Digital droplet micro-fluidic chip system and bacterium detection method

Abstract

The invention provides a digital liquid drop micro-fluidic chip system and a bacteria detection method, and relates to the technical field of microbiological detection, the digital liquid drop micro-fluidic chip system comprises a micro-fluidic liquid drop generation chip, a micro-fluidic liquid drop fusion chip and a micro-fluidic liquid drop reaction chip. The bacterium detection method comprises the step of carrying out bacterium detection on a sample by using the digital droplet micro-fluidic chip system. The digital droplet micro-fluidic chip system and the bacterium detection method have the advantages of high detection flux, high stability, high sensitivity, rapidness, low cost and the like, are suitable for simultaneous detection of various bacteria in unknown samples, and can be used in the fields of environmental microorganism detection, food-borne pathogenic bacterium detection, clinical pathogenic bacterium diagnosis and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganism detection, and in particular to a digital droplet microfluidic chip system and a bacteria detection method. Background Art

[0002] Pathogenic bacteria usually exist in food and the environment. After infection, they cause various diseases in the human body, which seriously threatens human health. Traditional methods based on bacterial culture and color development take a long time to detect, and cannot detect living but uncultivable bacteria, which easily produces false negative results. The currently used constant temperature nucleic acid amplification technology such as recombinase polymerase constant temperature amplification (RPA) avoids the temperature control problem of frequent temperature changes in PCR reactions, and can react at close to room temperature, but there are still problems such as low detection sensitivity, high reagent consumption, high detection cost, and low detection throughput. Digital nucleic acid amplification detection methods such as digital RPA have the advantage of low sample consumption. Although it improves the detection sensitivity, there are still some problems: the ability to detect multiple targets at the same time is low, and repeated operation procedures are required to distinguish different bacteria.

[0003] Microfluidic chip technology is called a laboratory on a chip. It has the advantages of high integration, small size, small reagent usage, low cost, high throughput, simple operation, and flexible design. It provides a new technology for high-sensitivity, high-throughput, and low-cost detection of bacteria. At present, droplet microfluidic chips can realize all aspects of digital bacterial detection, but droplet microfluidic chips for multi-target detection have low integration, low droplet encoding ability, the generated droplets require additional auxiliary structures for droplet mixing and other operations, and droplet observation requires image stitching, which is not conducive to the stable detection and analysis of multiple target bacteria. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the deficiencies of the prior art, the present invention provides a digital droplet microfluidic chip system and a bacteria detection method, which solves the technical problem that the prior microfluidic chip is not conducive to multi-target bacteria detection and analysis.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] A digital droplet microfluidic chip system comprises a microfluidic droplet generation chip, a microfluidic droplet fusion chip and a microfluidic droplet reaction chip connected by a PTFE tube. The microfluidic droplet generation chip, the microfluidic droplet fusion chip and the microfluidic droplet reaction chip can be organically combined in sequence and quantity.

[0009] Preferably, the microfluidic droplet generation chip comprises n (n>1) droplet generation units, and the n droplet generation units share one coded droplet collection port;

[0010] The droplet generation unit includes a dispersed phase 1 inlet, a dispersed phase 2 inlet, a dispersed phase mixing channel, and a droplet generation area;

[0011] The dispersed phase 1 inlet is fed with a droplet encoding reagent solution, and the dispersed phase 2 inlet is fed with a detection reaction reagent solution;

[0012] The dispersed phase mixing channel is a serpentine structure, a zigzag structure or a spiral structure;

[0013] The droplet generation area includes a cross intersection, which is designed as a closing structure. The closing structure is trapezoidal, and the length of the upper base is smaller than the length of the lower base, so that a large shear force is generated to quickly generate droplets.

[0014] Preferably, the microfluidic droplet generating chip further comprises a first inlet of continuous phase 1, a second inlet of continuous phase 1, a third inlet of continuous phase 1 and a fourth inlet of continuous phase 1, and the continuous phase 1 is injected from the first inlet of continuous phase 1 and transported to the second inlet of continuous phase 1, the third inlet of continuous phase 1 and the fourth inlet of continuous phase 1 respectively until it converges with the mixed dispersed phase in the dispersed phase mixing channel in the droplet generating area.

[0015] Preferably, the continuous phase 1 is mineral oil or fluorinated oil.

[0016] Preferably, the droplet coding reagent solution is a water-soluble dye reagent of different colors and intensities, a fluorescent reagent or a fluorescent nanomaterial solution of different fluorescent colors and intensities, or an orthogonal combination of a water-soluble dye reagent and a fluorescent reagent or a fluorescent nanomaterial solution, corresponding to different droplet encoding-decoding methods.

