Digital droplet micro-fluidic chip system

Abstract

The utility model provides a digital droplet micro-fluidic chip system 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 microfluidic liquid drop generation chip comprises n liquid drop generation units, wherein each liquid drop generation unit comprises a first dispersed phase inlet, a second dispersed phase inlet, a dispersed phase mixing channel and a liquid drop generation area; the dispersed phase mixing channel is of a snakelike structure, a broken line structure or a spiral structure; the liquid drop generation area comprises a cross intersection, and the cross intersection is of a closing-up structure which is in a trapezoid shape. The digital droplet micro-fluidic chip system disclosed by the utility model has the advantages of high detection flux, high stability, high sensitivity, rapidness, low cost and the like, is suitable for simultaneously detecting various bacteria in an unknown sample, and can be applied to the fields of environmental microorganism detection, food-borne pathogenic bacterium detection, clinical pathogenic bacterium diagnosis and the like.

Description

TECHNICAL FIELD

[0001] The utility model relates to microorganism detection technical field, concretely relates to a digital liquid drop microfluidic chip system. BACKGROUND

[0002] Pathogenic bacteria usually exist in food and environment, cause various diseases in human body after infection, and seriously threaten human health. Traditional detection methods based on bacterial culture and color development have long detection time, cannot detect live but unculturable bacteria, and are prone to false negative results. Currently used constant temperature nucleic acid amplification technologies such as recombinase polymerase constant temperature amplification (RPA) avoid the temperature control problem of frequent temperature change in PCR reaction, and can react at room temperature, but still have problems such as low detection sensitivity, large 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 the detection sensitivity is improved, there are still some problems: low simultaneous detection capability for multiple targets, and repeated operation procedures are needed to distinguish different bacteria.

[0003] Microfluidic chip technology is called lab-on-a-chip, and has the advantages of high integration, small volume, small reagent consumption, low cost, high throughput, simple operation, flexible design, etc., and provides a new technology for high sensitivity, high throughput, and low cost detection of bacteria. At present, the liquid drop microfluidic chip can realize each link of digital detection of bacteria, but the liquid drop microfluidic chip for detecting multiple targets has the problems of low integration, low liquid drop coding capacity, the need for additional auxiliary structure for liquid drop mixing and other operations, and the need for image stitching for liquid drop observation, which is not conducive to stable detection and analysis of multiple target bacteria. UTILITY MODEL CONTENT

[0004] (I) Technical problem solved

[0005] In view of the deficiencies of the prior art, the utility model provides a digital liquid drop microfluidic chip system, which solves the technical problem that the existing microfluidic chip is not conducive to the detection and analysis of multiple target bacteria.

[0006] (II) Technical scheme

[0007] To achieve the above purpose, the utility model realizes the following technical scheme:

[0008] The digital liquid drop microfluidic chip system comprises a microfluidic liquid drop generation chip, a microfluidic liquid drop fusion chip and a microfluidic liquid drop reaction chip connected through a PTFE tube, and the microfluidic liquid drop generation chip, the microfluidic liquid drop fusion chip and the microfluidic liquid drop 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 encoding droplet collection port;

[0010] The droplet generation unit comprises a first dispersed phase inlet, a second dispersed phase inlet, a dispersed phase mixing channel, and a droplet generation zone;

[0011] The first dispersed phase inlet is connected to a droplet encoding reagent solution, and the second dispersed phase inlet is connected to a detection reaction reagent solution;

[0012] The dispersed phase mixing channel has a serpentine structure, a polyline structure, or a spiral structure;

[0013] The droplet generation zone comprises a cross intersection, which is designed as a converging structure, and the converging structure is a trapezoid with an upper base length smaller than a lower base length, thereby generating a large shear force to rapidly generate droplets.

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

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

[0016] Preferably, the droplet encoding 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 the water-soluble dye reagent and the fluorescent reagent or the fluorescent nanomaterial solution, corresponding to different droplet encoding-decoding methods.

