Microfluidic devices and methods of making and using the same

Abstract

The present disclosure relates to a microfluidic device and methods of manufacturing and using the same, the microfluidic device comprising: a mixing device for mixing a plurality of sample liquids, the mixing device comprising a ring channel and a mixing valve, the ring channel comprising an inlet for inputting the sample liquids and an outlet for outputting a mixed liquid of the sample liquids, the mixing valve for controlling fluid flow in the ring channel; and a droplet generation valve connected to the outlet of the mixing device for generating droplets of the mixed liquid.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of microfluidics, and in particular to a microfluidic device and methods of making and using the same. BACKGROUND

[0002] Droplet-based microfluidic devices have emerged as a powerful alternative to traditional high-throughput screening platforms due to their operational flexibility, high-throughput characteristics, and ability to efficiently handle small fluid volumes. However, challenges related to performing custom operations on user-defined droplets often limit the utility of droplet-based microfluidic devices in screening applications involving complex workflows. Existing droplet-based microfluidic devices are difficult to quickly and accurately configure with user-desired compositions of droplets, failing to meet the needs of biochemical experiments such as enzyme-catalyzed biochemical reactions. SUMMARY

[0003] To solve the problems in the related art, the embodiments of the present disclosure provide a microfluidic device and methods of making and using the same, the microfluidic device comprising:

[0004] a mixing device for mixing a plurality of sample liquids, the mixing device comprising a ring channel and a mixing valve, the ring channel comprising an inlet and an outlet, the inlet for inputting the sample liquids, the outlet for outputting a mixed liquid of the sample liquids, the mixing valve for controlling fluid flow in the ring channel;

[0005] a droplet generation valve connected to the outlet of the mixing device for generating droplets of the mixed liquid.

[0006] According to an embodiment of the present disclosure, the microfluidic device further comprises an inlet channel, wherein:

[0007] the inlet channel is connected to the inlet of the ring channel through an inlet valve.

[0008] According to an embodiment of the present disclosure, the microfluidic device further comprises a plurality of first sample channels, wherein:

[0009] the first sample channels are for inputting first sample liquids for mixing, the plurality of first sample channels being connected to the inlet channel through a corresponding plurality of first sample valves.

[0010] According to an embodiment of the present disclosure, the microfluidic device further comprises a peristaltic pump disposed on the inlet channel for controlling an amount of sample liquid pumped into the ring channel, wherein:

[0011] the peristaltic pump is disposed between the plurality of first sample valves and the inlet valve.

[0012] According to an embodiment of the present disclosure, the microfluidic device further comprises a first waste channel and a buffer channel, wherein:

[0013] The first waste liquid channel is connected to the inlet channel through a first waste liquid valve.

[0014] The buffer liquid channel is connected to the inlet channel through a buffer liquid valve.

[0015] According to an embodiment of the present disclosure, wherein:

[0016] The peristaltic pump is arranged between the buffer liquid valve and the inlet valve, and the first waste liquid valve is arranged between the peristaltic pump and the inlet valve.

[0017] According to an embodiment of the present disclosure, wherein:

[0018] The plurality of first sample valves are arranged between the buffer liquid valve and the peristaltic pump.

[0019] According to an embodiment of the present disclosure, the microfluidic device further comprises:

[0020] At least two isolation valves are arranged on the annular channel for isolating the annular channel into at least two sections, wherein at least one section is connected to the inlet and outlet of the annular channel.

[0021] According to an embodiment of the present disclosure, the microfluidic device further comprises an outlet channel, a droplet output channel, a second waste liquid channel, wherein:

[0022] The outlet of the annular channel is connected to the outlet channel through an outlet valve;

[0023] The outlet channel is connected to the droplet output channel through the droplet generation valve;

[0024] The second waste liquid channel is connected to the outlet channel through a second waste liquid valve.

[0025] According to an embodiment of the present disclosure, the microfluidic device further comprises an oil channel, a second sample channel, a third waste liquid channel, wherein:

[0026] The oil channel is connected to the droplet output channel through an oil valve;

[0027] The second sample channel is connected to the droplet output channel through a droplet generation valve;

[0028] The third waste liquid channel is connected to the droplet output channel through a third waste liquid valve.

[0029] According to an embodiment of the present disclosure, the microfluidic device further comprises a first control circuit for controlling the valves in the microfluidic device.

[0030] According to an embodiment of the present disclosure, when a plurality of first sample liquids are configured, the first control circuit performs the following operations:

[0031] The inlet valve and a first sample valve corresponding to a designated first sample liquid among the plurality of first sample liquids are opened, and the peristaltic pump is controlled to inject the designated first sample liquid into the annular channel;

[0032] The inlet valve is closed, and the mixing valve is activated to uniformly distribute the designated first sample liquid in the mixing device;

[0033] The isolation valve is closed to enclose the designated first sample liquid in the first section, the inlet valve and the outlet valve are opened, and the designated first sample liquid in the second section is removed from the mixing device through the outlet;

[0034] The inlet valve and a first sample valve corresponding to another first sample liquid among the plurality of first sample liquids are opened, and the peristaltic pump is controlled to inject the another first sample liquid into the annular channel;

[0035] The inlet valve is closed, and the mixing valve is activated to mix the plurality of first sample liquids to obtain a mixed liquid.

[0036] According to an embodiment of the present disclosure, the first control circuit controls the droplet generation valve to alternately generate droplets of the mixed liquid and droplets of the second sample liquid.

[0037] According to an embodiment of the present disclosure, the microfluidic device further comprises a droplet storage array, the droplet storage array comprising a plurality of droplet storage units connected in sequence by a droplet channel, the droplet storage units having an upper cavity and a lower cavity communicating with each other, for filling a pair of droplets.

[0038] According to an embodiment of the present disclosure, in the working state, the upper cavity is located above the lower cavity:

[0039] The first droplet to arrive at an idle droplet storage unit enters the lower cavity of the idle droplet storage unit, and the next droplet to arrive at the idle droplet storage unit enters the upper cavity of the idle droplet storage unit;

[0040] When the upper cavity and the lower cavity of the droplet storage unit are both occupied by droplets, a subsequent droplet to arrive at the droplet storage unit is sent to the next idle droplet storage unit through the droplet channel.

[0041] According to an embodiment of the present disclosure, the pair of droplets filled in the droplet storage unit are respectively a droplet of the mixed liquid and a droplet of the second sample liquid.

[0042] According to an embodiment of the present disclosure, the droplet storage array further comprises an electrode channel for generating an electric field at the droplet storage unit to facilitate merging of the pair of droplets.

[0043] According to an embodiment of the present disclosure, the microfluidic device further comprises a second control circuit for applying square wave pulses or sinusoidal wave pulses to the electrode channel.

