Multi-channel synchronous sample injection droplet chip and analysis application thereof

Abstract

The invention relates to a multi-channel synchronous sample injection droplet chip and analysis application thereof. The chip comprises a multi-channel sample injection port structure arranged on a chip body, the multi-channel sample injection port structure is converged at the upstream of the liquid drop generation port through the mixing channel, and through the fluid resistance design of the multi-channel sample injection channel, multiple reaction components are introduced at the same time under the driving of a single pressure source, and the multiple reaction components have the same or different fluid properties; the liquid drops enter a downstream liquid drop generation structure according to the same or different volume proportions, so that high-flux micro-volume reaction with highly consistent reaction components and reaction conditions is formed; through a multi-channel sample inlet structure, the reaction component composition and the starting time of the droplet micro-volume multi-component reaction are controlled as required, and the accurate control of the initial state of the high-flux droplet micro-reaction is realized. The device is easy and convenient to control, does not need precise driving pressure control, and belongs to the field of biochemical reactions and detection and analysis thereof.

Description

Technical Field

[0001] The present invention relates to the field of biochemical reaction and detection and analysis thereof, and in particular to a multi-channel synchronous sampling droplet chip and analysis application thereof. Background Art

[0002] Digital analysis usually involves dispersing the molecules of the sample to be tested into a large number of parallel analysis units, so that each analysis unit contains at most one molecule. The analysis unit is binarized to "1" or "0" based on whether or not there is a molecular signal. The number or proportion of positive signals "1" is counted to determine the number or concentration of molecules in the sample to be tested. The signal reading of digital analysis is relatively simple and has higher analytical accuracy and absolute quantitative capabilities. Therefore, it plays an important role in precision analysis application fields including biochemistry, molecular biology research, and quantitative detection of nucleic acids, proteins, and enzyme activities.

[0003] Microdroplet technology is a powerful tool for digital analysis. Compared with reaction units in the form of microchambers, virtual reaction units such as microdroplets have higher analytical flexibility and spatiotemporal controllability. In particular, compared with the method of directly distributing the uniformly mixed reaction components to each chamber unit in the final reaction state, the components participating in the final reaction can be precisely controlled during the dynamic process of microdroplet formation, which can greatly expand the scope of application of such high-throughput reactions and analyses.

[0004] However, the traditional micro-droplet generation process generally requires at least precise flow rate or flow control of the continuous phase and the dispersed phase to ensure that the reaction unit of the desired size is generated on demand. These systems often require complex and bulky fluid drive control devices. What is more challenging is that when the dispersed phase of the micro-droplet involves a multi-component fluid that can be started by mixing, the traditional drive control mode requires independent and precise adjustment and control of the flow rate or flow of more reaction component fluids, which often causes asynchronous injection and mutual crosstalk due to slight differences or fluctuations in the respective pipelines or drive sources. This may lead to a series of problems such as the need for a more complex and time-consuming steady-state pressure establishment process, inconsistent droplet reaction components, component contamination, and sample waste, which greatly increases the difficulty of actual operation and uncontrollable random interference, and ultimately affects the reliability of such high-throughput reactions or the accuracy of high-precision measurements. For example, in some rapid reactions triggered by component mixing, different proportions and states of the reaction components when entering the micro-droplet may result in different reaction outcomes. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the purpose of the present invention is to provide a multi-channel synchronous sampling droplet chip that is easy to operate and does not require precise driving pressure control, and its analytical application.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A multi-channel synchronous injection droplet chip comprises a multi-channel injection port structure arranged on a chip body; the multi-channel injection port structure is merged at the upstream of a droplet generation port through a mixing channel, and a plurality of reaction components are introduced simultaneously under the drive of a single pressure source through the fluid resistance design of the multi-channel injection channel, wherein the plurality of reaction components have the same or different fluid properties and enter the downstream droplet generation structure in the same or different volume ratios, thereby forming a high-throughput micro-volume reaction with highly consistent reaction components and reaction conditions; the multi-channel injection port structure is used to realize on-demand control of the reaction component composition and start-up time of the droplet micro-volume multi-component reaction, thereby realizing precise control of the starting state of the high-throughput droplet micro-reaction and simplifying the operation.

[0008] As a preferred embodiment, a multi-channel synchronous sampling droplet chip also includes a mixing channel, a continuous phase channel, a droplet generation structure, a droplet distribution branch structure, a droplet detection area, an outlet branch structure, and a downstream sample outlet arranged on the chip body; the multi-channel sample outlet structure includes at least two dispersed phase channels, and the dispersed phase channel, the mixing channel, the droplet generation structure, the droplet distribution branch structure, the droplet detection area, the outlet branch structure, and the downstream sample outlet are arranged in sequence from upstream to downstream, and the continuous phase channel is connected to the droplet generation structure.

[0009] As a preference, the mixing channel is used for mixing and premature reaction of components input into the dispersed phase channel; or the mixing channel is extremely short to prevent premature reaction of components input into the dispersed phase channel.

[0010] As a preferred embodiment, the dispersed phase channel includes an injection port, a liquid storage tank, and a connecting channel, and the continuous phase channel includes a continuous phase liquid storage tank and a connecting channel. By setting the size of the connecting channel, the mixing ratio of the reaction system components is controlled, thereby achieving automatic injection of the reaction components on demand.