[0017] Preferably, the droplet encoding-decoding method comprises RGB mean filtering of a bright field image, Hough circle detection of the filtered image, color K-Means clustering of the detected circles (droplet regions) based on RGB values, and corresponding with a fluorescent image.

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

[0019] Preferably, the sample droplet generation unit comprises a second continuous phase inlet, a third dispersed phase inlet, a sample droplet generation zone, a second continuous phase channel, a third dispersed phase channel, and a sample droplet channel;

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

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

[0022] The second continuous phase channel and the third dispersed phase channel converge at the sample droplet generation zone;

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

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

[0025] Preferably, the encoded droplet injection unit comprises an encoded droplet library droplet buffer zone, an encoded droplet library droplet channel connected to the encoded droplet library droplet buffer zone, a third continuous phase inlet, and a third continuous phase channel connected to the third continuous phase inlet.

[0026] Preferably, the third continuous phase 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 of the flow channel conveying droplets to the droplet fusion port, so as to increase the electrode action area.

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

[0030] Preferably, a first filter structure is arranged behind the first dispersed phase inlet, the second dispersed phase inlet, the second continuous phase inlet, and the third dispersed phase inlet, to remove impurities and ensure 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 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 fusion droplet reaction unit comprises m (m>1) reaction chambers and a connecting channel, and the m reaction chambers are connected in series by the connecting channel.

[0035] The reaction chamber is in the shape of a "petal", with a size smaller than the field size of a 4X microscope objective, facilitating the observation of droplets in individual chambers without image stitching, ensuring the accuracy of counting, and enabling the reaction and counting of 2000-20000 droplets;

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

[0037] A bacterial detection method using any one of the above digital droplet microfluidic chip systems for bacterial detection of a sample, comprising:

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

[0039] The third dispersed phase inlet of the sample droplet generation unit is added with a sample solution to be detected;

[0040] The coding droplet library generation, sample droplet library generation, coding droplet and sample droplet fusion, fusion droplet reaction, and signal detection are sequentially controlled to obtain the detection result.

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

[0042] Step 1: n kinds of coding reagents and n kinds of detection reagents for target bacteria are respectively introduced into the first dispersed phase inlet and the second dispersed phase inlet of the n repeated droplet generation units on the microfluidic droplet generation chip, and fluorinated oil is introduced into the first continuous phase first inlet to prepare a coding droplet library under a certain continuous phase and flow phase flow rate ratio;

[0043] Step 2: a sample solution to be detected is introduced into the third dispersed phase inlet on the microfluidic droplet fusion chip, and fluorinated oil is introduced into the second continuous phase inlet to prepare sample droplets, while the coding droplet library droplets are injected into the coding droplet library droplet buffer area;

[0044] Step 3: adjust the flow rate of the third dispersed phase and the second continuous phase to ensure the arrangement order of sample droplets-coding droplet library droplets-sample droplets-coding droplet library droplets; turn on the high-voltage power supply and adjust the voltage to make the sample droplets and the coding droplet library droplets fuse into a large droplet in the droplet fusion unit;

[0045] Step 4: the fusion droplet is directly introduced into the microfluidic droplet reaction chip to perform a detection reaction and release a detection signal;

[0046] Step 5: place the microfluidic droplet reaction chip under a microscope and take pictures under bright field and fluorescence field, respectively, to obtain droplet coding pictures and signal pictures;

[0047] Step 6: droplet decoding and positive droplet counting are carried out, sample concentration calculation is carried out according to the Poisson distribution formula, a fitting curve with a theoretical value is made, absolute quantitative analysis of bacteria is carried out, and a detection limit is obtained.