[0044] The present disclosure also provides a method for manufacturing a microfluidic device, the microfluidic device comprising a mixing device for mixing a plurality of sample liquids, the mixing device comprising a ring-shaped channel comprising an inlet for inputting the sample liquids and an outlet for outputting mixed liquids of the sample liquids, and a mixing valve for controlling fluid flow in the ring-shaped channel, and a droplet generation valve connected to the outlet of the mixing device for generating droplets of the mixed liquids, the method comprising:

[0045] On a first wafer, a fluid channel layer mold is manufactured using a first photoresist, the fluid channel layer comprising the ring-shaped channel;

[0046] A fluid channel layer is formed on the first wafer by the fluid channel layer mold;

[0047] On a second wafer, a control channel layer mold is manufactured using a second photoresist;

[0048] A control channel layer is formed on the second wafer by the control channel layer mold;

[0049] The fluid channel layer is peeled off from the first wafer;

[0050] The fluid channel layer and the control channel layer are aligned and bonded, and the second wafer is removed.

[0051] According to an embodiment of the present disclosure, the method further comprises:

[0052] A droplet storage array and an electrode channel of the microfluidic device are 3D printed on the first wafer.

[0053] According to an embodiment of the present disclosure, the fluid channel layer further comprises any one or more of an inlet channel, a first sample channel, a buffer channel, a first waste channel, an outlet channel, a second sample channel, a second waste channel, an oil channel, a third waste channel, a droplet output channel, a droplet channel, a first electrode channel, and a second electrode channel of the microfluidic device.

[0054] The present disclosure also provides a method for using a microfluidic device, the microfluidic device comprising a mixing device and a droplet generation valve, the mixing device being configured to mix a plurality of sample liquids, the mixing device comprising a ring-shaped channel and a mixing valve, the ring-shaped channel comprising an inlet and an outlet, the inlet being configured to input the sample liquids, the outlet being configured to output a mixed liquid of the sample liquids, the mixing valve being configured to control fluid flow in the ring-shaped channel, the droplet generation valve being connected to the outlet of the mixing device and being configured to generate droplets of the mixed liquid, the method comprising:

[0055] inputting, through the inlet, a sample liquid to be mixed into the ring-shaped channel;

[0056] controlling, through the mixing valve, sample liquid flow in the ring-shaped channel to generate a mixed liquid of the sample liquids;

[0057] delivering, through the outlet of the ring-shaped channel, the mixed liquid to the droplet generation valve;

[0058] generating, through the droplet generation valve, droplets of the mixed liquid.

[0059] According to an embodiment of the present disclosure, the microfluidic device further comprises an inlet channel and a plurality of first sample channels, the inlet channel being connected to the inlet of the ring-shaped channel through an inlet valve, the first sample channels being configured to input first sample liquids for mixing, the plurality of first sample channels being connected to the inlet channel through a corresponding plurality of first sample valves, at least two isolation valves being provided on the ring-shaped channel and configured to isolate the ring-shaped channel into at least two sections, wherein at least one section is connected to the inlet and the outlet of the ring-shaped channel, and the inputting, through the inlet, a sample liquid to be mixed into the ring-shaped channel comprises:

[0060] opening the inlet valve and a first sample valve corresponding to a designated first sample liquid, and inputting the designated first sample liquid into the ring-shaped channel;

[0061] closing the inlet valve, and starting the mixing valve to uniformly spread the designated first sample liquid in the ring-shaped channel;

[0062] closing the isolation valves to enclose the designated first sample liquid in a first section, opening the inlet valve and the outlet valve, and removing the designated first sample liquid in a second section from the ring-shaped channel through the outlet;

[0063] opening the inlet valve and a first sample valve corresponding to another first sample liquid of the plurality of first sample liquids, and inputting the another first sample liquid into the ring-shaped channel.

[0064] According to an embodiment of the present disclosure, a peristaltic pump is arranged on the inlet channel, arranged between the plurality of first sample valves and the inlet valve, for controlling the amount of sample liquid pumped into the annular channel,

[0065] The inputting of the specified first sample liquid into the annular channel includes controlling the peristaltic pump to inject the specified first sample liquid into the annular channel.

[0066] The inputting of the another first sample liquid into the annular channel includes controlling the peristaltic pump to inject the another first sample liquid into the annular channel.

[0067] According to an embodiment of the present disclosure, the microfluidic device further comprises a buffer channel connected to the inlet channel through a buffer valve, the peristaltic pump is arranged between the buffer valve and the inlet valve, the plurality of first sample valves are arranged between the buffer valve and the peristaltic pump, and the removing of the specified first sample liquid in the second section from the annular channel through the outlet includes:

[0068] Opening the buffer valve, the inlet valve and the outlet valve, injecting buffer liquid into the annular channel through the peristaltic pump, and flushing the second section with the buffer liquid to remove the specified first sample liquid in the second section from the annular channel through the outlet.

[0069] According to an embodiment of the present disclosure, the microfluidic device further comprises a first waste liquid channel connected to the inlet channel through a first waste liquid valve, the first waste liquid valve is arranged between the peristaltic pump and the inlet valve, and the method further comprises:

[0070] After the inputting of the another first sample liquid into the annular channel, closing the inlet valve, opening the buffer valve and the first waste liquid valve, flushing the inlet channel with the buffer liquid, and the generated waste liquid flows out from the first waste liquid channel.

[0071] According to an embodiment of the present disclosure, the controlling of the sample liquid flow in the annular channel through the mixing valve to generate a mixed liquid of the sample liquid includes:

[0072] Opening the isolation valve, starting the mixing valve, mixing the sample liquid in the annular channel to generate a mixed liquid of the sample liquid.

[0073] According to an embodiment of the present disclosure, the transporting of the mixed liquid to the droplet generation valve through the outlet of the annular channel includes:

[0074] Inputting buffer liquid into the annular channel, the buffer liquid pushing the mixed liquid out of the outlet of the annular channel to the droplet generation valve.

[0075] According to an embodiment of the present disclosure, the microfluidic device further comprises an outlet channel and an oil channel, an outlet of the annular channel is connected to the outlet channel through an outlet valve, the outlet channel is connected to the droplet output channel through the droplet generation valve, the oil channel is connected to the droplet output channel through an oil valve, and the droplet of the mixed liquid is generated through the droplet generation valve, comprising:

[0076] The oil valve is opened, and the opening time of the droplet generation valve is controlled to inject the mixed liquid into the oil to generate droplets of the mixed liquid.

[0077] According to an embodiment of the present disclosure, the microfluidic device further comprises a second sample channel connected to the droplet output channel through a droplet generation valve, and the method further comprises:

[0078] The oil valve is opened, and the second sample liquid is input into the droplet generation valve from the second sample channel, and the opening time of the droplet generation valve is controlled to inject the second sample liquid into the oil to generate droplets of the second sample liquid.

[0079] According to an embodiment of the present disclosure, the microfluidic device further comprises a droplet storage array comprising a plurality of droplet storage units connected in sequence by a droplet channel, the droplet storage unit has an upper cavity and a lower cavity communicating with each other, for filling a pair of droplets, and the method further comprises:

[0080] Alternately generating droplets of the mixed liquid and droplets of the second sample liquid;

[0081] Sequentially transporting a pair of droplets to the droplet storage unit through the droplet output channel, and respectively storing in the upper cavity and the lower cavity, the pair of droplets are respectively droplets of the mixed liquid and droplets of the second sample liquid.