[0017] Preferably, the droplet encoding-decoding method includes performing RGB mean filtering on the bright field image, performing Hough circle detection on the filtered image, performing color K-Means clustering on the detected circles (droplet areas) based on RGB values, and corresponding to the fluorescence image.

[0018] Preferably, the microfluidic droplet fusion chip comprises a sample droplet generation unit, a coding droplet injection unit, a droplet fusion unit and a fusion droplet outlet.

[0019] Preferably, the sample droplet generating unit comprises a continuous phase 2 inlet, a dispersed phase 3 inlet, a sample droplet generating area, a continuous phase 2 channel, a dispersed phase 3 channel and a sample droplet channel;

[0020] The continuous phase 2 inlet is connected to the continuous phase 2 channel;

[0021] The dispersed phase 3 inlet is connected to the dispersed phase 3 channel;

[0022] The continuous phase 2 channel and the dispersed phase 3 channel converge in the sample droplet generation area;

[0023] The continuous phase 2 is mineral oil or fluorinated oil, and the dispersed phase 3 is a solution of a bacterial sample to be tested;

[0024] The sample droplet channel is connected between the sample droplet generation area and the encoding droplet injection unit, and the structure of the sample droplet generation area is the same as that of the droplet generation area.

[0025] Preferably, the coded droplet injection unit comprises a coded droplet library droplet buffer area, a coded droplet library droplet channel connected to the coded droplet library droplet buffer area, a continuous phase 3 inlet and a continuous phase 3 channel connected to the continuous phase 3 inlet.

[0026] Preferably, the continuous phase 3 is mineral oil or fluorinated oil.

[0027] Preferably, the droplet fusion unit comprises a droplet fusion port and a positive electrode and a negative electrode connected to the droplet fusion port.

[0028] Preferably, the channel width of the droplet fusion port is greater than the flow channel width for transporting the droplets to the droplet fusion port, so as to increase the electrode action area.

[0029] Preferably, the material of the positive electrode and the negative electrode is liquid metal, NaCl electrolyte solution or commercial ITO electrode; the distance between the positive electrode and the negative electrode and the droplet fusion port is preferably 50 μm.

[0030] Preferably, a first filtering structure is disposed behind the dispersed phase 1 inlet, the dispersed phase 2 inlet, the continuous phase 2 inlet, and the dispersed phase 3 inlet to remove impurities and ensure a stable flow rate.

[0031] Preferably, the microfluidic droplet reaction chip comprises a fusion droplet inlet, a fusion droplet reaction unit, a reaction unit connecting channel, a counting code and a fusion droplet outlet;

[0032] The fusion droplet inlet is connected to the fusion droplet reaction unit through a pipeline;

[0033] The fusion droplet reaction unit is connected to the fusion droplet outlet through the reaction unit connecting channel.

[0034] Preferably, the fused droplet reaction unit comprises m (m>1) reaction chambers and connecting channels, and the m reaction chambers are connected in series by the connecting channel.

[0035] The reaction chamber is in the shape of a "petal", and its size is smaller than the field of view of a 4X objective lens of a microscope, which facilitates the observation of droplets in an independent single chamber, and does not require image stitching processing, thereby ensuring accurate counting, and can achieve the reaction and counting of 2,000 to 20,000 droplets;

[0036] The width of the connecting channel is consistent with the size of the droplet flow channel.

[0037] A method for detecting bacteria, using any of the digital droplet microfluidic chip systems described above to detect bacteria on a sample, comprising:

[0038] The number of droplet generating units in the microfluidic droplet generating chip, i.e., the specific value of n, is determined according to the number of species of bacteria to be detected, and then the coding reagent and the reaction reagent are respectively added to the dispersed phase 1 inlet and the dispersed phase 2 inlet of the droplet generating unit;

[0039] Adding the sample solution to be tested into the dispersed phase 3 inlet of the sample droplet generating unit;

[0040] The generation of the coded droplet library, the generation of the sample droplet library, the fusion of the coded droplet and the sample droplet, the reaction of the fused droplet and the signal detection are controlled in sequence to obtain the detection result.

[0041] Preferably, the method specifically comprises the following steps:

[0042] Step 1: n encoding reagents and n detection reagents for target bacteria are introduced into the dispersed phase 1 inlet and dispersed phase 2 inlet of n repeated droplet generating units on the microfluidic droplet generating chip, respectively, and fluorinated oil is introduced into the first inlet of the continuous phase 1, and a coded droplet library is prepared under a certain flow rate ratio of the continuous phase and the mobile phase;

[0043] Step 2: introducing the sample solution to be tested into the dispersed phase 3 inlet on the microfluidic droplet fusion chip, and introducing the fluorinated oil into the continuous phase 2 inlet to prepare sample droplets. While preparing the sample droplets, injecting the coded droplet library droplets into the coded droplet library droplet buffer area;