[0048] (Three) beneficial effects

[0049] The utility model provides a kind of digital droplet microfluidic chip system.Compared with prior art, it has the following beneficial effects:

[0050] 1, the digital droplet microfluidic chip system of the utility model, by the organic combination of microfluidic droplet generation chip, microfluidic droplet fusion chip and microfluidic droplet reaction chip in system in sequence and quantity, realize the preparation of mixed uniform coding droplet library, carry out droplet reaction after directly with sample droplet droplet fusion, release detection signal;While droplet coding realizes the specific marking of multiple different target objects, guarantees the detection result of multiple target objects obtained in one detection, and image splicing is not needed when positive droplet is judged, and image acquisition can be carried out to droplet in each chamber;Chip system has certain flexibility, and the same chip can prepare droplet library of different sizes, and droplet merging ratio can also be adjusted, which expands its application range.

[0051] 2, the digital droplet microfluidic chip system of the utility model has the advantages of high detection flux, high stability, high sensitivity, rapidness, low cost and the like, is suitable for simultaneous detection of multiple bacteria in unknown sample, and can be used in the fields of environmental microorganism detection, foodborne pathogenic bacteria detection and clinical pathogenic bacteria diagnosis. DRAWING DESCRIPTION

[0052] In order to more clearly illustrate the technical scheme in the embodiment of the utility model or prior art, the following will briefly introduce the drawings needed to be used in embodiment or prior art description, and obviously, the drawings in the following description are only some embodiments of the utility model, and for those skilled in the art, other drawings can also be obtained according to these drawings without creating labor.

[0053] Figure 1 It is the structure schematic view of the digital droplet microfluidic chip system in embodiment 1 of the application;

[0054] Figure 2 It is the structure schematic view of microfluidic droplet generation chip in embodiment 1 of the application;

[0055] Figure 3 It is the structure schematic view of microfluidic droplet fusion chip in embodiment 1 of the application;

[0056] Figure 4is a structural schematic diagram of a microfluidic droplet reaction chip in embodiment 1 of the present application;

[0057] Figure 5 is a structural schematic diagram of a droplet generation area in embodiment 1 of the present application;

[0058] Figure 6 is a principle diagram of a droplet encoding-decoding process in embodiment 1 of the present application;

[0059] Figure 7 is a flow chart of a bacterial detection method in embodiment 4 of the present application;

[0060] Figure 8 is a principle diagram of a bacterial detection in embodiment 4 of the present application.

[0061] wherein 1, a microfluidic droplet generation chip;

[0062] 101, a droplet generation unit; 102, an encoding droplet collection port; 103, a first continuous phase first inlet; 104, a first continuous phase second inlet; 105, a first continuous phase third inlet; 106, a first continuous phase fourth inlet; 107, a dispersed phase channel; 108, a first continuous phase channel; 109, an encoding library droplet channel;

[0063] 1011, a first dispersed phase inlet; 1012, a second dispersed phase inlet; 1013, a first filtering structure; 1014, a dispersed phase mixing channel; 1015, a droplet generation area;

[0064] 2, a microfluidic droplet fusion chip;

[0065] 201, a sample droplet generation unit;

[0066] 2011, a second continuous phase inlet; 2012, a third dispersed phase inlet; 2013, a sample droplet generation area; 2014, a second filtering structure; 2015, a second continuous phase channel; 2016, a third dispersed phase channel; 2017, a sample droplet channel;

[0067] 202, an encoding droplet injection unit;

[0068] 2021, an encoding droplet library droplet buffer area; 2022, a third continuous phase inlet; 2023, an encoding droplet library droplet channel; 2024, a third continuous phase channel;

[0069] 203, a droplet fusion unit;

[0070] 2031, a droplet fusion port; 2032, a positive electrode; 2033, a negative electrode;

[0071] 204, a fused droplet outlet;

[0072] 3. Microfluidic droplet reaction chip;

[0073] 301. Fusion droplet inlet; 302. Fusion droplet outlet; 303. Fusion droplet reaction unit; 304. Reaction unit connecting channel; 305. Counting code;

[0074] 3031. Reaction chamber; 3032. Connecting channel;

[0075] 4. PTFE connecting tube. DETAILED DESCRIPTION

[0076] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme in the embodiments of the present application is described clearly and completely. Obviously, the described embodiments are part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0077] The embodiments of the present application provide a digital droplet microfluidic chip system and a bacterial detection method, and solve the technical problem that the existing microfluidic chip is not conducive to multi-target bacterial detection analysis.