[0011] As a preference, the mixing state of the reaction system before droplet generation is regulated by setting the relative position and geometric configuration of the mixing channel.

[0012] As a preferred embodiment, a single pressure or driving source is applied to the injection port or downstream outlet of the multi-channel synchronous injection droplet chip to achieve the regulation of the ratio of the flow resistance of the dispersed phase and the continuous phase, and generate micro-droplets of uniform size; the single pressure or driving source drive ensures that before the droplets are formed, the system components remain separated in their respective injection channels in the chip and are always injected according to the expected mixing ratio until entering the droplet generation structure. Applying a single pressure or driving source can also avoid the use of independent source drives in the process of establishing steady-state equilibrium, which often leads to crosstalk between different fluids and the influence of unexpected mixing ratios due to uneven pressure distribution or fluctuations.

[0013] As a preferred embodiment, a multi-channel synchronous sampling droplet chip also includes a droplet guiding structure, which is arranged downstream inside the droplet detection area and is used to guide the generated droplets; the droplet guiding structure includes a plurality of rounded long rectangular structures, and the plurality of rounded long rectangular structures are parallel to each other and arranged in a symmetrical herringbone shape.

[0014] As a preferred embodiment, the outlet branch structure includes a plurality of shunt channels connected in parallel, the upstream of the shunt channel is connected to the droplet detection area, and the downstream is collected at the downstream sample outlet, ensuring that the droplets are discharged from the chip through a unified outlet. Multiple shunt channels are set to accelerate the discharge of droplets and prevent problems such as blockage. One outlet is used to facilitate subsequent collection and analysis. In practical applications, the design of this structure can also be flexibly adjusted according to specific needs, such as changing the number, size or layout of branches.

[0015] As a preference, the length of the extremely short mixing channel is less than or equal to the width of the mixing channel.

[0016] An analytical application of a multi-channel synchronous injection droplet chip is applied to biochemical processes that need to be started on demand, including enzymatic reactions, isothermal amplification technologies (such as RPA, LAMP), CRISPR-Cas reactions, and single-cell analysis.

[0017] The principle of the present invention is: multiple independent injection channels designed according to fluid resistance allow multiple different reaction components or samples to be input simultaneously and separately, enter the downstream droplet generation structure in different volume ratios according to the reaction or analysis requirements, and form a high-throughput micro-volume reaction unit with highly consistent reaction components and reaction conditions under the drive of the same pressure source. This design can avoid triggering unexpected reactions due to early mixing of components and can control the reaction state before droplet formation on demand. It is particularly suitable for reaction or analysis application scenarios involving multi-component reactions involving different fluid viscosities, where conventional methods are difficult to effectively control the start-up time of multi-component reactions. The multi-channel synchronous injection droplet chip can adjust the size of the multi-channel dispersed phase channel and the continuous phase channel upstream of the droplet generation port, and simultaneously realize the regulation of the mixing ratio of the reaction system components and the flow resistance ratio of the dispersed phase to the continuous phase, thereby simply using a single pressure or driving source to generate uniform droplets on demand.

[0018] The present invention has the following advantages:

[0019] 1. Provide a precise spatiotemporal control method for droplet multi-component reactions: ensure that components with different fluid properties participating in the droplet reaction can be independently and synchronously injected under the drive of the same pressure source, control the reaction time and reaction volume ratio of different reaction components as needed, and control the reaction state of various components that finally enter the droplet reaction system (whether to react in advance, how long or to what extent the reaction occurs, and the combination of early reactions between different components), thereby providing a simple and precise control method for accurately controlling droplet multi-component reactions.

[0020] 2. Provide a "foolproof" droplet analysis platform that avoids multi-channel pressure-driven precise adjustment: the multi-channel pressure-driven adjustment parameters are "solidified" in the form of corresponding injection channel flow resistance parameter design, so that it can be used immediately, avoiding the operational difficulties of driving pressure adjustment and steady-state equilibrium establishment in the current conventional way, preventing the resulting crosstalk of sample components, reaction group distribution ratio deviation and sample waste before steady-state establishment, thereby affecting the reliability of high-throughput reactions or the accuracy of high-precision measurements.

[0021] 3. Improve the integration of chip operations: Integrating the functions required for reaction component mixing, droplet generation, manipulation and detection on a single chip greatly simplifies the complexity of such systems and improves the convenience of operation and the versatility of the control platform.

[0022] 4. Reduce contamination risk: Improve chip design to reduce the risk of exposure to the environment during operations such as mixing reaction components and sample transfer, thereby reducing the possibility of contamination and improving the accuracy and reliability of experimental results.

[0023] 5. Set up a mixing channel, and control the mixing state of the reaction system before droplet generation through the geometric configuration of the mixing channel, and whether to react in advance in the mixing channel.

[0024] 6. Set up a droplet diversion structure to ensure stable droplet generation, smooth flow and prevent stacking and blockage.

