping the microbeads into a detection area through a sample adding hole, flushing and pressurizing the microbeads by PBS after the microbeads enter the detection area, ensuring that each microtopop is provided with one microbead, adding bacteria to be detected into the detection area from an inlet, capturing bacterial liquid by the microbeads, flushing excess bacterial liquid by the PBS, sucking excess bacterial liquid from a waste liquid port 1, adding corresponding rabbit primary anti-working liquid and FITC-labeled goat anti-rabbit fluorescent secondary antibody working liquid according to the detected bacteria, flushing excess unreacted liquid by the PBS after reaction is finished, sucking excess liquid from the waste liquid port 1, observing the detection area of a chip under a fluorescent microscope, analyzing the average fluorescent color development concentration of the observed result by Image Pro software, judging the concentration of the bacteria to be detected according to a standard curve, and performing the whole step in a portable constant temperature box at 37 ℃;

Step S3: if the fluorescence reaction in the detection area is negative, the result of the detection bacteria is proved to be negative, and the drug sensitivity experiment is not carried out; if the fluorescence reaction in the detection area is positive, the drug sensitive test is continued.

9. The use of claim 8, wherein the step of susceptibility testing comprises,

Step S31: washing a liquid channel by PBS (phosphate buffer solution), pumping a medicament into a medicament sample adding hole, pumping diluent into a diluent/buffer solution sample adding hole, forming medicaments with different concentration gradients in each row of holes through a concentration gradient generator, and removing redundant liquid medicaments flowing out of a waste liquid hole after a sterile filter paper sheet in a medicament sensitive culture tank is completely soaked in the medicaments;

Step S32: and pulling out the PVDF membrane on one side of the sample adding hole of the sample to be detected, adding bacterial liquid containing pathogenic bacteria confirmed in the step S2 into the sample adding hole of the sample to be detected, removing excessive bacterial liquid flowing out of the waste liquid hole after all the drug sensitive culture tanks are filled with the bacterial liquid, placing the chip into a warm box for culture, and judging the minimum inhibitory concentration MIC according to the color depth.

10. The use according to claim 8, wherein the standard curve in step S2 is drawn by adding bacterial solutions having concentrations of 1.5 × 108cfu/ml,1.5 × 107cfu/ml,1.5 × 106cfu/ml,1.5 × 105cfu/ml,1.5 × 104cfu/ml,1.5 × 103cfu/ml,1.5 × 102cfu/ml and 1.5 × 101cfu/ml to the detection area, respectively, with 0.5MCF equivalent to 1.5 × 108cfu/ml of escherichia coli as a reference, washing excess unreacted bacterial solution after passing through the detection area, observing the solution with a fluorescence microscope, analyzing the observed result with Image Pro software to determine the average fluorescence development concentration, and drawing the standard curve.

Technical Field

The invention relates to the technical field of microbial detection, in particular to a method for quantitatively detecting bacteria and performing drug sensitivity experiments by using a microfluidic chip.

Background

The traditional gold-labeled method for identifying bacteria is a culture method, which is accurate, but time-consuming, complex to operate and expensive in price due to the use of large instruments. Dilution-based susceptibility testing can be used to quantitatively determine the in vitro activity of an antibacterial agent against a particular bacterium and is classified into agar dilution and broth dilution. In an experiment, the concentration of an antibacterial drug is usually diluted by multiple times, the lowest drug concentration capable of inhibiting the visible growth of the flesh and eyes of a bacterium to be tested becomes the Minimum Inhibitory Concentration (MIC), and the test concentration range of a specific antibacterial drug should contain the concentration of an explanatory break point (sensitive, intermediate and drug-resistant) capable of detecting the bacterium and also should contain the MIC of a quality control reference strain. Factors influencing drug sensitive results comprise a culture medium and drug concentration, wherein the culture medium is prepared according to the nutritional requirements of test bacteria, the concentration and the total amount of drugs directly influence the results of bacteriostatic tests, the drugs are required to be accurately prepared, and the commercial drugs are required to be prepared according to the recommended treatment amount strictly.

The microfluidic method is a new technology in recent years, and is applied to bacteria detection and drug sensitivity experiments, so that the detection time can be greatly shortened, the material consumption is reduced, and the detection equipment is small and convenient. The parallel flow type concentration gradient generator is common, and utilizes the laminar flow principle to transfer mass between parallel adjacent liquid flows through diffusion action so as to form a concentration gradient perpendicular to the liquid flow direction. The features (e.g., shape, concentration, spacing, etc.) that form the gradient are related only to the species, flow rate, and contact time. Common parallel flow type concentration gradient generators have a T configuration, a Christmas tree configuration, a universal type and the like. The Christmas tree structure manufactured by the same principle has a complex structure, initial liquid flows are divided and converged for many times, the formed resultant gradient is finer and discontinuous, and the gradient forming device and the reaction analysis are separated, so that the analysis area is enlarged. However, the current microfluidic chip structure can only realize the drug sensitivity experiment of bacteria alone, and cannot realize the detection of pathogenic bacteria and the drug sensitivity experiment of the pathogenic bacteria at the same time.

disclosure of Invention

In order to solve the technical problems, the invention provides a method for quantitatively detecting bacteria by combining a micro-fluidic chip with micro-beads.