[0078] In order to better understand the above technical scheme, the above technical scheme will be described in detail in combination with the drawings of the specification and specific embodiments.

[0079] The first continuous phase, the second continuous phase and the third continuous phase are mineral oil or fluorinated oil, and the first continuous phase, the second continuous phase and the third continuous phase can be completely identical or not completely identical.

[0080] The first dispersed phase is a droplet coding reagent solution, the second dispersed phase is a detection reaction reagent solution, and the third dispersed phase is a to-be-detected sample solution.

[0081] Further, the droplet coding reagent solution is a dye water-soluble 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 the dye water-soluble reagent and the fluorescent reagent / fluorescent nanomaterial solution, corresponding to different droplet coding-decoding methods.

[0082] Further, the droplet coding-decoding method includes RGB mean filtering of a bright field image, Hough circle detection of the filtered image, color K-Means clustering of the detected circle (droplet region) based on RGB values, and corresponding with a fluorescent image.

[0083] Still further, the droplet coding-decoding method specifically includes:

[0084] First, the RGB mean filter is performed on the bright field droplet image;

[0085] Hough circle detection is performed on the filtered image;

[0086] K-Means clustering is performed on the detected circle (droplet region) based on color;

[0087] Labeling and splitting are performed on different clusters to obtain a plurality of single type droplet region mask images;

[0088] The single type droplet mask image obtained in the previous step and the droplet fluorescence image are used to obtain the fluorescence image corresponding to the single type droplet;

[0089] The droplet encoding-decoding process is completed.

[0090] The material of the positive electrode and the negative electrode is liquid metal, NaCl electrolyte solution or commercial ITO electrode.

[0091] Embodiment 1 - Digital droplet microfluidic chip system

[0092] As shown in Figure 1 , the digital droplet microfluidic chip system comprises a microfluidic droplet generation chip 1, a microfluidic droplet fusion chip 2 and a microfluidic droplet reaction chip 3, and the chips are connected through PTFE tubes.

[0093] As shown in Figure 2 , the microfluidic droplet generation chip 1 comprises a droplet generation unit 101, an encoded droplet collection port 102, a first continuous phase first inlet 103, a first continuous phase second inlet 104, a first continuous phase third inlet 105, a first continuous phase fourth inlet 106, a dispersed phase channel 107, a first continuous phase channel 108 and an encoded library droplet channel 109.

[0094] The droplet generation unit 101 has a total of 8, and the 8 droplet generation units 101 are arranged around to form a ring structure, and the 8 droplet generation units 101 can be used to simultaneously prepare 8 different types of encoded library droplets. The 8 droplet generation units 101 share one encoded droplet collection port 102, and the encoded droplet collection port 102 is located at the center of the 8 droplet generation units 101, and the two are connected through the encoded library droplet channel 109.

[0095] The droplet generation unit 101 comprises a first dispersed phase inlet 1011, a second dispersed phase inlet 1012, a first filter structure 1013, a dispersed phase mixing channel 1014 and a droplet generation area 1015.

[0096] The first dispersed phase inlet 1011 is connected with the second dispersed phase inlet 1012 through the dispersed phase mixing channel 1014, and the dispersed phase mixing channel 1014 is in a serpentine structure, a polyline structure or a spiral structure, and the structure is determined according to the actual situation of the sample to be detected.

[0097] The dispersed phase channel 107 is connected to the end of the dispersed phase mixing channel 1014 away from the first dispersed phase inlet 1011 and the second dispersed phase inlet 1012.

[0098] The first filtering structure 1013 is arranged behind the first dispersed phase inlet 1011 and the second dispersed phase inlet 1012, and is used for filtering impurities in the solution to ensure smooth flow of the solution.