[0025] 7. The multi-channel synchronous injection droplet chip provided by the present invention has a unique design of multiple independent injection channels and can input multiple samples. This design allows the reaction system components to be split, achieving rapid mixing of reactants and precise control of reaction initiation. It also uses a single pressure source to ensure that the system components remain separated in the chip before droplets are formed, preventing the reaction from starting prematurely. Therefore, the multi-channel synchronous injection droplet chip technology provided by the present invention can be widely used in biochemical processes that require rapid reaction initiation.

[0026] The present invention provides a multi-channel synchronous injection droplet chip, which mainly realizes the control of the reaction sequence and reaction effect of various different components participating in the droplet reaction before the droplet is generated, including whether to react in advance, the time or degree of early reaction, the reaction ratio of the components participating in the reaction and the component combination. The precise control provides the necessary basis for the refined droplet multi-component reaction and further expands the application potential of micro-droplets such as high-throughput reactions and digital analysis. In particular, in rapid biochemical multi-component reactions that can be triggered by mixing some components, the reaction effect and application flexibility can be significantly improved. In general, this multi-channel synchronous injection droplet chip can improve the reliability of droplet high-throughput reactions or the accuracy of high-precision measurements. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of the multi-channel synchronous injection droplet chip of the first embodiment.

[0028] Figure 2 These are some typical methods of simultaneous injection dispersed phase channel design and related flow resistance design requirements as well as the differences from conventional single-channel injection dispersed channel design.

[0029] Figure 3 It is a schematic diagram of the multi-channel synchronous injection droplet chip of Example 2.

[0030] Figure 4 It is a partial enlarged view of the upstream of the multi-channel synchronous injection droplet chip of Example 2.

[0031] Figure 5 yes Figure 4 Further enlargement of the image.

[0032] Figure 6 It is a partial enlarged view of the droplet guiding structure of Example 2.

[0033] Figure 7 It is a partial enlarged view of the downstream of the multi-channel synchronous injection droplet chip of Example 2.

[0034] Figure 8 It is a schematic diagram of the structure of the multi-channel synchronous injection droplet chip of the second embodiment.

[0035] Fig. 9 This is a typical result of the enzymatic fluorescence reaction of the multi-channel synchronous injection droplet chip of Example 3.

[0036] Fig.10 This is a typical result of the recombinase amplification (RPA) reaction performed by the multiplex synchronous injection droplet chip of Example 3.

[0037] Fig.11It is a schematic diagram and typical experimental results of the effect of dispersed phase channel design in multi-channel synchronous injection droplet chip on the subsequent reaction outcome of CRISPR-Cas12a droplets.

[0038] In the figure, 1-dispersed phase inlet and liquid storage tank, 2-dispersed phase connecting channel, 3-mixing channel, 4-continuous phase connecting channel, 5-droplet generation structure, 6-droplet distribution branch structure, 7-droplet detection area, 8-droplet guiding structure, 9-outlet branch structure, 10-downstream sample outlet. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with specific implementation methods.

[0040] Embodiment 1

[0041] A multi-channel synchronous injection droplet chip is designed with multiple key components and functions, aiming to achieve synchronous and independent introduction of multiple reaction component fluids participating in the droplet reaction under the drive of the same pressure source, on-demand matching, robust high-throughput and uniform droplet generation, thereby achieving simple and precise control of droplet multi-component reactions.

[0042] The chip of the present invention includes a dispersed phase channel, a mixing channel, a continuous phase channel, a droplet generation structure, a droplet distribution branch structure, a droplet detection area, an outlet branch structure, and a downstream sample outlet. The dispersed phase channel includes an inlet and a liquid storage tank, and a connecting channel, the continuous phase channel includes a continuous phase liquid storage tank, and a connecting channel, and a droplet diversion structure is arranged downstream inside the droplet detection area.

[0043] In this embodiment, Figure 1 As shown, the dispersed phase channel, mixing channel, droplet generation structure, droplet distribution branch structure, droplet detection area, outlet branch structure, and downstream sample outlet are arranged in sequence from upstream to downstream, and the continuous phase channel is connected to the left and right ends of the droplet generation structure. The mixing channel is set as a longer channel for mixing and early reaction of the input components of the dispersed phase channel.

[0044] Several important structures will be highlighted below.