In a first aspect, the present invention provides a microfluidic chip, which includes a top layer, a middle layer and a bottom layer, wherein the middle layer is a PDMS structure, the bottom layer is a glass slide, and the middle layer includes four regions: the device comprises an inlet area, a detection area, a concentration gradient generator and a waste liquid hole, wherein the inlet area is positioned between the detection area and the concentration gradient generator, the concentration gradient generator is of a Christmas tree type and comprises a drug sensitive culture pool, wherein n is equal to 1, 2, 3 and 4 … …;

Optionally, the inlet area comprises sample adding holes for samples to be tested, diluent/buffer solution adding holes and drug adding holes, and the bottom layer of the inlet area is 2-4 μm higher than the bottom layers of other areas, so that the samples can be added quickly and conveniently under the action of gravity.

optionally, a detachable PVDF film with a thickness of 90-110 μm is disposed on one side of the concentration gradient generator of the sample addition hole to be tested, so that the bacteria liquid is prevented from flowing into the concentration gradient generator during bacteria liquid detection, and can be pulled out to form a bacteria liquid flow channel during drug sensitive test and flow into the concentration gradient generator.

Optionally, the diameter of the drug sensitive culture tank is 2-5mm, and the diameter of the waste liquid hole is 0.5-1 mm.

Optionally, the detection zone comprises a microtorr and a microbead; the micro support is V-shaped or semicircular, the diameter of the micro bead is 60-90 μm, and the micro support is slightly wider than the micro bead to support the micro bead.

Optionally, the waste liquid hole includes detection zone waste liquid hole (1) and concentration gradient generator waste liquid hole (9), waste liquid hole (1) is linked together with the detection zone through liquid channel, waste liquid hole (9) pass through liquid channel with drug sensitive culture pond low reaches are linked together.

Optionally, a curved channel (10) is arranged between the inlet region and the concentration gradient generator and between each drug sensitive culture tank, the width of the channel between the detection region and the waste liquid hole (1) is 40 μm, and the width of all other channels is 100 μm.

In a second aspect, the present invention provides a method for preparing the microfluidic chip, including: sealing the middle layer of the microfluidic chip with the bottom layer of the microfluidic chip, respectively placing sterile filter paper sheets in the drug sensitive culture tank, carrying out plasma treatment, and sealing the middle layer of the microfluidic chip with the top layer of the microfluidic chip.

Optionally, the layers are baked for 2h at 90 ℃ before the chip is sealed, and irradiated overnight by an ultraviolet lamp.

In a third aspect, the invention also provides an application of the microfluidic chip in pathogen identification and drug sensitivity experiments, which comprises the following steps:

Step S1: sterilizing each layer of the chip under high pressure, sealing the bottom layer and the middle layer, adding a culture medium into a drug sensitive culture tank, adding a sterile filter paper sheet into the drug sensitive culture tank, sealing the upper layer, storing the sealed whole chip in a refrigerator at 4 ℃, and irradiating the surface of the chip for 30min by using an ultraviolet lamp before use;

step S2: preparing antibody-coated microbeads, pumping the microbeads into a detection area through a sample adding hole, flushing and pressurizing the microbeads by PBS after the microbeads enter the detection area, ensuring that each microtopop is provided with one microbead, adding bacteria to be detected into the detection area from an inlet, capturing bacteria liquid by the microbeads, flushing excess bacteria liquid by the PBS, sucking excess bacteria liquid from a waste liquid port 1, adding corresponding rabbit primary anti-working liquid and FITC-labeled goat anti-rabbit fluorescent secondary antibody working liquid according to the bacteria to be detected, flushing excess unreacted liquid by the PBS after reaction is completed, sucking excess liquid from the waste liquid port 1, observing the detection area of a chip under a fluorescent microscope, analyzing the average fluorescent color development concentration of the observation result by Image Pro software, judging the concentration of the bacteria to be detected according to a standard curve, putting the chip into a portable incubator for culturing in the whole process, wherein the size of the portable incubator is 50 multiplied by 30 cm.

Step S3: if the fluorescence reaction in the detection area is negative, the result of the detection bacteria is proved to be negative, and the drug sensitivity experiment is not carried out; if the fluorescence reaction in the detection area is positive, the drug sensitive test is continued.