[0099] The first dispersed phase inlet 1011, the second dispersed phase inlet 1012 and the dispersed phase mixing channel 1014 are covered in the first continuous phase channel 108, and the first continuous phase channel 108 in this area is in a regular hexagonal structure. The first continuous phase channel 108 and the dispersed phase channel 107 meet at the droplet generation area 1015. Due to the shearing force, the mixed phase of the first continuous phase and the first dispersed phase and the second dispersed phase generates the coding library droplet at the droplet generation area 1015.

[0100] Combining Figure 5 As shown in the figure, the droplet generation area 1015 includes a cross intersection, and the cross intersection is in a tapered structure. The tapered structure is a trapezoid with a lower bottom of 40 μm, an upper bottom of 80 μm and a height of 40 μm. The design of the tapered structure is more conducive to the formation of the coding library droplet.

[0101] The first continuous phase second inlet 104 has eight places, respectively located on each droplet generation unit 101, and can be used to simultaneously prepare eight different types of coding library droplets. The first continuous phase third inlet 105 is arranged at the end of the first continuous phase second inlet 104 away from the droplet generation unit 101, and the first continuous phase third inlet 105 has four places. Each two adjacent first continuous phase second inlets 104 share one first continuous phase third inlet 105. The first continuous phase fourth inlet 106 is arranged at the end of the first continuous phase third inlet 105 away from the first continuous phase second inlet 104, and the first continuous phase fourth inlet 106 has two places. Each two adjacent first continuous phase third inlets 105 share one first continuous phase fourth inlet 106. The first continuous phase is injected from the first continuous phase first inlet 103 and transported to the first continuous phase second inlet 104, the first continuous phase third inlet 105 and the first continuous phase fourth inlet 106, respectively, until it is collected with the mixed dispersed phase in the dispersed phase mixing channel 1014 at the droplet generation area 1015.

[0102] The first filtering structure 1013 is arranged behind the first continuous phase first inlet 103, and is used for filtering impurities in the first continuous phase, so as to ensure smooth liquid flow.

[0103] In combination Figure 3 As shown in the figure, the microfluidic droplet fusion chip 2 comprises a sample droplet generation unit 201, an encoding droplet injection unit 202, a droplet fusion unit 203, and a fused droplet outlet 204.

[0104] The sample droplet generation unit 201 comprises a second continuous phase inlet 2011, a third dispersed phase inlet 2012, a sample droplet generation area 2013, a second filtering structure 2014, a second continuous phase channel 2015, a third dispersed phase channel 2016, and a sample droplet channel 2017.

[0105] The second continuous phase inlet 2011 is connected with the second continuous phase channel 2015, the second continuous phase channel 2015 surrounds the third dispersed phase inlet 2012, and the third dispersed phase inlet 2012 is connected with the third dispersed phase channel 2016.

[0106] The second continuous phase channel 2015 and the third dispersed phase channel 2016 converge at the sample droplet generation area 2013, the sample droplet channel 2017 is used for connecting the sample droplet generation area 2013 with the encoding droplet injection unit 202, and the sample droplet generated is transmitted to the encoding droplet injection unit 202 through the sample droplet channel 2017, and the structure of the sample droplet generation area 2013 is the same as that of the droplet generation area 1015.

[0107] The second filtering structure 2014 is a four-row circular array, the first row is composed of three circular micropillars with a diameter of 500 μm arranged at equal intervals, the second row is composed of six circular micropillars with a diameter of 300 μm arranged at equal intervals, the third row is composed of eight circular micropillars with a diameter of 300 μm arranged at equal intervals, and the fourth row is composed of twelve circular micropillars with a diameter of 200 μm arranged at equal intervals.

[0108] The encoding droplet injection unit 202 comprises an encoding droplet library droplet buffer area 2021, an encoding droplet library droplet channel 2023 connected with the encoding droplet library droplet buffer area 2021, a third continuous phase inlet 2022, and a third continuous phase channel 2024 connected with the third continuous phase inlet 2022.