[0044] Step 3: Adjust the flow rates of dispersed phase 3 and continuous phase 2 to ensure the order of sample droplet-coded droplet library droplet-sample droplet-coded droplet library droplet; turn on the high-voltage power supply and adjust the voltage so that the sample droplet and the coded droplet library droplet fuse into a large droplet in the droplet fusion unit;

[0045] Step 4: The fused droplets are directly introduced into the microfluidic droplet reaction chip to perform detection reaction and release detection signals;

[0046] Step 5: Place the microfluidic droplet reaction chip under a microscope and take photos in bright field and fluorescence field to obtain droplet coding images and signal images;

[0047] Step 6: Perform droplet decoding and positive droplet counting, calculate the sample concentration according to the Poisson distribution formula, and make a fitting curve between it and the theoretical value, perform absolute quantitative analysis of bacteria, and obtain the detection limit.

[0048] (III) Beneficial effects

[0049] The present invention provides a digital droplet microfluidic chip system and a bacteria detection method. Compared with the prior art, it has the following beneficial effects:

[0050] 1. The digital droplet microfluidic chip system of the present invention realizes the preparation of a uniformly mixed coded droplet library by organically combining the order and quantity of the microfluidic droplet generation chip, the microfluidic droplet fusion chip and the microfluidic droplet reaction chip in the system, directly performs droplet fusion with the sample droplet and then reacts with the droplet to release the detection signal; at the same time, the droplet coding realizes the specific marking of a variety of different targets, ensuring that the detection results of a variety of targets are obtained in one detection, and there is no need to perform image stitching when judging positive droplets, and only needs to collect images of the droplets in each chamber; the chip system has a certain flexibility, and the same chip can prepare droplet libraries of different sizes, and the droplet merging ratio can also be adjusted, which expands its application range.

[0051] 2. The bacterial detection method of the present invention can realize the labeling, detection reaction and positive droplet judgment of multiple target bacteria at the same time. By encapsulating different reagents in the continuous phase, the coded droplet library and the sample droplet library can be prepared, thereby realizing the digital detection of multiple targets.

[0052] 3. The digital droplet microfluidic chip system and bacteria detection method of the present invention have the advantages of high detection throughput, high stability, high sensitivity, rapidity and low cost. They are suitable for the simultaneous detection of multiple bacteria in unknown samples and can be used in the fields of environmental microbial detection, foodborne pathogenic bacteria detection, clinical pathogenic bacteria diagnosis and so on. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0054] Figure 1 is a schematic structural diagram of the digital droplet microfluidic chip system in Example 1 of the present invention;

[0055] Figure 2 is a schematic structural diagram of a microfluidic droplet generation chip in Example 1 of the present invention;

[0056] Figure 3 is a schematic structural diagram of the microfluidic droplet fusion chip in Example 1 of the present invention;

[0057] Figure 4 is a schematic structural diagram of the microfluidic droplet reaction chip in Example 1 of the present invention;

[0058] Figure 5 is a schematic structural diagram of a droplet generating region in Example 1 of the present invention;

[0059] Figure 6 is a schematic diagram of the droplet encoding-decoding process in Example 1 of the present invention;

[0060] Figure 7 is a flow chart of the bacteria detection method in Example 4 of the present invention;

[0061] Figure 8 This is a schematic diagram of the bacteria detection principle in Example 4 of the present invention.

[0062] Among them: 1. Microfluidic droplet generation chip;

[0063] 101, droplet generation unit; 102, coded droplet collection port; 103, first inlet of continuous phase 1; 104, second inlet of continuous phase 1; 105, third inlet of continuous phase 1; 106, fourth inlet of continuous phase 1; 107, dispersed phase channel; 108, continuous phase 1 channel; 109, coded library droplet channel;

[0064] 1011, dispersed phase 1 inlet; 1012, dispersed phase 2 inlet; 1013, first filtering structure; 1014, dispersed phase mixing channel; 1015, droplet generation area;

[0065] 2. Microfluidic droplet fusion chip;

[0066] 201. Sample droplet generating unit;

[0067] 2011, continuous phase 2 inlet; 2012, dispersed phase 3 inlet; 2013, sample droplet generation area; 2014, second filtering structure; 2015, continuous phase 2 channel; 2016, dispersed phase 3 channel; 2017, sample droplet channel;

[0068] 202, a coding droplet injection unit;

[0069] 2021, coded droplet library droplet buffer area; 2022, continuous phase 3 inlet; 2023, coded droplet library droplet channel; 2024, continuous phase 3 channel;

[0070] 203. Droplet fusion unit;

[0071] 2031, droplet fusion port; 2032, positive electrode; 2033, negative electrode;

[0072] 204, fusion droplet outlet;

[0073] 3. Microfluidic droplet reaction chip;

[0074] 301, fusion droplet inlet; 302, fusion droplet outlet; 303, fusion droplet reaction unit; 304, reaction unit connecting channel; 305, counting code;

[0075] 3031, reaction chamber; 3032, connecting channel;

[0076] 4. PTFE connecting tube. DETAILED DESCRIPTION

[0077] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0078] The embodiments of the present invention solve the technical problem that the existing microfluidic chips are not conducive to multi-target bacteria detection and analysis by providing a digital droplet microfluidic chip system and a bacteria detection method.