Further, the drug sensitivity test in step S3 includes,

step S31: washing a liquid channel by PBS (phosphate buffer solution), pumping a medicament into a medicament sample adding hole, pumping diluent into a diluent/buffer solution sample adding hole, forming medicaments with different concentration gradients in each row of holes through a concentration gradient generator, and removing redundant liquid medicaments flowing out of a waste liquid hole after sterile filter paper in a medicament sensitive culture tank completely infiltrates the medicaments;

Step S32: pulling out the PVDF membrane on one side of the sample adding hole of the sample to be detected, adding bacterial liquid containing pathogenic bacteria confirmed in the step S2 into the sample adding hole of the sample to be detected, removing excessive bacterial liquid flowing out of the waste liquid hole after all the drug sensitive culture tanks are filled with the bacterial liquid, placing the chip into a portable incubator for culture, wherein the size of the portable incubator is 50 multiplied by 30cm, and judging the minimum inhibitory concentration MIC according to the color depth.

Further, the standard curve in step S2 is drawn by adding bacterial liquids having concentrations of 1.5X 108cfu/ml, 1.5X 107cfu/ml, 1.5X 106cfu/ml, 1.5X 105cfu/ml, 1.5X 104cfu/ml, 1.5X 103cfu/ml, 1.5X 102cfu/ml and 1.5X 101cfu/ml to a detection area, respectively, with reference to Escherichia coli having a concentration of 0.5MCF corresponding to 1.5X 108cfu/ml, washing excess unreacted bacterial liquids after passing through the detection area, observing the cells with a fluorescence microscope, and analyzing the observation result with Image Pro software to determine the average fluorescence developing concentration.

further, in step S2, the method for preparing antibody-coated microbeads includes the steps of,

Treating the microbeads: accurately weighing 10mg of glass microspheres, soaking the glass microspheres in Piranha solution overnight, washing the glass microspheres with sterile ultrapure water for 5 times, drying the glass microspheres at 70 ℃ for 30min, adding 2% of APTES acetone solution for reaction for 1min, and sequentially washing the glass microspheres with acetone and sterile ultrapure water for 5 times;

Preparing an antibody working solution: mu.L of MES buffer, 8. mu.L of 4 mg. mL-1EDC (about 2 mmol. mL-1) and 12. mu.L of 4 mg. mL-1NHS (about 2 mmol. mL-1) were added to 10. mu.L of 1 mg. mL-1 antibody working solution, and after reaction at room temperature for 15min, 120. mu.L of 0.1 mol. L-1PBS buffer was added to terminate the NHS-antibody activated ester formation reaction, and thus an antibody reaction solution having an antibody concentration of 50. mu.g. mL-1 was obtained.

antibody coating with beads: adding the treated glass beads into the antibody reaction solution, and reacting for 2 hours at room temperature. After the reaction is finished, washing the mixture for 3 times by using 0.1 mol.L-1 PBS to obtain the immune microbeads modified by the antibody, and placing the immune microbeads at 4 ℃ for later use.

Compared with the prior art, the invention has the beneficial effects that:

1. The micro-fluidic chip can realize the simultaneous execution of the quantitative detection of bacteria and the drug sensitive experiment, and meanwhile, MIC can be observed in any column in the concentration gradient generator, so that the micro-fluidic chip is more convenient and faster compared with the traditional method or other micro-fluidic drug sensitive experiment methods.

2. The detection area of the micro-fluidic chip provided by the invention adopts an antigen-antibody reaction method to realize rapid and accurate detection of bacteria, has high sensitivity and high specificity, is simple and convenient in detection steps, and is driven by a plasma pump to avoid manual operation. The dosage of the drug sensitive reagent is small, and the MIC is determined by the optical density collector according to the change of the color depth, so that the drug sensitive reagent is quick and effective.

3. The paper-based technology is used for the drug sensitivity experiment in the microfluidic chip, and compared with the drug sensitivity experiment of the traditional concentration gradient generator, the paper-based technology not only shortens the detection time, improves the detection sensitivity, simplifies the operation steps, but also realizes automatic control and is more beneficial to observing the experiment result.

Drawings

FIG. 1 is a schematic diagram of the overall structure of a microfluidic chip according to the present invention

Wherein, 1-waste liquid hole 1, 2-detection area, 3-micro bead, 4-micro support, 5-drug sample adding hole, 6-sample adding hole to be tested, 7-diluent/buffer solution sample adding hole, 8-concentration gradient generator, 9-waste liquid hole 2, 10-curve channel.

FIG. 2 is a schematic view of a portable incubator used in the assay of the present invention.

FIG. 3 is a standard curve diagram of the detection zone of the microfluidic chip of the present invention.

FIG. 4 is the concentration ratio of the final concentration in the drug sensitive culture tank of the concentration gradient generator of the present invention.

FIG. 5 shows the color development intensity of different concentration gradients in the drug sensitivity test of the present invention.

Detailed Description

Hereinafter, embodiments of the present invention will be described in detail. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby. It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.