[0082] According to an embodiment of the present disclosure, in the working state, the upper cavity is located above the lower cavity, and the sequentially transporting a pair of droplets to the droplet storage unit through the droplet output channel and respectively storing in the upper cavity and the lower cavity, comprising:

[0083] The first droplet to arrive at an idle droplet storage unit enters the lower cavity of the idle droplet storage unit, and the next droplet to arrive at the droplet storage unit enters the upper cavity of the idle droplet storage unit.

[0084] When the upper cavity and the lower cavity of the droplet storage unit are both occupied by droplets, the droplet coming to the droplet storage unit goes to the next idle droplet storage unit through the droplet channel.

[0085] According to an embodiment of the present disclosure, the droplet storage array further comprises an electrode channel, and the method further comprises:

[0086] generating an electric field at the droplet storage unit through the electrode channel to facilitate merging of the pair of droplets.

[0087] According to an embodiment of the present disclosure, the generating an electric field at the droplet storage unit through the electrode channel comprises:

[0088] applying a square wave pulse or a sine wave pulse to the electrode channel to generate an electric field at the droplet storage unit through the electrode channel.

[0089] The present disclosure also provides a method for measuring enzyme properties using a microfluidic device, the microfluidic device comprising a mixing device and a droplet generation valve, the mixing device being configured to mix a plurality of sample liquids, the mixing device comprising a ring-shaped channel and a mixing valve, the ring-shaped channel comprising an inlet and an outlet, the inlet being configured to input the sample liquids, the outlet being configured to output a mixed liquid of the sample liquids, the mixing valve being configured to control fluid flow in the ring-shaped channel, the droplet generation valve being connected to the outlet of the mixing device and configured to generate droplets of the mixed liquid, the method comprising:

[0090] mixing the plurality of sample liquids using the microfluidic device to generate a substrate mixture, the substrate being configured to react with an enzyme to test properties of the enzyme;

[0091] outputting the substrate mixture to the droplet generation valve to generate substrate droplets through the droplet generation valve.

[0092] According to an embodiment of the present disclosure, the substrate comprises any one or more of: a fluorescent substrate, a non-fluorescent substrate, an inhibitor.

[0093] According to an embodiment of the present disclosure, the microfluidic device further comprises a second sample channel, the second sample channel being connected to the droplet generation valve, the method further comprising:

[0094] outputting an enzyme solution to the droplet generation valve through the second sample channel to generate enzyme droplets from the droplet generation valve.

[0095] According to an embodiment of the present disclosure, the substrate mixture and the solution containing the enzyme are output alternately to the droplet generation valve to generate the substrate droplets and the enzyme droplets alternately.

[0096] According to an embodiment of the present disclosure, the microfluidic device further comprises a droplet storage array, the droplet storage array comprising a plurality of droplet storage units connected in sequence by droplet channels, the droplet storage units having upper and lower cavities communicating with each other and being configured to fill a pair of droplets, the method further comprising:

[0097] The pair of droplets generated by the droplet generation valve are sequentially transported to the droplet storage unit and stored in the upper chamber and the lower chamber, respectively, and the pair of droplets are a substrate droplet and an enzyme droplet, respectively.

[0098] According to an embodiment of the present disclosure, in the working state, the upper chamber is located above the lower chamber, and the sequentially transporting the pair of droplets generated by the droplet generation valve to the droplet storage unit and storing them in the upper chamber and the lower chamber, respectively, comprises:

[0099] The droplet that first reaches the idle droplet storage unit enters the lower chamber of the idle droplet storage unit, and the next droplet that reaches the droplet storage unit enters the upper chamber of the idle droplet storage unit.

[0100] When both the upper chamber and the lower chamber of the droplet storage unit are occupied by droplets, the droplet that reaches the droplet storage unit goes to the next idle droplet storage unit through the droplet channel.

[0101] According to an embodiment of the present disclosure, the droplet storage array further comprises an electrode channel, and the method further comprises:

[0102] Generating an electric field at the droplet storage unit through the electrode channel to facilitate the merging of the pair of droplets.

[0103] The microfluidic device according to the embodiment of the present disclosure can generate a mixed liquid of sample liquids by coupling a plurality of sample liquid inputs to the mixing device, accurately generate droplets of the mixed liquid by the droplet generation valve, thereby mixing a plurality of sample liquids in a fast manner and generating droplets required for experiments, and effectively meet the needs of various biochemical experiments including enzyme processes and related kinetics research.

[0104] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0105] Other features, objects, and advantages of the present disclosure will become more apparent from the following detailed description of the non-limiting embodiments, taken in conjunction with the accompanying drawings. In the drawings:

[0106] Figure 1 A structural block diagram of a microfluidic device according to an embodiment of the present disclosure is shown.

[0107] Figure 2 A process of mixing two first sample liquids according to an embodiment of the present disclosure is shown.

[0108] Figure 3 A structural schematic diagram of a droplet generation valve according to an embodiment of the present disclosure is shown.

[0109] Figures 4a to 4fA process of generating droplets and a mixture is shown according to an embodiment of the present disclosure.

[0110] Figures 5a to 5e A process of loading droplets into a droplet storage array is shown according to an embodiment of the present disclosure.

[0111] Figure 6 A process of facilitating merging of a droplet in an upper chamber and a droplet in a lower chamber by applying a voltage on an electrode channel is shown according to an embodiment of the present disclosure.

[0112] Figure 7 A flowchart of fabricating a microfluidic device is shown according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0113] Hereinafter, exemplary embodiments of the present disclosure will be described in detail with reference to the accompanying drawings so as to be easily implemented by those skilled in the art. Also, portions unrelated to describing the exemplary embodiments are omitted in the accompanying drawings for the sake of clarity.

[0114] In the present disclosure, it should be understood that terms such as "include" or "have" are intended to indicate that there are features, numbers, steps, actions, components, parts, or combinations thereof disclosed in the specification, and do not exclude the possibility that one or more other features, numbers, steps, actions, components, parts, or combinations thereof exist or are added.

[0115] It is also necessary to note that the embodiments in the present disclosure and the features in the embodiments can be combined with each other without conflict. The present disclosure will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0116] As described above, droplet-based microfluidic devices have become a powerful alternative to traditional high-throughput screening platforms due to their operational flexibility, high-throughput characteristics, and ability to efficiently handle small fluid volumes. However, challenges related to performing custom operations on user-defined droplets often limit the utility of droplet-based microfluidic devices in screening applications involving complex workflows. Existing droplet-based microfluidic devices are difficult to quickly and accurately configure droplets of user-desired components, failing to meet the needs of biochemical experiments such as enzyme-catalyzed biochemical reactions.

[0117] The inventors have found that if a microfluidic device can mix various samples in a fast manner and accurately generate droplets required for experiments, the needs of enzyme processes and their related kinetics research can be effectively met. The microfluidic device according to an embodiment of the present disclosure can also be used in scenarios such as studying the behavior of enzyme-catalyzed reactions in the presence of inhibitors, digital polymerase chain reaction (PCR), single-cell sequencing, directed evolution, and high-throughput single-cell analysis.