[0079] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0080] The continuous phase 1, continuous phase 2 and continuous phase 3 described in the present invention are mineral oil or fluorinated oil. The continuous phase 1, continuous phase 2 and continuous phase 3 may be completely the same or may not be completely the same.

[0081] The dispersed phase 1 of the present invention is a droplet encoding reagent solution, the dispersed phase 2 is a detection reaction reagent solution, and the dispersed phase 3 is a sample solution to be tested.

[0082] Furthermore, the droplet coding reagent solution is a water-soluble dye reagent of different colors and intensities, a fluorescent reagent or a fluorescent nanomaterial solution of different fluorescent colors and intensities, or an orthogonal combination of a water-soluble dye reagent and a fluorescent reagent / fluorescent nanomaterial solution, corresponding to different droplet encoding-decoding methods.

[0083] Furthermore, the droplet encoding-decoding method includes performing RGB mean filtering on the bright field image, performing Hough circle detection on the filtered image, performing color K-Means clustering on the detected circles (droplet areas) based on RGB values, and corresponding to the fluorescence image.

[0084] Furthermore, the droplet encoding-decoding method is specifically as follows:

[0085] First, the bright field droplet image is subjected to RGB mean filtering;

[0086] Perform Hough circle detection on the filtered image;

[0087] Perform K-Means clustering on the detected circles (droplet areas) based on color;

[0088] Different clusters are marked and split to obtain multiple single-type droplet area mask images;

[0089] Use the single-type droplet mask image and droplet fluorescence image obtained in the previous step to obtain the fluorescence image corresponding to the single-type droplet;

[0090] Complete the droplet encoding-decoding process.

[0091] The material of the positive electrode and the negative electrode of the present invention is liquid metal, NaCl electrolyte solution or commercial ITO electrode.

[0092] Example 1 - Digital droplet microfluidic chip system

[0093] like Figure 1 As shown, the digital droplet microfluidic chip system includes a microfluidic droplet generation chip 1, a microfluidic droplet fusion chip 2 and a microfluidic droplet reaction chip 3, and the chips are connected by PTFE tubes.

[0094] Combination Figure 2 As shown, the microfluidic droplet generating chip 1 includes a droplet generating unit 101, a coded droplet collecting port 102, a first inlet 103 of continuous phase 1, a second inlet 104 of continuous phase 1, a third inlet 105 of continuous phase 1, a fourth inlet 106 of continuous phase 1, a dispersed phase channel 107, a continuous phase 1 channel 108 and a coded library droplet channel 109.

[0095] There are 8 droplet generating units 101 in total, which are arranged in a circle to form a ring structure, and the 8 droplet generating units 101 can be used to simultaneously prepare 8 different types of coding library droplets. The 8 droplet generating units 101 share a coding droplet collecting port 102, and the coding droplet collecting port 102 is located at the center of the 8 droplet generating units 101, and the two are connected through a coding library droplet channel 109.

[0096] The droplet generating unit 101 includes a dispersed phase 1 inlet 1011 , a dispersed phase 2 inlet 1012 , a first filtering structure 1013 , a dispersed phase mixing channel 1014 and a droplet generating area 1015 .

[0097] The dispersed phase 1 inlet 1011 is introduced into the droplet coding reagent solution, and the dispersed phase 2 inlet 1012 is introduced into the detection reaction reagent solution; the dispersed phase 1 inlet 1011 and the dispersed phase 2 inlet 1012 are connected through a dispersed phase mixing channel 1014, and the dispersed phase mixing channel 1014 is a serpentine structure, a zigzag structure or a spiral structure, and its structure is determined according to the actual situation of the sample to be detected.

[0098] The dispersed phase channel 107 is connected to one end of the dispersed phase mixing channel 1014 away from the dispersed phase 1 inlet 1011 and the dispersed phase 2 inlet 1012 .

[0099] The first filtering structure 1013 is disposed behind the dispersed phase 1 inlet 1011 and the dispersed phase 2 inlet 1012 to filter impurities in the solution to ensure smooth flow of the solution.