[0045] Dispersed phase channel, the dispersed phase channel is located upstream of the mixing channel, and is connected to each dispersed phase sample injection port and the liquid storage tank and the upstream of the mixing channel. In order to realize the synchronous independent injection of dispersed phase components with different fluid properties including different viscosity coefficients, the corresponding connecting channel is set by a reasonable length Ld and cross-sectional area Sd to balance its injection flow resistance Ri, referring to the formula Ri=ηdL / Sd (where ηd is the fluid dynamic viscosity of each dispersed phase). For example, a completely symmetrical geometric design can be adopted for two dispersed phase fluids with viscosity coefficients close to each other. Its total flow resistance is equivalent to the parallel effect of the flow resistance of each independent dispersed phase injection channel. This design allows the independent introduction of multiple different reaction components participating in the droplet reaction, and completes the synchronous injection of multiple reaction components with different fluid properties under the same pressure source, avoiding the need to perform independent and accurate flow rate or flow control on each reaction component separately in conventional operation, which causes the operational difficulties of driving pressure regulation and steady-state equilibrium establishment, as well as sample component crosstalk, reaction component distribution ratio deviation and sample waste caused before steady-state establishment. At the same time, by planning the fluid path of the dispersed phase injection channel, the components involved in the droplet multi-component reaction are pre-reacted and the component volume ratio is controlled according to the needs, so as to achieve precise time and space control of the multi-components before entering the droplet, so as to meet more precise high-throughput droplet reaction and analysis. Compared with the single-channel dispersed phase droplet formation method in the premixed state, this independent synchronous injection can minimize the quantitative error and background interference caused by the unexpected start of component mixing during the droplet formation process, especially in some cases where the reaction rate is relatively fast and the complete micro-division of the entire premixed reaction system is not completed instantly, it can ensure the consistency of the experimental starting conditions and the reliability of the results. The dispersed phase channel can also be provided with a mixing channel as needed, which is located upstream of the droplet generation structure. Several branches of the multi-channel dispersed phase channels converge in the mixing channel. The mixing channel is used to mix the converged reaction components to provide trigger conditions for the subsequent corresponding reaction initiation. The number of mixing channels is not limited, and can be designed as any series-parallel combination with several injection channels and mixing channels as needed, and finally all the multi-channel input dispersed phases are converged and input into the droplet generation structure. Its geometric configuration mainly depends on when the droplet reaction system converges and fully mixes the multiple dispersed phases participating in the reaction to start the reaction. The length of the mixing channel can control whether the multiple dispersed phases participating in the reaction can effectively start the reaction before the droplets are formed and the degree of progress of the reaction. The flow resistance of the mixing channel and the total flow resistance of the dispersed phase channel mentioned above are used in series as the total flow resistance of the dispersed phase channel. Specifically, Figure 2 Assume that Figure 2 The dispersed phase channel resistance of the single dispersed phase injection droplet chip shown on the left is R d =R dA , then in Figure 2 The total flow resistance of the dispersed phase injection channel in the parallel dual dispersed phase component synchronous injection droplet chip shown in the middle is Rd =0.5R dA =0.5R dB . Similarly, in Figure 2 In the multi-channel dispersed phase synchronous injection droplet chip shown on the right, in order to ensure that the three different dispersed phases are injected synchronously under the same pressure difference, the dispersed phase A and dispersed phase B located upstream of the mixing channel are required to have the same flow resistance, that is, R dA =R dB ; At the same time, the dispersed phase C injection channel flow resistance R dC Equal to the series mixing channel flow resistance R dM Plus the parallel flow resistance of dispersed phase A and B injection channels, that is, R dC =R dM +0.5R dA , the overall flow resistance of the corresponding dispersed phase injection channel is equal to the parallel effect of two dispersed phase C injection channels, that is, R d =0.5R dC In terms of the droplet reaction effect achieved, Figure 2 The single dispersed phase injection channel droplet chip shown on the left is limited to reactions that hardly react under normal conditions after some components are mixed or that occur extremely slowly and can be ignored before the mixed liquid completely forms droplets, such as most hot-start PCR reactions, or droplet reactions triggered by thermal effects, light fields, electric fields, magnetic fields, etc. Otherwise, the reaction state entering the droplet may be inconsistent or difficult to control accurately due to the premature reaction before the droplet is formed. For example, if some isothermal amplification reactions are started in advance before the droplet is formed, the template subsequently distributed into the droplet may not be the original target but its amplification product, which may cause the subsequent quantitative measurement results to be biased high. Figure 2 The droplet chip shown in the middle, which contains a dual dispersed phase injection channel, can be further used to separate the dispersed phase components A and B that trigger premature reactions due to mixing. The extremely short mixing channel allows the components to merge without mixing, thereby ensuring that the initial states of the droplet reactions are highly consistent during the process of forming droplets of all samples. This is conducive to achieving better control of reaction conditions and the accuracy and reliability of reaction results, while also avoiding the need for specialized instruments and equipment outside the chip for reaction triggering control. Figure 2 The droplet chip shown on the right, which contains three dispersed phase injection channels, can be used for more complex multi-component droplet reaction condition control. The mixing channel is used to regulate the degree of mixing reaction of dispersed phase components A and B and merge with the third dispersed phase component C to form droplets. This ensures that the reaction participants entering the droplets are always the intermediate reaction products of dispersed phases A and B and dispersed phase C. This is suitable for some multi-component droplet reactions or droplet synthesis applications that require precise control of the reaction sequence. Figure 2The structure and flow resistance equivalent method shown are only used to illustrate the design concept of the present invention. In specific applications, they can be flexibly changed, combined or replaced based on the design concept.

[0046] Continuous phase channel, one end of the continuous phase connection channel is connected to the continuous phase reservoir, and the other end of the continuous phase connection channel is connected to the droplet generation structure. The flow resistance entering the droplet generation structure is adjusted by accurately controlling the cross-sectional area (Sc) and length (Lc) of the flow resistance adjustment channel, maintaining a specific ratio of the dispersed phase and continuous phase flow resistances, thereby achieving precise control of the proportion of different components in the droplet reaction, the size of the droplets formed, and the generation rate. The ratio of the dispersed phase flow resistance Rd to the continuous phase flow resistance Rc ranges from 0.5 to 2. Specifically, it is preferably 0.8-1.2, so that droplets of the expected size can be formed under the same driving pressure difference ΔP.