[0118] To this end, the present disclosure provides a microfluidic device, comprising: a mixing device for mixing a plurality of sample liquids, the mixing device comprising a ring channel and a mixing valve, the ring channel comprising an inlet for inputting the sample liquids and an outlet for outputting a mixed liquid of the sample liquids, the mixing valve for controlling fluid flow in the ring channel; and a droplet generation valve connected to the outlet of the mixing device for generating droplets of the mixed liquid.

[0119] The microfluidic device according to the embodiments of the present disclosure can utilize the input of a plurality of sample liquids coupled to the mixing device to produce a mixed liquid of the sample liquids, and precisely generate droplets of the mixed liquid through the droplet generation valve, thereby enabling the mixing of a plurality of sample liquids in a fast manner and the generation of droplets required for experiments, effectively meeting the needs of a variety of biochemical experiments including enzyme processes and related kinetics research.

[0120] Figure 1 A structural block diagram of the microfluidic device according to the embodiments of the present disclosure is shown, wherein, Figure 1 The middle part shows the overall structural block diagram of the microfluidic device, Figure 1 The left side shows the detailed block diagram of the mixing device, the droplet generation valve and other components, Figure 1 The right side shows the detailed block diagram of the droplet storage array.

[0121] As Figure 1 shown, the microfluidic device comprises a mixing device 100 for mixing a plurality of sample liquids and a droplet generation valve 200 connected to the outlet of the mixing device 100 for generating droplets of the mixed liquid. The mixing device 100 comprises a ring channel 101 and a plurality of mixing valves 102. The ring channel 101 comprises an inlet for inputting sample liquids and an outlet for outputting a mixed liquid of the sample liquids. The mixing valves 102 are used to control fluid flow in the ring channel 101. For example, the mixing valves 102 can be used to circulate and mix the fluid in the ring channel 101. The driving of the mixing valves 102 enhances mixing by rapidly changing the direction of fluid flow. After mixing is completed, the mixing valves 102 push the mixed liquid to the outlet of the ring channel 101, and then generate droplets through the droplet generation valve 200.

[0122] Figure 1 Three mixing valves 102 are shown as an example, but more or fewer mixing valves can be provided according to actual needs.

[0123] According to embodiments of the present disclosure, the annulus of the annular channel 101 can be a circular ring or an elliptical ring, and the cross section of the channel can be rectangular or semicircular. The microfluidic device further comprises an inlet channel 310 connected to the inlet of the annular channel through an inlet valve 311, and an outlet channel 320 connected to the outlet of the annular channel through an outlet valve 321.

[0124] According to embodiments of the present disclosure, the microfluidic device further comprises a plurality of first sample channels 400 connected to the inlet channel 310 through first sample valves 401. The first sample channels 400 are used to input first sample liquids for mixing. Figure 1 Four first sample channels 400 are shown in FIG. 4, but more or fewer first sample channels can be provided according to actual needs. According to embodiments of the present disclosure, different first sample channels are used to input different first sample liquids, and each first sample channel is independently addressable. By controlling the first sample valve 401 corresponding to the first sample channel 400, the first sample liquid to be delivered into the mixing device 100 can be selected.

[0125] According to embodiments of the present disclosure, the microfluidic device further comprises a peristaltic pump, such as Figure 1 As shown in FIG. 5, the peristaltic pump comprises a plurality of pumping valves 500. Figure 1 The peristaltic pump shown in FIG. 5 comprises three pumping valves 500, but more or fewer pumping valves can be provided according to actual needs.

[0126] The peristaltic pump can be provided on the inlet channel 310 for controlling the amount of sample liquid pumped into the annular channel 101. For example, as shown in Figure 1 The peristaltic pump can be provided between the plurality of first sample valves 401 and the inlet valve 311 of the annular channel 101. According to embodiments of the present disclosure, the peristaltic pump is capable of pumping sample liquid into the annular channel 101 with a resolution of picoliters (pL).

[0127] According to embodiments of the present disclosure, the microfluidic device further comprises a first waste channel 600 connected to the inlet channel 310 through a first waste valve 601, and a buffer channel 700 connected to the inlet channel 310 through a buffer valve 701. The buffer is used to wash the inlet channel 310 and the mixing device 100 during the mixing liquid configuration process to remove residual fluids and potential contaminants.

[0128] According to embodiments of the present disclosure, the peristaltic pump is further provided between the buffer valve 701 and the inlet valve 311 of the annular channel 101, and the first waste valve 601 is provided between the peristaltic pump and the inlet valve 311.

[0129] According to an embodiment of the present disclosure, the plurality of first sample valves 401 are arranged between the buffer valve 701 and the peristaltic pump.

[0130] Between each dispensing step, the mixing device 100 is repeatedly washed with buffer to remove residual fluids and potential contaminants. Valve actuation allows accurate metering of user-defined sample liquid volumes into droplets.

[0131] When pumping the specified first sample liquid into the mixing device 100, the corresponding first sample valve 401 and the inlet valve 311 of the annular channel 101 are opened, and the peristaltic pump circulates a user-defined number of cycles. To assess the accuracy and precision of fluid delivery into the mixing device 100, the accurate volume of a single shot performed by the peristaltic pump consisting of a sequence of pumping valves 500 can be determined first, which controls the minimum volume of the first sample liquid that can be pumped into the mixing device 100. By controlling the amount of first sample liquid pumped into the annular channel 101, the concentration of the mixed liquid can be accurately controlled.

[0132] According to an embodiment of the present disclosure, the microfluidic device further comprises at least two isolation valves 103 arranged on the annular channel 101 for isolating the annular channel 101 into at least two sections, wherein at least one section is connected to the inlet and outlet of the annular channel 101.

[0133] As an example, Figure 1 Two isolation valves 103 are shown in FIG. 1 to isolate the annular channel 101 into a first section and a second section, wherein the second section is connected to the inlet and outlet of the annular channel 101. According to actual needs, more isolation valves can be arranged to isolate the annular channel 101 into more sections, wherein at least one section is connected to the inlet and outlet of the annular channel 101. When the isolation valves are closed, the annular channel 101 is divided into multiple sections, different first sample liquids can be input in each section, and then the isolation valves are opened to achieve mixing of the first sample liquids.

[0134] Figure 2 The process of mixing two first sample liquids according to an embodiment of the present disclosure is shown in FIG. 2.