[0100] The dispersed phase 1 inlet 1011, the dispersed phase 2 inlet 1012 and the dispersed phase mixing channel 1014 are covered by the continuous phase 1 channel 108. The continuous phase 1 channel 108 in this area has a regular hexagonal structure. The continuous phase 1 channel 108 and the dispersed phase channel 107 intersect in the droplet generation area 1015. Due to the shear force, the continuous phase 1 and the mixed phase of the dispersed phase 1 and the dispersed phase 2 generate encoding library droplets in the droplet generation area 1015.

[0101] Combination Figure 5 As shown, the droplet generation area 1015 includes a cross intersection, and the cross intersection is a closing structure. The closing structure is trapezoidal, with a lower base of 40 μm, an upper base of 80 μm, and a height of 40 μm. The design of the closing structure is more conducive to the formation of encoding library droplets.

[0102] There are eight second inlets 104 of continuous phase 1, which are respectively located on each droplet generating unit 101, and can be used to prepare 8 different types of encoding library droplets at the same time. The third inlet 105 of continuous phase 1 is arranged at one end of the second inlet 104 of continuous phase 1 away from the droplet generating unit 101. There are four third inlets 105 of continuous phase 1, and every two adjacent second inlets 104 of continuous phase 1 share one third inlet 105 of continuous phase 1. The fourth inlet 106 of continuous phase 1 is arranged at one end of the third inlet 105 of continuous phase 1 away from the second inlet 104 of continuous phase 1. There are two fourth inlets 106 of continuous phase 1, and every two adjacent third inlets 105 of continuous phase 1 share one fourth inlet 106 of continuous phase 1. The continuous phase 1 is injected from the first inlet 103 of continuous phase 1, and is respectively transported to the second inlet 104 of continuous phase 1, the third inlet 105 of continuous phase 1, and the fourth inlet 106 of continuous phase 1, until it is collected in the droplet generating area 1015 with the mixed dispersed phase in the dispersed phase mixing channel 1014.

[0103] A first filtering structure 1013 is also disposed behind the first inlet 103 of the continuous phase 1 to filter impurities in the continuous phase 1 to ensure smooth liquid flow.

[0104] Combination Figure 3 As shown, the microfluidic droplet fusion chip 2 includes a sample droplet generation unit 201 , a coded droplet injection unit 202 , a droplet fusion unit 203 and a fusion droplet outlet 204 .

[0105] The sample droplet generating unit 201 includes a continuous phase 2 inlet 2011 , a dispersed phase 3 inlet 2012 , a sample droplet generating region 2013 , a second filtering structure 2014 , a continuous phase 2 channel 2015 , a dispersed phase 3 channel 2016 and a sample droplet channel 2017 .

[0106] The continuous phase 2 inlet 2011 is connected to the continuous phase 2 channel 2015 , the continuous phase 2 channel 2015 surrounds the dispersed phase 3 inlet 2012 , and the dispersed phase 3 inlet 2012 is connected to the dispersed phase 3 channel 2016 .

[0107] The continuous phase 2 channel 2015 and the dispersed phase 3 channel 2016 converge in the sample droplet generation area 2013. The sample droplet channel 2017 is used to connect the sample droplet generation area 2013 with the coded droplet injection unit 202. After the sample droplets are generated, they are transmitted to the coded droplet injection unit 202 through the sample droplet channel 2017. The structure of the sample droplet generation area 2013 is the same as that of the droplet generation area 1015.

[0108] The second filtering structure 2014 is a four-row circular array, the first row is composed of three circular microcolumns with a diameter of 500 μm arranged evenly, the second row is composed of six circular microcolumns with a diameter of 300 μm arranged evenly, the third row is composed of eight circular microcolumns with a diameter of 300 μm arranged evenly, and the fourth row is composed of twelve circular microcolumns with a diameter of 200 μm arranged evenly.

[0109] The coded droplet injection unit 202 includes a coded droplet library droplet buffer area 2021 , a coded droplet library droplet channel 2023 connected to the coded droplet library droplet buffer area 2021 , a continuous phase 3 inlet 2022 , and a continuous phase 3 channel 2024 connected to the continuous phase 3 inlet 2022 .

[0110] The coded droplet library droplet buffer area 2021 is in the shape of a "petal" and is used for temporary storage of droplets.

[0111] The droplet fusion unit 203 includes a droplet fusion port 2031 and a positive electrode 2032 and a negative electrode 2033 connected to the droplet fusion port 2031 .

[0112] The droplet fusion port 2031 is a square structure with a side length of 200 μm. Its channel width is greater than the flow channel width for transporting droplets to the droplet fusion port 2031, so as to increase the electrode action area.

[0113] The positive electrode 2032 and the negative electrode 2033 are respectively disposed on both sides of the droplet fusion opening 2031 and are 50 μm away from the droplet fusion opening 2031 to ensure a uniform electric field.