[0047] The droplet diversion structure is located downstream of the droplet detection area. Its main function is to reduce the resistance of the generated droplets to the downstream flow, prevent the accumulation of droplets, and prevent interference with the subsequent droplet generation and discharge. Before the droplets are stably generated, some droplets of different sizes or irregular shapes may be generated. The diversion structure helps to guide and discharge these uneven droplets to prevent them from affecting the subsequent droplet generation and detection and analysis process; secondly, if the droplet generation speed is too fast and the flow speed is too slow, it may cause the droplets to accumulate downstream of the detection area. The droplet diversion structure can reduce or avoid this accumulation phenomenon, ensuring that the droplets are stably generated and discharged smoothly; the droplet diversion structure can also effectively balance the pressure difference in the chip, prevent the generation and circulation of droplets from being affected by excessive or small pressure differences, and maintain the stable operation of the micro-droplet chip. The design of the droplet diversion structure can reduce the problems that may occur during the chip operation and improve the reliability of the micro-droplet chip.

[0048] The outlet branch structure is an important design to ensure the smooth discharge of droplets. This structure contains several branches, which balance the flow resistance near the upstream of the branch outlet through multiple parallel diversion channels to prevent the local flow resistance from being too high, resulting in insufficient flow of droplets inside the chip and uneven distribution of droplets. Multiple diversion channels can significantly increase the flux of the micro-droplet chip, thereby accelerating the discharge of droplets from the droplet detection area and shortening the residence time of droplets from generation to discharge, that is, inside the chip. In general, the outlet branch structure improves the overall performance of the micro-droplet chip, including the stability of droplet generation, the efficiency of discharge, and the reliability of experimental results.

[0049] Embodiment 2

[0050] In this embodiment, Figure 3-8As shown, the dispersed phase channel, mixing channel, continuous phase channel, droplet generation structure, droplet distribution branch structure, droplet detection area, outlet branch structure, and downstream sample outlet are arranged in sequence from upstream to downstream. The mixing channel is set to be extremely short to prevent premature reaction of the input components of the dispersed phase channel.

[0051] Each component will be introduced below.

[0052] Dual-channel injection port and liquid reservoir: Figure 3 As shown, it is directly connected to the external space and is used to store the sample to be dispersed. The opening diameter of the injection port structure is 2.5 mm, and the injection port structure is cylindrical or conical with a wide top and a narrow bottom, so that samples or reagents can be added.

[0053] Dispersed phase connecting channel: located upstream of the mixing channel, connecting each dual-path inlet and the liquid storage tank with the upstream of the mixing channel. The viscosity coefficients of the two dispersed phase fluids involved in this experiment are almost the same, so a completely symmetrical geometric design is adopted.

[0054] Mixing channel: connects the downstream of the dispersed phase connection channel with the upstream of the droplet generation structure. In this experiment, the mixing channel is extremely short, 25 μm, and is mainly used to control the equal volumes of the two reaction components before droplet formation to merge into the formed droplets, avoiding premature mixing and causing the reaction to start prematurely.

[0055] Continuous phase reservoir: two symmetrical ones are arranged, the continuous phase reservoir is communicated with the external space and is used for loading and storing the continuous phase reagent, and the opening diameter of the continuous phase reservoir is 3.5 mm.

[0056] Continuous phase connecting channel: one end is connected to the continuous phase reservoir, and the other end is connected to the droplet generation structure.

[0057] Droplet generation structure: Figure 4 and Figure 5 As shown, the upper end of the droplet generation structure is connected to the multi-way injection channel through the mixing channel, the left and right ends of the droplet generation structure are connected to the continuous phase storage tank through the continuous phase connection channel, and the lower end of the droplet generation structure is connected to the droplet distribution branch structure.

[0058] Droplet distribution branch structure: The upstream of the droplet distribution branch structure is connected to the downstream of the droplet generation structure, and the downstream of the droplet distribution branch structure is connected to the droplet detection area, which is used to decelerate the newly generated high-speed droplets and evenly distribute them to the downstream droplet detection area. In this embodiment, the droplet distribution branch structure adopts a binary split branch cascade configuration. The droplet distribution branch structure has 8 levels of branches. The narrow width of the main channel of the droplet distribution branch structure is 20μm, and the wide width is 130μm. The widths of the first to seventh level branch channels are 65μm, 60μm, 55μm, 50μm, 45μm, 40μm, and 35μm, respectively. The number of branch channels of the last level generated is 64.

[0059] Droplet detection area: The width is 11.5mm, the length is 14.2mm, and the droplet detection pool is equipped with a microcolumn array with a diameter of 60μm and a center spacing of 160μm, which is used to construct a high-density single-layer droplet array for in situ imaging analysis or to form multiple parallel droplet queues for in situ incubation, reaction, and detection integration.