[0109] The encoding droplet library droplet buffer area 2021 is in the shape of a petal, and is used for temporary storage of droplets.

[0110] The droplet fusion unit 203 comprises a droplet fusion port 2031, a positive electrode 2032, and a negative electrode 2033 connected with the droplet fusion port 2031.

[0111] The droplet fusion port 2031 is a square structure with a side length of 200 μm, and the channel width is greater than the flow channel width for transporting the droplets to the droplet fusion port 2031, so as to increase the electrode acting area.

[0112] The positive electrode 2032 and the negative electrode 2033 are respectively arranged on the two sides of the droplet fusion port 2031, and the distance from the droplet fusion port 2031 is 50 μm, so as to ensure the uniformity of the electric field.

[0113] Combination Figure 4 As shown in the figure, the microfluidic droplet reaction chip 3 includes a fused droplet inlet 301, a fused droplet outlet 302, a fused droplet reaction unit 303, a reaction unit connecting channel 304, and a counting code 305.

[0114] 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 the reaction unit connecting channel 304.

[0115] The fused droplet reaction unit 303 includes reaction chambers 3031 and connecting channels 3032, and the reaction chambers 3031 are eight in number and are connected in series by the connecting channels 3032.

[0116] The reaction chambers 3031 are in the shape of a whole "petal", and the single structure is a rounded rhombus with diagonal lengths of 1.9 mm and 1.5 mm, respectively. The size is smaller than the field size of the 4X objective lens of the microscope, so as to facilitate the observation of the droplets in the single chamber, without image splicing processing, to ensure the accuracy of counting, and to realize the reaction and counting of 2000-20000 droplets.

[0117] The connecting channels 3032 have a width of 100 μm, which is consistent with the size of the reaction unit connecting channel 304.

[0118] Example 2 - Combination mode of digital droplet microfluidic chip system

[0119] The microfluidic droplet generation chip, the microfluidic droplet fusion chip, and the microfluidic droplet reaction chip can be organically combined in sequence and quantity according to the types and quantities of bacteria to be detected.

[0120] Combination mode 1:

[0121] The digital droplet microfluidic chip system includes one microfluidic droplet generation chip, one microfluidic droplet fusion chip, and one microfluidic droplet reaction chip connected in sequence through PTFE tubes.

[0122] Combination mode 2:

[0123] The digital droplet microfluidic chip system comprises one microfluidic droplet generation chip, n (n>1) microfluidic droplet fusion chips and one microfluidic droplet reaction chip connected in sequence through PTFE tubes.

[0124] Combination mode 3:

[0125] The digital droplet microfluidic chip system comprises n (n>1) microfluidic droplet fusion chips and one microfluidic droplet reaction chip connected in sequence through PTFE tubes.

[0126] The above combination modes are only three of the various combination modes of the digital droplet microfluidic chip system of the present application, which can be adjusted according to actual conditions.

[0127] Example 3 - Preparation method of digital droplet microfluidic chip system

[0128] Step 1: The master of the microfluidic droplet generation chip, the microfluidic droplet fusion chip and the microfluidic droplet reaction chip all adopts a silicon wafer with a diameter of 75 mm, and is produced using standard photolithography; the production material of each microfluidic chip is polydimethylsiloxane and initiator, and the weight ratio of the two is 10:1.

[0129] Step 2: Punch each inlet and outlet of the microfluidic droplet generation chip, and bond with a glass sheet (43 mm x 43 mm); punch each inlet and outlet of the microfluidic droplet fusion chip and the microfluidic droplet reaction chip, and bond with a glass slide (75 mm x 25 mm);

[0130] Step 3: During the bonding process, the pattern surface of each chip faces upwards and is plasma oxidized together with the glass sheet / slide for 1 min (the relevant parameters are: 100 mW, 1% O2), then the chip is quickly attached to the corresponding glass sheet / slide, and placed in an oven at 75°C for 20 min;

[0131] 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, ultraviolet curing glue is applied at the interface, and curing is carried out under ultraviolet lamp irradiation, and finally the microfluidic droplet fusion chip is transferred to room temperature for cooling.