[0135] As Figure 2As shown, in step S1, inlet valve 311 opens, the first sample valve corresponding to the first sample solution A opens, and the peristaltic pump sprays the first sample solution A into the annular channel 101. In step S2, the peristaltic pump sprays a first specified number of times to input a first specified volume of the first sample solution A into the annular channel 101. In step S3, inlet valve 311 is closed to seal the annular channel 101, and mixing valve 102 is activated to evenly distribute the first sample solution A in the annular channel 101. In step S4, isolation valve 103 is closed to seal the first sample solution A in the first section, and inlet valve 311 and outlet valve 321 are opened to purge the first sample solution A in the second section out of the annular channel 101 through the outlet. For example, by opening buffer valve 701, inlet valve 311, and outlet valve 321, buffer solution is sprayed into the mixing device 100 by the peristaltic pump, and the second section is rinsed with buffer solution, thereby purging the first sample solution A in the second section out of the annular channel 101 through the outlet. In step S5, inlet valve 311 is opened, buffer valve 701 is closed, and the first sample valve corresponding to the first sample solution B is opened. The peristaltic pump sprays the first sample solution B into the annular channel 101 a second specified number of times to input a second specified volume of the first sample solution B into the annular channel 101. In step S6, inlet valve 311 is closed to seal the annular channel 101, isolation valve 103 is opened, and mixing valve 102 is activated to mix the first sample solution A and the first sample solution B to obtain a mixture. Buffer valve 701 and first waste liquid valve 601 are opened, and buffer solution is used to clean inlet channel 310 to remove residual sample solution. The generated waste liquid flows out from first waste liquid channel 600.

[0136] like Figure 1 As shown, according to an embodiment of this disclosure, the microfluidic device further includes a droplet output channel 800 and a second waste liquid channel 900. The outlet channel 320 is connected to the droplet output channel 800 via a droplet generation valve 200, and the second waste liquid channel 900 is connected to the outlet channel 320 via a second waste liquid valve 901. When rinsing the second stage with buffer solution, the second waste liquid valve 901 is opened, allowing waste liquid to be discharged through the second waste liquid channel 900.

[0137] During droplet generation, the mixture within the annular channel 101 is gradually replaced by a buffer solution. This buffer solution propels the mixture through the outlet channel 320 to the droplet generation valve 200. By controlling the opening time of the droplet generation valve 200, the volume and number of droplets can be controlled. For example, in one instance, when the opening time of the droplet generation valve 200 is set to 100 ms, up to 35 identical droplets can be formed. When the opening time of the droplet generation valve 200 is set to 150 ms and 200 ms, smaller numbers (28 and 16, respectively) of larger droplets are generated.

[0138] According to an embodiment of the present disclosure, the second waste liquid channel 900 is used to discharge excess mixed liquid and waste liquid generated during the mixing process of the first sample liquid.

[0139] According to an embodiment of the present disclosure, the microfluidic device further comprises an oil channel 1000, a second sample channel 1100, and a third waste liquid channel 1200. The second sample channel 1100 is connected to the droplet output channel 800 through the droplet generation valve 200.

[0140] According to an embodiment of the present disclosure, the droplet generation valve 200 allows two droplets to be generated from different inputs. One droplet is formed by the mixed liquid, and the second droplet is formed by the second sample liquid (e.g., enzyme substrate, etc.) input by the second sample channel 1100. According to an embodiment of the present disclosure, the droplet generation valve alternately generates droplets of the mixed liquid and droplets of the second sample liquid.

[0141] The oil channel 1000 is connected to the droplet output channel 800 through an oil valve 1001, which is used to input oil into the microfluidic device. The mixed liquid or the second sample liquid ejected by the droplet generation valve enters the oil phase to form droplets, and the oil carrying the droplets is output through the droplet output channel 200. The third waste liquid channel 1200 is connected to the droplet output channel 800 through a third waste liquid valve, which is used to discharge waste liquid in the droplet output channel.

[0142] Figure 3 A structural schematic diagram of a droplet generation valve according to an embodiment of the present disclosure is shown.

[0143] As shown in Figure 3 , the droplet generation valve comprises a first droplet valve and a second droplet valve. The outlet channel is connected to the oil channel through the first droplet valve, and the second sample channel is connected to the oil channel through the second droplet valve. The first droplet valve and the second droplet valve are alternately opened to alternately inject the mixed liquid in the outlet channel and the second sample liquid in the second sample channel into the oil in the oil channel, thereby alternately forming droplets of the mixed liquid and droplets of the second sample liquid. By controlling the opening time of the first droplet valve and the second droplet valve, the size of the corresponding droplet can be accurately controlled.

[0144] Figures 4a to 4f A mixed liquid and a droplet generation process of the mixed liquid according to an embodiment of the present disclosure are shown.

[0145] As shown in Figure 4a and Figure 4b , the first sample valve corresponding to the first sample liquid to be mixed is opened, and the first sample liquid to be mixed is pumped into the mixing device. Then, as shown in Figure 4c , the outlet and the inlet of the annular channel of the mixing device are closed, and as shown in Figure 4d , the first sample liquid is mixed, and the waste liquid in the inlet channel is discharged through the first waste liquid channel. After the mixing is completed, as shown in Figure 4eAs shown, the outlet valve is opened, and the mixture is pumped into the droplet generation valve shown in the rectangular dashed box. As shown, Figure 4f As shown, the droplet generation valve is kept open for a specified duration, allowing the mixture to enter the oil phase, thereby forming a droplet of the mixture and being output via the droplet output channel.

[0146] According to embodiments of the present disclosure, the minimum volume that can be metered from the droplet generation valve forms a droplet that occupies most of the cross-sectional area of the droplet output channel. Therefore, the axial length of the droplet is longer than its cross-sectional diameter, and the droplet is separated from the walls of the droplet output channel by an oil film during droplet transport.

[0147] According to embodiments of the present disclosure, the microfluidic device comprises a first control circuit for controlling the valves in the microfluidic device to operate in the above-described method. According to embodiments of the present disclosure, the valves in the microfluidic device are pneumatic valves suitable for mass production and capable of controlling fluid flow in a fast and precise manner.

[0148] Experimental results show that when using a sample of less than 1 μL, the microfluidic device according to embodiments of the present disclosure can prepare up to 100 droplets in a fast and precise manner, and the dead volume is significantly smaller (about tens of nL) compared to the syringe pump driving method.

[0149] According to embodiments of the present disclosure, the microfluidic device further comprises a droplet storage array 1300 comprising a plurality of droplet storage units 1302 connected in sequence by a droplet channel 1301, the droplet storage units 1302 having upper and lower portions communicating with each other, namely upper chamber 1302a and lower chamber 1302b, for filling a pair of droplets. In operation, the upper chamber 1302a is located above the lower chamber 1302b.

[0150] Figures 5a to 5e The process of loading droplets into the droplet storage array according to embodiments of the present disclosure is shown, wherein, Figures 5a to 5d The bright field image of a droplet storage unit and the droplet channel.

[0151] As shown, Figure 5a and Figure 5b As shown, the first droplet entering the droplet storage unit enters the lower chamber with lower piezoresistance. As shown, Figure 5c and Figure 5d As shown, when the next droplet comes to the droplet storage unit, since the lower chamber has been occupied by the previous droplet, the droplet will be loaded into the upper chamber. As shown, Figure 5e As shown, when both the upper chamber and the lower chamber of the droplet storage unit are occupied, the pressure changes in such a way that the subsequent droplet will tend to go to the next free droplet storage unit through the droplet channel.

[0152] According to an embodiment of the present disclosure, the height of the droplet channel is set to make the droplet in the droplet channel have a cyst shape, for example, 30 μm. The height of the upper cavity and the lower cavity is set to make the droplet have a spherical shape for easy capture, for example, 80 μm.