[0060] Droplet diversion structure: The droplet diversion structure is located downstream of the droplet detection area. Its main function is to guide and stabilize the flow of droplets, ensuring the smooth generation and circulation of droplets. Droplets can smoothly pass through the droplet detection area and be discharged from the sample outlet. The droplet diversion structure is a rounded rectangular structure, such as Figure 6 As shown, each rectangular strip has a length of 900 μm and a width of 90 μm, and a spacing of 2.04 mm between each strip, forming a symmetrical herringbone layout.

[0061] Outlet branch structure: The outlet branch structure is connected to the droplet detection area upstream. Figure 7 As shown, the outlet branch structure includes 5 diversion channels, of which the width of the channels on both sides is 200μm and the length is 3.6mm, and the width of the central channel is 150μm and the length is 2.6mm. The outlet branch structure is used to merge the upstream fluid. The total width of the diversion channels of the outlet branch structure is not less than the width of the main channel of the droplet distribution network. The width of each independent diversion channel is not less than 1.5 times the droplet diameter, and the independent diversion channel selects appropriate length and width parameters to ensure that the flow resistance (length-width ratio) of each diversion channel tends to be equal; the design of the outlet branch structure improves the overall performance of the micro-droplet chip, including the stability of droplet generation, the efficiency of discharge, and the reliability of experimental results.

[0062] Downstream outlet: Figure 7 As shown, the downstream sample outlet is located at the most downstream of the chip. In this embodiment, the downstream sample outlet is connected to a negative pressure generator, and the negative pressure generator acts on the micro-droplet chip, so that the pressure in the sample inlet is greater than the pressure of the downstream sample outlet.

[0063] The multi-channel synchronous injection droplet chip provided in this embodiment generates droplets by negative pressure drive. The negative pressure generator can be a simple syringe, that is, a device that generates negative pressure. The negative pressure generator used in this embodiment is a 5mL plastic syringe, and the downstream sample outlet is connected to the syringe needle section through a section of Teflon tube. Compared with positive pressure drive, a simple negative pressure generator can get rid of the positive pressure drive mode's dependence on an additional precision pump / valve control system, reducing the difficulty of operation; the most important point is that positive pressure drive requires a certain response time to adjust the pressure. During the pressure adjustment process, the incoordination of pressure is likely to cause the cross-flow of liquids, causing the sample to mix, and the purpose of controlling the reaction start time cannot be achieved. It cannot be used for rapid start of biochemical detection reactions, and the use of negative pressure drive can ensure that before the droplets are formed, the system components remain separated in the chip to prevent the reaction from starting prematurely.

[0064] The multi-channel synchronous injection droplet chip provided in the embodiment of the present invention has a design of multiple independent injection channels, which can input multiple samples simultaneously. This design allows the reaction system components to be split, and rapid mixing of reactants and precise control of reaction initiation can be achieved. On the other hand, the multi-channel synchronous injection droplet chip of the present invention integrates the operating units required for the entire process of digital analysis such as reagent storage, sample mixing, droplet generation, incubation / reaction, and droplet detection. It does not require open tube operation or droplet transfer, is easy to use, and has no pollution risk.

[0065] The parts not mentioned in this embodiment are the same as those in the first embodiment.

[0066] Embodiment 3

[0067] A general workflow for using a multi-channel synchronous injection droplet chip:

[0068] (1) Figure 8 As shown, one end of the Teflon tube is connected to a 5 mL syringe, and a small amount of continuous phase (droplet generation oil: paraffin oil + 3% ABILEM 90 (v / v) + 0.1% Triton X-100) is sucked from the other end and pushed into the chip channel from the tail outlet. The purpose of pre-wetting the channel with the oil phase is to avoid bubbles trapped in the channel gap and to facilitate the circulation of droplets.

[0069] (2) When the oil phase passes through the outlet branch structure, reaches the droplet detection area, then flows to the droplet distribution branch structure, and then flows into the continuous phase connection channel through the droplet generation structure and infiltrates into the continuous phase storage pool structure, 30 μL of continuous phase is added to the continuous phase inlet of the chip.

[0070] (3) When the oil further infiltrates from the dispersed phase connecting channel to the injection port, the reaction system components are separated and added to different dispersed phase injection chambers, or the sample and the detection reagent are respectively added to their respective inlet chambers.

[0071] (4) The downstream sample outlet is connected to the syringe needle section through a section of Teflon tube, and the piston handle is fixed at a scale of about 1.2 mL using a double tail clamp. The injection pressure gauge applies pressure to the outlet chamber of the micro-droplet chip to create a pressure difference. The dispersed phase in the inlet chamber passes through its connecting channel, and the continuous phase reagent in the continuous phase reservoir passes through its connecting channel to reach the droplet generation structure to start droplet generation.