[0114] Combination Figure 4 As shown, the microfluidic droplet reaction chip 3 includes a fusion droplet inlet 301 , a fusion droplet outlet 302 , a fusion droplet reaction unit 303 , a reaction unit connecting channel 304 and a counting code 305 .

[0115] The fused droplet inlet 301 is connected to the fused droplet reaction unit 303 through a channel, and the fused droplet reaction unit 303 is connected to the fused droplet outlet 302 through a reaction unit connecting channel 304 .

[0116] The fused droplet reaction unit 303 includes reaction chambers 3031 and connecting channels 3032 . There are eight reaction chambers 3031 , and the eight reaction chambers 3031 are connected in series one by one through the connecting channels 3032 .

[0117] The reaction chamber 3031 is in the shape of a "petal", and a single structure is a rounded diamond with diagonal lengths of 1.9 mm and 1.5 mm respectively. The size is smaller than the field of view of the 4X objective lens of the microscope, which is convenient for observing droplets in a single chamber. There is no need for image stitching processing to ensure accurate counting, and the reaction and counting of 2000-20000 droplets can be achieved.

[0118] The width of the connecting channel 3032 is 100 μm, which is consistent with the size of the reaction unit connecting channel 304 .

[0119] Example 2 - Combination of digital droplet microfluidic chip system

[0120] Microfluidic droplet generation chips, microfluidic droplet fusion chips, and microfluidic droplet reaction chips can be organically combined in sequence and quantity according to the types and quantities of bacteria that need to be detected.

[0121] Combination 1:

[0122] The digital droplet microfluidic chip system includes a microfluidic droplet generation chip, a microfluidic droplet fusion chip and a microfluidic droplet reaction chip which are sequentially connected through PTFE tubes.

[0123] Combination 2:

[0124] The digital droplet microfluidic chip system includes a microfluidic droplet generation chip, n (n>1) microfluidic droplet fusion chips and a microfluidic droplet reaction chip which are sequentially connected through PTFE tubes.

[0125] Combination 3:

[0126] The digital droplet microfluidic chip system includes n (n>1) microfluidic droplet fusion chips and one microfluidic droplet reaction chip which are sequentially connected through PTFE tubes.

[0127] The above-mentioned combinations are only three of the various combinations of the digital droplet microfluidic chip system of the present invention, and can be adjusted according to actual conditions.

[0128] Example 3 - Preparation method of digital droplet microfluidic chip system

[0129] Step 1. The master plates of the microfluidic droplet generation chip, the microfluidic droplet fusion chip, and the microfluidic droplet reaction chip are all made of a 75 mm diameter silicon wafer using a standard photolithography method; the manufacturing materials of each microfluidic chip are polydimethylsiloxane and an initiator, and the weight ratio of the two is 10:1.

[0130] Step 2: Punch holes at each inlet and outlet of the microfluidic droplet generation chip and bond them to a glass sheet (43 mm × 43 mm); punch holes at each inlet and outlet of the microfluidic droplet fusion chip and the microfluidic droplet reaction chip and bond them to a glass slide (75 mm × 25 mm);

[0131] Step 3: The bonding process is to plasma oxidize each chip pattern side upward with the glass sheet / slide for 1 minute (related parameters: 100 mW, 1% O2), and then quickly bond the chip to the corresponding glass sheet / slide, put it in an oven, and place it at 75°C for 20 minutes;

[0132] Step 4: After bonding, the microfluidic droplet fusion chip is taken out and placed on a heating plate at 90°C; at the same time, the liquid metal is melted, and the liquid metal is sucked into the positive electrode and the negative electrode using a syringe; a copper wire with a diameter of 1 mm is inserted at the outlet of the two electrode channels, and UV-curable glue is applied to the interface, and cured under UV light, and finally the microfluidic droplet fusion chip is transferred to room temperature for cooling.

[0133] Example 4 - Bacteria Detection Method

[0134] like Figure 6 , Figure 7 , Figure 8 As shown:

[0135] Step 1: Eight coding reagents and eight detection reagents for target bacteria were introduced into the dispersed phase 1 inlet and dispersed phase 2 inlet of eight repeated droplet generating units on the microfluidic droplet generating chip, respectively. HFE-7500 fluorinated oil containing >0.2% surfactant was introduced into the first inlet of the continuous phase 1. The flow rate ratios of dispersed phase 1, dispersed phase 2 and continuous phase 1 were all 1:5, and a coded droplet library with a diameter of 80 μm was prepared.

[0136] The coding reagent solution is a solution of 8 different colors obtained by mixing 50 mg / mL of amaranth, Prussian blue, and fluorescent yellow in different proportions;

[0137] The detection reagents are template-free RPA reaction reagents and CRISPR / Cas13a reagents designed for the target gene structure of the target bacteria.