[0153] According to an embodiment of the present disclosure, the droplet storage array 1300 further comprises an electrode channel and a second control circuit. The electrode channel is arranged near the droplet storage unit, and the second control circuit applies a voltage on the electrode channel to generate an electric field in the droplet storage unit to facilitate the merging of the droplet in the upper cavity and the droplet in the lower cavity. According to an embodiment of the present disclosure, the electrode channel is filled with a 4M sodium chloride solution, constituting a liquid salt electrode.

[0154] As shown in Figure 1 The electrode channel includes a first electrode channel 1303a and a second electrode channel 1303b, which are arranged alternately in parallel, and the rows of the droplet storage units are arranged between adjacent first electrode channel 1303a and second electrode channel 1303b. After the droplets are loaded into the droplet storage array 1300, the second control circuit applies a voltage on the first electrode channel 1303a and the second electrode channel 1303b to generate an electric field at the droplet storage unit.

[0155] Figure 6 The process of facilitating the merging of the droplet in the upper cavity and the droplet in the lower cavity by applying a voltage on the electrode channel according to an embodiment of the present disclosure is shown.

[0156] As shown in Figure 6 When 100 square wave pulses with a frequency of 10 kHz and a voltage of 120 V are applied on the electrode channel, the two droplets achieve good merging, and when 100 sine wave pulses with a frequency of 10 kHz and a voltage of 120 V are applied on the electrode channel, the two droplets achieve merging, but discrete droplets appear, which shows that the merging effect of square wave pulses is more stable.

[0157] According to an embodiment of the present disclosure, since the microfluidic device integrates features of different heights, the master mold is manufactured using a multi-step photoresist coating and developing technology.

[0158] The method comprises: inputting sample liquids to be mixed into the ring-shaped channel through the inlet; controlling the flow of the sample liquids in the ring-shaped channel through the mixing valve to generate mixed liquids of the sample liquids; delivering the mixed liquids to the droplet generation valve through the outlet of the ring-shaped channel; and generating droplets of the mixed liquids through the droplet generation valve.

[0159] According to an embodiment of the present disclosure, the microfluidic device further comprises an inlet channel and a plurality of first sample channels, the inlet channel is connected to the inlet of the ring-shaped channel through an inlet valve, the first sample channels are used for inputting first sample liquids for mixing, the plurality of first sample channels are connected to the inlet channel through a plurality of corresponding first sample valves, at least two isolation valves are arranged on the ring-shaped channel to isolate the ring-shaped channel into at least two sections, wherein at least one section is connected to the inlet and the outlet of the ring-shaped channel, and the inputting of the sample liquids to be mixed into the ring-shaped channel through the inlet comprises: opening the inlet valve and a first sample valve corresponding to a specified first sample liquid, inputting the specified first sample liquid into the ring-shaped channel; closing the inlet valve, starting the mixing valve, and uniformly distributing the specified first sample liquid in the ring-shaped channel; closing the isolation valve, closing the specified first sample liquid in the first section, opening the inlet valve and the outlet valve, and removing the specified first sample liquid in the second section from the ring-shaped channel through the outlet; opening the inlet valve and a first sample valve corresponding to another first sample liquid in the plurality of first sample liquids, and inputting the another first sample liquid into the ring-shaped channel.

[0160] According to an embodiment of the present disclosure, the controlling of the flow of the sample liquids in the ring-shaped channel through the mixing valve to generate the mixed liquids of the sample liquids comprises: opening the isolation valve, starting the mixing valve, mixing the sample liquids in the ring-shaped channel, and generating the mixed liquids of the sample liquids.

[0161] As Figure 1As shown, according to embodiments of the present disclosure, a specified first sample liquid (e.g., first sample liquid A) is input through any one of the first sample channels, and the first sample liquid is enclosed in the first section. Another first sample liquid (e.g., first sample liquid B) is input through another first sample channel to the second section, and then the isolation valve is opened, and the two first sample liquids are mixed in the annular channel to generate a mixed liquid. Further, before inputting another first sample liquid, the other first sample liquid can be enclosed in the second section through the isolation valve, the other first sample liquid not enclosed is cleared, and then another first sample liquid (e.g., first sample liquid C) is input through still another first sample channel to the third section, and then the isolation valve is opened, and the three first sample liquids are mixed in the annular channel to generate a mixed liquid, and so on.

[0162] According to embodiments of the present disclosure, a peristaltic pump is arranged on the inlet channel and arranged between the plurality of first sample valves and the inlet valve, for controlling the amount of sample liquid pumped into the annular channel, the inputting of the specified first sample liquid into the annular channel comprises controlling the peristaltic pump to inject the specified first sample liquid into the annular channel, and the inputting of the another first sample liquid into the annular channel comprises controlling the peristaltic pump to inject the another first sample liquid into the annular channel.

[0163] According to embodiments of the present disclosure, by controlling the number of injection cycles of the peristaltic pump, the amount of each first sample liquid pumped into the annular channel can be controlled, so that the concentration of the mixed liquid can be accurately controlled.

[0164] According to embodiments of the present disclosure, the microfluidic device further comprises a buffer channel connected to the inlet channel through a buffer valve, the peristaltic pump is arranged between the buffer valve and the inlet valve, and the plurality of first sample valves are arranged between the buffer valve and the peristaltic pump, and the clearing of the specified first sample liquid in the second section from the annular channel through the outlet comprises: opening the buffer valve, the inlet valve and the outlet valve, injecting a buffer liquid into the annular channel through the peristaltic pump, and flushing the second section with the buffer liquid, so as to clear the specified first sample liquid in the second section from the annular channel through the outlet.

[0165] According to embodiments of the present disclosure, the microfluidic device further comprises a first waste liquid channel connected to the inlet channel through a first waste liquid valve, and the first waste liquid valve is arranged between the peristaltic pump and the inlet valve, and the method further comprises: after inputting the another first sample liquid into the annular channel, closing the inlet valve, opening the buffer valve and the first waste liquid valve, flushing the inlet channel with the buffer liquid, and the generated waste liquid flows out from the first waste liquid channel.

[0166] For example, according to an embodiment of the present disclosure, the buffer can be used to wash the mixing device and the fluid channels to remove residual fluid and potential contaminants, and the resulting waste liquid is discharged via the corresponding waste liquid channels. For example, the inlet valve 311 and each first sample valve 401 are closed, the first waste liquid valve 601 is opened, and the inlet channel 310 is flushed with the buffer, and the waste liquid is discharged from the first waste liquid channel 600. The inlet valve 311, the outlet valve 321, and the second waste liquid valve 901 are opened, and the annular channel is flushed with the buffer, and the waste liquid is discharged from the second waste liquid channel 900. The inlet valve 311, the outlet valve 321, and the third waste liquid valve are opened, and the outlet channel 320 is flushed with the buffer, and the waste liquid is discharged from the third waste liquid channel 1200.

[0167] According to an embodiment of the present disclosure, the transporting the mixed liquid to the droplet generation valve through the outlet of the annular channel comprises: inputting a buffer to the annular channel, the buffer pushing the mixed liquid out of the outlet of the annular channel to the droplet generation valve.