[0072] (5) The generated droplets are decelerated by the droplet distribution branch structure and evenly distributed to the downstream droplet detection area; when the droplet detection area is filled, or the pressure difference is removed to put the droplets in a static state for in situ incubation / reaction and detection. If the reaction requires incubation, the entire chip can be incubated under suitable temperature conditions, and then the droplet detection area of ​​the microdroplet chip is moved into the focal plane of the fluorescence microscopy imaging to collect the droplet fluorescence image and the corresponding bright field image. Under this condition, the total number of droplets N is counted in the field of view of the bright field microscopy image, and the number of positive droplets (droplets containing fluorescence) in the corresponding field of view of the fluorescence image is N. P ; Further by counting the total number of droplets N in the image field of view and the number of positive droplets N containing fluorescent signals p , combined with the average droplet volume V, the nucleic acid content level C in the original sample can be calculated by the following formula. The calculation formula is as follows:

[0073]

[0074] The use of multi-channel synchronous injection micro-droplet chips can achieve precise control of the time from reaction initiation to droplet generation, minimize the quantitative error caused by time differences, and ensure the reliability of experimental results. It is suitable for some commonly used rapid reaction start-up biochemical processes in the laboratory, including but not limited to enzymatic reactions, isothermal amplification technology (such as RPA), CRISPR, etc.

[0075] 1. Enzyme-catalyzed reaction

[0076] Enzymatic detection reactions usually use the catalytic properties of enzymes to detect the presence or activity of specific molecules. Typical enzymatic reactions include: 1) β-galactosidase detection, using fluorescein-β-D-galactoside as a substrate, β-galactosidase catalyzes its hydrolysis to produce fluorescein, and the fluorescence intensity of fluorescein is proportional to the enzyme activity. 2) Enzyme-linked immunosorbent assay (ELISA), using antigen-antibody reaction and enzyme-labeled antibodies or antigens, quantitatively detecting antigens or antibodies by enzyme-catalyzing the substrate to produce a detectable signal (such as color or fluorescence). Fig. 9As shown, the enzyme and substrate of the enzymatic reaction are separated, the enzyme (streptavidin-β-galactosidase conjugate (SβG)) is added to one sample mouth, and the enzyme substrate fluorescein-β-D-galactoside (FDG) is added to the other sample mouth. The enzymatic reaction is controlled by multiple synchronous injection channels and the reaction is restarted when the mixture is packaged into the droplet, ensuring that the positive signal in each microdroplet is generated by a single independent enzymatic reaction, thereby ensuring the accuracy of the quantitative results.

[0077] 2. RPA response

[0078] RPA (recombinase polymerase amplification) reaction is a recombinase-based nucleic acid detection technology that can quickly start and replicate the target sequence at room temperature (about 25-42°C). In the RPA reaction, magnesium ions (Mg 2+ ) is an agonist of the RPA reaction. Once added to the system, the RPA reaction will start, so magnesium acetate particles are generally added last to avoid the reaction starting early and affecting the experimental results. However, the current existing digital quantitative methods have no way to better control the interval from the start of the reaction to the generation of droplets. For the quantitative detection results, the early start of amplification will produce a large quantitative error. Using the multi-channel sampling micro-droplet chip of the present invention, the components of the reaction system can be separated: first, the freeze-dried particles are dissolved in a solution consisting of 29.5μL rehydration buffer, 2.1μL forward primer (10μM), 2.1μL reverse primer (10μM), 0.6μL external probe (10μM) and 5.7μL sterile water. The mixture was then divided into two equal parts: one part was added with 5 μL of the DNA sample to be tested, and the other part was added with 500 μL of 140 mM magnesium acetate. The two solutions containing the target and magnesium acetate were then added to the two injection ports of the chip respectively. The reaction would only start when negative pressure was applied to generate droplets, that is, when the two components were mixed (v / v = 1:1). The typical droplet results are shown in Fig.10 The multi-channel sampling chip provided by the present invention can realize an automatic mixing system, reduce the risk of exposure to the environment during operations such as reaction component mixing and sample transfer, thereby reducing the possibility of contamination and improving the accuracy and reliability of experimental results.

[0079] 3. CRISPR reaction

[0080] Cas12a is an enzyme of the CRISPR-Cas system and belongs to the Class II CRISPR / Cas system. It has RNA-mediated DNA cleavage activity and can be used for nucleic acid detection. crRNA binds to Cas12a to form a complex, and the complementary pairing of crRNA is used to guide the Cas12a enzyme to recognize the target DNA sequence. Once crRNA guides Cas12a to recognize and bind to the target DNA, Cas12a will cis-cleave the target DNA double strand, and then activate its trans-cleavage activity to cut the fluorescently labeled single-stranded DNA reporter probe, thereby generating a fluorescent signal and realizing the detection of specific nucleic acid sequences.