[0132] Example 4 - Bacterial detection method

[0133] As shown in Figure 6 , Figure 7 , Figure 8

[0134] ​Step 1: Eight coded reagents and eight detection reagents targeting target bacteria are introduced into the first and second dispersed phase inlets of eight repeating droplet generation units on the microfluidic droplet generation chip, respectively. HFE-7500 fluorinated oil containing >0.2% surfactant is introduced into the first inlet of the first continuous phase. The flow rate ratio of the first dispersed phase, the second dispersed phase and the first continuous phase is 1:5. A library of coded droplets with a diameter of 80 μm is prepared.

[0135] The coding reagent solution is a solution of 8 different colors obtained by mixing 50 mg / mL amaranth red, Prussian blue and fluorescein solutions in different proportions;

[0136] The detection reagents are template-free DNA RPA reaction reagents and CRISPR / Cas13a reagents designed for the target gene structure of the target bacteria.

[0137] Step 2: The sample solution to be tested is introduced into the third dispersed phase inlet on the microfluidic droplet fusion chip, and HFE-7500 fluorinated oil containing >0.2% surfactant is introduced into the second continuous phase inlet. The flow rate ratio of the third dispersed phase to the second continuous phase is 1:5 to prepare a sample droplet library with a diameter of 80 μm. At the same time as the sample droplet is prepared, the encoded droplet library droplets are injected into the encoded droplet library droplet buffer area through a PTFE tube.

[0138] Step 3: Adjust the flow rates of the third dispersed phase and the second continuous phase to ensure the arrangement order of sample droplet-encoded droplet library droplet-sample droplet-encoded droplet library droplet; turn on the high voltage power supply and adjust the voltage (DC, >600 V, 10 kHz) to fuse one sample droplet and one encoded droplet into one fused droplet.

[0139] Step 4: The fusion droplets are directly introduced into the fusion droplet inlet on the microfluidic droplet reaction chip, filling the reaction chambers of all droplet reaction units; the fusion droplet inlet and outlet are blocked with polyacrylamide micropillars; the microfluidic droplet reaction chip is placed at 39℃-43℃ for 30 min to detect the reaction and signal release.

[0140] Step 5: Place the microfluidic droplet reaction chip under a microscope and take pictures under bright field and fluorescence field to obtain droplet-coded images and signal images; the pictures are taken one by one according to the counting code.

[0141] Step 6: Perform droplet decoding and positive droplet counting. Using ImageJ software, extract RGB values ​​from the fluorescence image, determine the number of positive droplets based on the defined threshold of G value, calculate the sample concentration according to the Poisson distribution formula, and create a fitting curve between the concentration and the theoretical value. Perform absolute quantitative analysis of bacteria and obtain the detection limit.

[0142] Compared with the prior art, the following beneficial effects are achieved:

[0143] 1、The digital droplet microfluidic chip system of the utility model, through organic combination of the microfluidic droplet generation chip, the microfluidic droplet fusion chip and the microfluidic droplet reaction chip in the system, realizes preparation of mixed uniform coding droplet library, carries out droplet fusion after directly with sample droplet, carries out droplet reaction, releases detection signal; At the same time, droplet coding realizes specific marking of multiple different target objects, guarantees to obtain multiple target object detection results in one detection, and when judging positive droplet, does not need to carry out image splicing, only needs to carry out image acquisition to the droplet in each chamber; The chip system has certain flexibility, and the same chip can prepare droplet library of different sizes, and the droplet merging ratio can also be adjusted, thereby expanding its application range.