[0168] According to an embodiment of the present disclosure, the microfluidic device further comprises an outlet channel and an oil channel, the outlet of the annular channel is connected to the outlet channel through an outlet valve, the outlet channel is connected to the droplet output channel through the droplet generation valve, the oil channel is connected to the droplet output channel through an oil valve, and the generating the droplet of the mixed liquid through the droplet generation valve comprises: opening the oil valve, and controlling the opening time of the droplet generation valve, and ejecting the mixed liquid into the oil to generate the droplet of the mixed liquid.

[0169] According to an embodiment of the present disclosure, the microfluidic device further comprises a second sample channel, the second sample channel is connected to the droplet output channel through a droplet generation valve, and the method further comprises: opening the oil valve, inputting a second sample liquid to the droplet generation valve from the second sample channel, controlling the opening time of the droplet generation valve, and ejecting the second sample liquid into the oil to generate the droplet of the second sample liquid.

[0170] For example, after obtaining the mixed liquid, the outlet valve 321, the oil valve 1001, and the first droplet valve of the droplet generation valve 200 are opened, the opening time of the first droplet valve is controlled, and the mixed liquid is ejected into the oil to form a droplet of the mixed liquid of a specified size in the oil. Then, the first droplet valve is closed, the second droplet valve is opened, the opening time of the second droplet valve is controlled, and the second sample liquid is ejected into the oil to form a droplet of the second sample liquid of a specified size in the oil.

[0171] According to an embodiment of the present disclosure, the microfluidic device further comprises a droplet storage array comprising a plurality of droplet storage units connected in sequence by a droplet channel, the droplet storage unit having an upper chamber and a lower chamber in communication with each other for filling a pair of droplets, and the method further comprises: alternately generating droplets of the mixed liquid and droplets of the second sample liquid; and sequentially transporting a pair of droplets to the droplet storage unit through the droplet output channel and storing the pair of droplets in the upper chamber and the lower chamber, respectively.

[0172] According to an embodiment of the present disclosure, in the working state, the upper chamber is located above the lower chamber, and the sequentially transporting a pair of droplets to the droplet storage unit through the droplet output channel and storing the pair of droplets in the upper chamber and the lower chamber, respectively, comprises: when the upper chamber and the lower chamber of the droplet storage unit are both idle, the first droplet arriving at the idle droplet storage unit enters the lower chamber of the idle droplet storage unit, and the next droplet arriving at the droplet storage unit enters the upper chamber of the idle droplet storage unit; and when the upper chamber and the lower chamber of the droplet storage unit are both occupied by droplets, the droplet arriving at the droplet storage unit goes to the next idle droplet storage unit through the droplet channel.

[0173] For example, the droplet storage unit 1302 comprises an upper chamber 1302a and a lower chamber 1302b, when the upper chamber and the lower chamber of the droplet storage unit 1302 are both idle, the first droplet arriving at the droplet storage unit 1302 enters the lower chamber, and the next droplet arriving at the droplet storage unit 1302 enters the upper chamber. When the upper chamber and the lower chamber of the droplet storage unit 1302 are both occupied by droplets, the droplet arriving at the droplet storage unit 1302 goes to the next droplet storage unit adjacent to the droplet storage unit 1302 through the droplet channel 1301. According to an embodiment of the present disclosure, according to the generation order of the droplets, the droplet entering the upper chamber and the droplet entering the lower chamber can be a droplet of the mixed liquid and a droplet of the second sample liquid, respectively, or the droplet entering the upper chamber and the droplet entering the lower chamber can be a droplet of the second sample liquid and a droplet of the mixed liquid, respectively.

[0174] According to an embodiment of the present disclosure, the droplet storage array further comprises an electrode channel, and the method further comprises: generating an electric field at the droplet storage unit through the electrode channel to facilitate merging of the pair of droplets. The generating an electric field at the droplet storage unit through the electrode channel comprises: applying a square wave pulse or a sine wave pulse to the electrode channel to generate an electric field at the droplet storage unit through the electrode channel.

[0175] The method for measuring enzyme properties using a microfluidic device, the microfluidic device comprising a mixing device and a droplet generation valve, the mixing device being used for mixing a plurality of sample liquids, the mixing device comprising a ring-shaped channel and a mixing valve, the ring-shaped channel comprising an inlet and an outlet, the inlet being used for inputting the sample liquids, the outlet being used for outputting a mixed liquid of the sample liquids, the mixing valve being used for controlling fluid flow in the ring-shaped channel, the droplet generation valve being connected to the outlet of the mixing device and being used for generating droplets of the mixed liquid, the method comprising: mixing the plurality of sample liquids using the microfluidic device to generate a substrate mixed liquid, the substrate being used for reacting with an enzyme to test properties of the enzyme; outputting the substrate mixed liquid to the droplet generation valve to generate substrate droplets through the droplet generation valve.

[0176] According to embodiments of the present disclosure, a plurality of sample liquids can be mixed by a mixing device to obtain a substrate mixed liquid with a specified concentration, and then a substrate droplet can be generated by a droplet generation valve. By precisely controlling the amount of sample liquid entering the mixing device, the concentration of the substrate mixed liquid can be precisely controlled.

[0177] According to embodiments of the present disclosure, the microfluidic device further comprises a second sample channel connected to the droplet generation valve, and the method further comprises: outputting an enzyme solution to the droplet generation valve through the second sample channel to generate enzyme droplets from the droplet generation valve.

[0178] According to embodiments of the present disclosure, the substrate mixed liquid and the solution containing the enzyme are alternately output to the droplet generation valve to alternately generate the substrate droplets and enzyme droplets.

[0179] According to embodiments of the present disclosure, the microfluidic device further comprises a droplet storage array, the droplet storage array comprising a plurality of droplet storage units connected in sequence by a droplet channel, the droplet storage unit having an upper cavity and a lower cavity communicating with each other, and being used for filling a pair of droplets, and the method further comprises: sequentially transporting a pair of droplets generated by the droplet generation valve to the droplet storage unit and storing them in the upper cavity and the lower cavity respectively, the pair of droplets being a substrate droplet and an enzyme droplet respectively.

[0180] According to embodiments of the present disclosure, in a working state, the upper cavity is located above the lower cavity, and the sequentially transporting a pair of droplets generated by the droplet generation valve to the droplet storage unit and storing them in the upper cavity and the lower cavity respectively comprises: a first droplet arriving at an idle droplet storage unit entering the lower cavity of the idle droplet storage unit, and a next droplet arriving at the droplet storage unit entering the upper cavity of the idle droplet storage unit; when the upper cavity and the lower cavity of the droplet storage unit are both occupied by droplets, a droplet arriving at the droplet storage unit goes to a next idle droplet storage unit through the droplet channel.

[0181] According to an embodiment of the present disclosure, the droplet storage array further comprises an electrode channel, and the method further comprises: generating an electric field at the droplet storage unit through the electrode channel to facilitate the merging of the pair of droplets.