[0081] Since the target is mixed with the CRISPR-Cas12a system under room temperature conditions, the cleavage activity of Cas12a can be quickly triggered - the cis-cleavage target sequence and then the trans-cleavage of the single-stranded reporter probe, especially in the multi-target crRNAs system to increase the trans-cleavage in the droplet, this cleavage activity that is initiated in advance before the droplet is formed is likely to cause the fragmentation of the target to be randomly distributed to different droplets as the droplets are formed one by one, causing the detection results to deviate. At the same time, the cut reporter probe will also be distributed to subsequent droplets to form a certain droplet fluorescence background. If an extremely short mixing channel design is used in the injection channel design of the droplet chip, it can hinder the mixing of the target and the Cas12a reaction system before the droplet is formed, thereby avoiding premature activation, so that each target can only be fully mixed with the Cas12a reaction system after being wrapped in the droplet, thereby bringing into play the advantages of the multi-target crRNA in enhancing the overall trans-cleavage activity in the droplet, so as to "light up" the positive droplets faster while ensuring the accuracy of the final measurement results. In order to demonstrate the corresponding design and its droplet reaction outcome, such as Fig.11 The 10 microliters of liquid added to the dispersed phase injection port on the left side of the multi-channel synchronous injection droplet chip shown contain 2μL Cas12a (3μM), 2μL crRNAs (4.5μM) and 1μL 10×Buffer, and the 10 microliters of liquid added to the dispersed phase injection port on the right side contain 2μL FQ-6C probe (20μM), 2μL DNA sample to be tested and 1μL 10×Buffer. Among them, the dispersed phase injection channel of the first droplet chip (above) is designed with a longer mixing channel, and the dispersed phase injection channel of the second droplet chip (below) is designed with an extremely short mixing channel. From the final droplet reaction results, if CRISPR-Cas12a is fully mixed with the target and activated in advance, it may eventually cause the number and brightness of positive droplets "lit up" to decrease to varying degrees relative to the case of no early activation.

[0082] The parts not mentioned in this embodiment are the same as those in the second embodiment.

[0083] In summary, the present invention provides a highly efficient integrated multi-channel synchronous sampling droplet chip, which can solve the limitations of existing detection technologies in the application of rapidly started biochemical detection reactions, achieve precise control of the time from reaction start to droplet generation, minimize quantitative errors caused by time differences, and ensure the reliability of experimental results.

[0084] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A multi-channel synchronous injection droplet chip, characterized in that: The invention comprises a multi-channel injection port structure arranged on the chip body; the multi-channel injection port structure is converged at the upstream of the droplet generation port through a mixing channel, and the fluid resistance design of the multi-channel injection channel allows multiple reaction components to be introduced simultaneously under the drive of a single pressure source, and the multiple reaction components have the same or different fluid properties and enter the downstream droplet generation structure in the same or different volume ratios, so as to form a high-throughput micro-volume reaction with highly consistent reaction components and reaction conditions; the reaction component composition and the start time of the droplet micro-volume multi-component reaction can be controlled on demand through the multi-channel injection port structure, so as to realize the precise control of the starting state of the high-throughput droplet micro-reaction.

2. A multi-channel synchronous injection droplet chip according to claim 1, characterized in that: It also includes a mixing channel, a continuous phase channel, a droplet generation structure, a droplet distribution branch structure, a droplet detection area, an outlet branch structure, and a downstream sample outlet arranged on the chip body; the multi-channel inlet structure includes at least two dispersed phase channels, the dispersed phase channel, the mixing channel, the droplet generation structure, the droplet distribution branch structure, the droplet detection area, the outlet branch structure, and the downstream sample outlet are arranged in sequence from upstream to downstream, and the continuous phase channel is connected to the droplet generation structure.

3. A multi-channel synchronous injection droplet chip according to claim 2, characterized in that: The mixing channel is used for mixing and early reaction of the components input into the dispersed phase channel; or the mixing channel is extremely short, which is used to prevent the early reaction of the components input into the dispersed phase channel.

4. A multi-channel synchronous injection droplet chip according to claim 2, characterized in that: The dispersed phase channel includes an injection port, a liquid storage tank, and a connecting channel. The continuous phase channel includes a continuous phase liquid storage tank and a connecting channel. By setting the size of the connecting channel, the mixing ratio of the reaction system components is controlled, thereby realizing automatic injection of the reaction components on demand.

5. A multi-channel synchronous injection droplet chip according to claim 1, characterized in that: By setting the relative position and geometric configuration of the mixing channel, the mixing state of the reaction system before droplet generation can be controlled.

6. A multi-channel synchronous injection droplet chip according to claim 2, characterized in that: A single pressure or driving source is applied at the injection port or downstream outlet of the multi-channel synchronous injection droplet chip to control the flow resistance ratio of the dispersed phase and the continuous phase, thereby generating micro-droplets of uniform size; the single pressure or driving source ensures that before the droplets are formed, the system components remain separated in their respective injection channels in the chip and are always injected according to the expected mixing ratio until entering the droplet generation structure.

7. A multi-channel synchronous injection droplet chip according to claim 2, characterized in that: It also includes a droplet guiding structure, which is arranged downstream inside the droplet detection area and is used to guide the generated droplets; the droplet guiding structure includes a plurality of rounded long rectangular structures, and the plurality of rounded long rectangular structures are parallel to each other and arranged in a symmetrical herringbone shape.

8. A multi-channel synchronous injection droplet chip according to claim 2, characterized in that: The outlet branch structure includes a plurality of mutually parallel shunt channels, the upstream of which is connected to the droplet detection area, and the downstream is gathered at the downstream sample outlet, ensuring that the droplets are discharged from the chip through a unified outlet.

9. A multi-channel synchronous injection droplet chip according to claim 3, characterized in that: The extremely short mixing channel length is less than or equal to the mixing channel width.

10. The analytical application of a multi-channel synchronous injection droplet chip according to any one of claims 1 to 9, characterized in that: Applied to biochemical processes that need to be started on demand, including enzymatic reactions, isothermal amplification technology, CRISPR-Cas reactions, and single-cell analysis.

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