[0138] Step 2: The sample solution to be tested is introduced into the dispersed phase 3 inlet on the microfluidic droplet fusion chip, and the HFE-7500 fluorinated oil containing >0.2% surfactant is introduced into the continuous phase 2 inlet. The flow rate ratio of the dispersed phase 3 and the continuous phase 2 is 1:5, and a sample droplet library with a diameter of 80 μm is prepared; while the sample droplets are being prepared, the coded droplet library droplets are injected into the coded droplet library droplet buffer area through the PTFE tube.

[0139] Step 3: Adjust the flow rates of dispersed phase 3 and continuous phase 2 to ensure the arrangement order of sample droplet-coded droplet library droplet-sample droplet-coded droplet library droplet; turn on the high-voltage power supply and adjust the voltage (DC, >600V, 10kHz) to make one sample droplet and one coding droplet merge into one fused droplet.

[0140] Step 4: The fused droplets are directly introduced into the fused droplet inlet on the microfluidic droplet reaction chip to fill the reaction chambers of all droplet reaction units; polyacrylamide microcolumns are used to block the fused droplet inlet and fused droplet outlet; the microfluidic droplet reaction chip is placed at 39°C-43°C for 30 minutes for detection reaction and signal release.

[0141] Step 5: Place the microfluidic droplet reaction chip under a microscope and take photos in the bright field and fluorescence field to obtain droplet coding images and signal images; the shooting sequence is performed one by one according to the counting code.

[0142] Step 6: Perform droplet decoding and positive droplet counting. Use Image J software to extract RGB values ​​from the fluorescence image, determine the number of positive droplets based on the defined G value threshold, calculate the sample concentration based on the Poisson distribution formula, and make a fitting curve with the theoretical value to perform absolute quantitative analysis of bacteria and obtain the detection limit.

[0143] In summary, compared with the prior art, the present invention has the following beneficial effects:

[0144] 1. The digital droplet microfluidic chip system of the present invention realizes the preparation of a uniformly mixed coded droplet library by organically combining the order and quantity of the microfluidic droplet generation chip, the microfluidic droplet fusion chip and the microfluidic droplet reaction chip in the system, directly performs droplet fusion with the sample droplet and then reacts with the droplet to release the detection signal; at the same time, the droplet coding realizes the specific marking of a variety of different targets, ensuring that the detection results of a variety of targets are obtained in one detection, and there is no need to perform image stitching when judging positive droplets, and only needs to collect images of the droplets in each chamber; the chip system has a certain flexibility, and the same chip can prepare droplet libraries of different sizes, and the droplet merging ratio can also be adjusted, which expands its application range.

[0145] 2. The bacterial detection method of the present invention can realize the labeling, detection reaction and positive droplet judgment of multiple target bacteria at the same time. By encapsulating different reagents in the continuous phase, the coded droplet library and the sample droplet library can be prepared, thereby realizing the digital detection of multiple targets.

[0146] 3. The digital droplet microfluidic chip system and bacteria detection method of the present invention have the advantages of high detection throughput, high stability, high sensitivity, rapidity and low cost. They are suitable for the simultaneous detection of multiple bacteria in unknown samples and can be used in the fields of environmental microbial detection, foodborne pathogenic bacteria detection, clinical pathogenic bacteria diagnosis and so on.

[0147] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0148] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A digital droplet microfluidic chip system, characterized in that: It includes a microfluidic droplet generation chip, a microfluidic droplet fusion chip and a microfluidic droplet reaction chip.

2. The digital droplet microfluidic chip system according to claim 1, characterized in that: The microfluidic droplet generation chip includes n (n>1) droplet generation units, and the n droplet generation units share a coded droplet collection port; The droplet generation unit includes a dispersed phase 1 inlet, a dispersed phase 2 inlet, a dispersed phase mixing channel and a droplet generation area; A droplet encoding reagent solution is introduced into the inlet of the dispersed phase 1, and a detection reaction reagent solution is introduced into the inlet of the dispersed phase 2; The dispersed phase mixing channel is a serpentine structure, a zigzag structure or a spiral structure; The droplet generation area includes a cross intersection, the cross intersection is a closing structure, and the closing structure is trapezoidal.

3. The digital droplet microfluidic chip system according to claim 2, characterized in that: The microfluidic droplet generation chip also includes a first inlet of continuous phase 1, a second inlet of continuous phase 1, a third inlet of continuous phase 1 and a fourth inlet of continuous phase 1. Continuous phase 1 is injected from the first inlet of continuous phase 1 and transported to the second inlet of continuous phase 1, the third inlet of continuous phase 1 and the fourth inlet of continuous phase 1 respectively until it is gathered in the droplet generation area with the mixed dispersed phase in the dispersed phase mixing channel.