[0144] 2、The digital droplet microfluidic chip system has the advantages of high detection flux, high stability, high sensitivity, rapidness and low cost, is suitable for simultaneous detection of multiple bacteria in unknown samples, and can be applied to the fields of environmental microorganism detection, foodborne pathogenic bacteria detection and clinical pathogenic bacteria diagnosis.

[0145] It should be noted that the relational terms herein such as first and second and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any such actual relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element preceded by "comprises... a" does not, without more constraints, foreclose the existence of additional identical elements in the process, method, article, or apparatus that comprises the recited element.

[0146] The above embodiments are only used to illustrate the technical solutions of the utility model, and not to limit them; although the utility model has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement to part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the utility model.

Claims

1. A digital droplet microfluidic chip system, characterized in that, This includes microfluidic droplet generation chips, microfluidic droplet fusion chips, and microfluidic droplet reaction chips; The microfluidic droplet generation chip includes n droplet generation units, and the n droplet generation units share a single coded droplet collection port; where n>1; The droplet generation unit includes a first dispersed phase inlet, a second dispersed phase inlet, a dispersed phase mixing channel, and a droplet generation region. A droplet-encoding reagent solution is introduced into the first dispersed phase inlet, and a detection reaction reagent solution is introduced into the second dispersed phase inlet; The dispersed phase mixing channel has a serpentine structure, a zigzag structure, or a spiral structure; The droplet generation area includes a cross-shaped intersection, which has a tapering structure that is trapezoidal.

2. The digital droplet microfluidic chip system as described in claim 1, characterized in that, The microfluidic droplet generation chip further includes a first continuous phase first inlet, a first continuous phase second inlet, a first continuous phase third inlet, and a first continuous phase fourth inlet. The first continuous phase is injected from the first continuous phase first inlet and delivered to the first continuous phase second inlet, the first continuous phase third inlet, and the first continuous phase fourth inlet, respectively, until the mixed dispersed phase in the dispersed phase mixing channel gathers in the droplet generation area.

3. The digital droplet microfluidic chip system as described in claim 2, characterized in that, The microfluidic droplet fusion chip includes a sample droplet generation unit, an coded droplet injection unit, a droplet fusion unit, and a fused droplet outlet.

4. The digital droplet microfluidic chip system as described in claim 3, characterized in that, The sample droplet generation unit includes a second continuous phase inlet, a third dispersed phase inlet, a sample droplet generation zone, a second continuous phase channel, a third dispersed phase channel, and a sample droplet channel; The second continuous phase inlet is connected to the second continuous phase channel; The third dispersed phase inlet is connected to the third dispersed phase channel; The second continuous phase channel and the third dispersed phase channel converge in the sample droplet generation region; The sample droplet channel is connected between the sample droplet generation area and the coded droplet injection unit, and the structure of the sample droplet generation area is the same as that of the droplet generation area.

5. The digital droplet microfluidic chip system as described in claim 3, 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 third continuous phase inlet, and a third continuous phase channel connected to the third continuous phase inlet.

6. The digital droplet microfluidic chip system as described in claim 3, 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.

7. The digital droplet microfluidic chip system as described in claim 4, characterized in that, A first filter structure is provided after the first dispersed phase inlet, the second dispersed phase inlet, and the first continuous phase inlet; a second filter structure is provided after the second continuous phase inlet and the third dispersed phase inlet.

8. The digital droplet microfluidic chip system as described in claim 1, characterized in that, The microfluidic droplet reaction chip includes a fusion droplet inlet, a fusion droplet reaction unit, a reaction unit connection channel, a counting code, and a fusion droplet outlet. The fusion droplet inlet is connected to the fusion droplet reaction unit via a pipe; The fusion droplet reaction unit is connected to the fusion droplet outlet through the reaction unit connection channel.

9. The digital droplet microfluidic chip system as described in claim 8, characterized in that, The fusion droplet reaction unit includes m reaction chambers and connecting channels, wherein the m reaction chambers are connected in series by the connecting channels; where m>1.

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