4. The digital droplet microfluidic chip system according to claim 3, characterized in that: The microfluidic droplet fusion chip includes a sample droplet generation unit, a coding droplet injection unit, a droplet fusion unit and a fusion droplet outlet.

5. The digital droplet microfluidic chip system according to claim 4, characterized in that: The sample droplet generation unit includes a continuous phase 2 inlet, a dispersed phase 3 inlet, a sample droplet generation area, a continuous phase 2 channel, a dispersed phase 3 channel and a sample droplet channel; The continuous phase 2 inlet is connected to the continuous phase 2 channel; The dispersed phase 3 inlet is connected to the dispersed phase 3 channel; The continuous phase 2 channel and the dispersed phase 3 channel converge in the sample droplet generation area; The sample droplet channel is connected between the sample droplet generation area and the encoding droplet injection unit, and the structure of the sample droplet generation area is the same as that of the droplet generation area.

6. The digital droplet microfluidic chip system according to claim 4, characterized in that: The coded droplet injection unit includes a coded droplet library droplet inlet, a coded droplet library droplet channel connected to the coded droplet library droplet inlet, a continuous phase 3 inlet, and a continuous phase 3 channel connected to the continuous phase 3 inlet.

7. The digital droplet microfluidic chip system according to claim 4, characterized in that: The droplet fusion unit includes a droplet fusion port and a positive electrode and a negative electrode connected to the droplet fusion port.

8. The digital droplet microfluidic chip system according to claim 4, characterized in that: A first filtering structure is disposed behind the dispersed phase 1 inlet, the dispersed phase 2 inlet, and the first continuous phase 1 inlet, and a second filtering structure is disposed behind the continuous phase 2 inlet and the dispersed phase 3 inlet.

9. The digital droplet microfluidic chip system according to claim 1, characterized in that: The microfluidic droplet reaction chip includes a fusion droplet inlet, a fusion droplet reaction unit, a reaction unit connecting channel, a counting code and a fusion droplet outlet; The fusion droplet inlet is connected to the fusion droplet reaction unit through a pipeline; The fusion droplet reaction unit is connected to the fusion droplet outlet through the reaction unit connecting channel.

10. The digital droplet microfluidic chip system according to claim 9, characterized in that: The fused droplet reaction unit includes m (m>1) reaction chambers and connecting channels, and the m reaction chambers are connected in series by the connecting channel.

11. A method for detecting bacteria, using the digital droplet microfluidic chip system according to any one of claims 1 to 10 to perform bacteria detection on a sample, characterized in that: include: The number of droplet generating units in the microfluidic droplet generating chip, i.e., the specific value of n, is determined according to the number of species of bacteria to be detected, and then the coding reagent and the reaction reagent are respectively added to the dispersed phase 1 inlet and the dispersed phase 2 inlet of the droplet generating unit; Adding the sample to be tested into the dispersed phase 3 inlet of the sample droplet generating unit; The generation of the coded droplet library, the generation of the sample droplet library, the fusion of the coded droplet and the sample droplet, the reaction of the fused droplet and the signal detection are controlled in sequence to obtain the detection result.

12. The bacteria detection method according to claim 11, characterized in that: The specific steps include: Step 1: n encoding reagents and n detection reagents for target bacteria are introduced into the dispersed phase 1 inlet and dispersed phase 2 inlet of n repeated droplet generating units on the microfluidic droplet generating chip, respectively, and fluorinated oil is introduced into the first inlet of the continuous phase 1 to prepare a coded droplet library; Step 2: The sample solution to be tested is introduced into the dispersed phase 3 inlet on the microfluidic droplet fusion chip, and the fluorinated oil is introduced into the continuous phase 2 inlet to prepare sample droplets. While preparing the sample droplets, the coded droplet library droplets are injected into the coded droplet library droplet buffer area; Step 3: Adjust the flow rates of dispersed phase 3 and continuous phase 2 to ensure the order of sample droplet-coded droplet library droplet-sample droplet-coded droplet library droplet; turn on the high-voltage power supply and adjust the voltage so that the sample droplet and the coded droplet library droplet fuse into a large droplet in the droplet fusion unit; Step 4: The fused droplets are directly introduced into the microfluidic droplet reaction chip to perform detection reaction and release detection signals; Step 5: Place the microfluidic droplet reaction chip under a microscope and take photos in bright field and fluorescence field to obtain droplet coding images and signal images; Step 6: Perform droplet decoding and positive droplet counting, calculate the sample concentration according to the Poisson distribution formula, and make a fitting curve between it and the theoretical value, perform absolute quantitative analysis of bacteria, and obtain the detection limit.

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