[0182] According to an embodiment of the present disclosure, the substrate comprises any one or more of: a fluorescent substrate, a non-fluorescent substrate, an inhibitor. Using a fluorescent substrate droplet or a non-fluorescent substrate droplet to react with an enzyme droplet can perform enzyme kinetics testing. Using a droplet containing an inhibitor and a fluorescent substrate to react with an enzyme droplet, or a droplet containing an inhibitor and a non-fluorescent substrate to react with an enzyme droplet, can test the behavior of the enzymatic reaction in the presence of an inhibitor.

[0183] Figure 7 A flowchart showing the process of manufacturing a microfluidic device according to an embodiment of the present disclosure is shown.

[0184] According to an embodiment of the present disclosure, the microfluidic device comprises a mixing device for mixing a plurality of sample liquids, the mixing device comprising a ring-shaped channel comprising an inlet for inputting the sample liquids and an outlet for outputting a mixed liquid of the sample liquids, and a mixing valve for controlling fluid flow in the ring-shaped channel; and a droplet generation valve connected to the outlet of the mixing device for generating droplets of the mixed liquid.

[0185] According to an embodiment of the present disclosure, a microfluidic channel with a semi-circular cross-section is generated by melting and reflowing of positive AZ 4620 photoresist, and a microfluidic channel with a rectangular cross-section is manufactured using SU-8 photoresist. According to an embodiment of the present disclosure, the microfluidic channel comprises any one or more of the channels for transporting fluid in the microfluidic device, such as any one or more of Figure 1 The shown inlet channel, first sample channel, buffer channel, first waste channel, outlet channel, second sample channel, second waste channel, oil channel, third waste channel, droplet output channel, ring-shaped channel, droplet channel, first electrode channel, and second electrode channel.

[0186] According to an embodiment of the present disclosure, the fluid channel layer comprises a microfluidic channel (with a rectangular or semi-circular cross-section) of 20 pm in height, a cavity of a droplet storage unit (80 pm in height), and an electrode channel of 40 pm in height.

[0187] As Figure 7As shown in section (a), a fluid channel layer mold was created by spin-coating AZ 4620 photoresist onto a silicon wafer (also known as the “first wafer”) at 3750 rpm for 40 seconds. The AZ 4620 photoresist-coated wafer was soft-baked at 126°C for 8 minutes and then cooled to ambient temperature over 10 minutes. The photoresist was patterned using a UV-KUB 3 mask aligner and exposed for an appropriate time at an incident intensity of 400 megajoules per square centimeter. The exposed wafer was then baked again at 105°C. Next, the wafer was developed in AZ300 MIF developer until the desired microstructure was visible to the naked eye. The wafer was washed in deionized water and dried under a nitrogen stream.

[0188] like Figure 7 As shown in section (b), a semi-circular cross-sectional channel feature is created by melting and reflowing AZ 4620 photoresist. In this step, the temperature is increased from ambient temperature to 65°C, heated to 150°C at a rate of 10°C / hour, and then cooled to ambient temperature.

[0189] like Figure 7 As shown in section (c), a droplet storage array and electrode channels were printed on a fluid channel layer wafer using a high-precision two-photon polymerization 3D printer. The wafer was then developed in SU-8 developer for 12 minutes, washed in isopropanol for 5 minutes, and baked in an oven at 200°C for 2 hours.

[0190] like Figure 7 As shown in section (d), a master mold for fabricating a control channel layer on a silicon wafer (also known as a "second wafer") is used with SU-8 photoresist. The control channel layer is used to control the fluid flow in the fluid channel layer. For example, the control channel has a valve at a position corresponding to the microfluidic channel that can control the opening or closing of the microfluidic channel.

[0191] According to embodiments of this disclosure, the microfluidic device is made of polydimethylsiloxane (PDMS). The fluid channel layer wafer and the control channel layer wafer are treated with trimethylchlorosilane in a vacuum chamber for 2 hours to inhibit PDMS adhesion to the silicon wafer surface. The fluid channel layer is made using a 5:1 ratio of alkali to crosslinking agent, while the control channel layer is made using a 20:1 ratio of alkali to crosslinking agent. In both cases, the alkali and crosslinking agent are thoroughly mixed together and then degassed in a vacuum dryer for 30 minutes.

[0192] like Figure 7 As shown in section (e), the 5:1 mixture was poured onto the fluid channel layer wafer and degassed. The mixture was then cured at 70°C for 15 minutes.

[0193] like Figure 7As shown in part (f) of FIG. 1, a 20: 1 mixture was spin-coated on the control layer wafer at 2600 rpm for 40 seconds and left to stand at room temperature for 2 hours.

[0194] As shown in part (g) of FIG. 1, after the mixture was cured, the fluidic channel layer was peeled off from the support wafer. Individual devices were cut out using a scalpel. The structured devices were carefully aligned with the control channel layer under a microscope, and trapped air bubbles were removed by applying pressure. After alignment, the wafer was cured again at 70 °C for 4 to 5 hours. This ensured a strong bond between the two layers due to the diffusion of the cured mixture from the control channel layer into the fluidic channel layer. Figure 7 As shown in part (h) of FIG. 1, the entire microfluidic device was then removed from the silicon wafer and holes were punched in the microfluidic device to form the required inlets and outlets. After treating both surfaces of the microfluidic device in an air plasma, the resulting microfluidic device was plasma bonded to a glass slide and kept in an oven at 70 °C for 4 to 5 hours.

[0195] ​ According to embodiments of the present disclosure, all pressure control channels of the microfluidic device were pre-filled with water and connected to solenoid valves, providing a pressure of 2 bar. Fluid flow in the fluidic channel layer was controlled using a pressure pump. The electrode channels were filled with a 4 M NaCl solution by applying a constant pressure of 0.5 bar. Copper wires were soldered to steel tubes and inserted into the electrode channel ports, connecting the electrode channels to a TREK 220 high-voltage amplifier.

[0196] It will be appreciated that, although the above describes the manufacturing process of the microfluidic device according to embodiments of the present disclosure using exemplary materials and processes, the manufacturing process of the microfluidic device according to embodiments of the present disclosure can be carried out using any suitable alternative materials and processes.

[0197] It will be appreciated that, although the above describes the manufacturing process of the microfluidic device according to embodiments of the present disclosure using exemplary materials and processes, the manufacturing process of the microfluidic device according to embodiments of the present disclosure can be carried out using any suitable alternative materials and processes.

[0198] The above description is merely exemplary of preferred embodiments of the present disclosure and of the technical principles employed. It should be understood that the scope of the application involved in the present disclosure is not limited to the technical solutions formed by the specific combinations of the above technical features, and should also cover other technical solutions formed by any combinations of the above technical features or equivalent features thereof without departing from the inventive concept. For example, the above technical features can be replaced with technical features disclosed in the present disclosure (but not limited to) having similar functions to form technical solutions.​

Claims

1. A microfluidic device, comprising: A mixing device for mixing multiple first sample solutions, the mixing device including an annular channel and a mixing valve, the annular channel including an inlet and an outlet, the inlet for inputting the first sample solutions, the outlet for outputting the mixture of the first sample solutions, and the mixing valve for controlling the fluid flow in the annular channel; The second sample channel is used to deliver the second sample solution; Oil passage, used for conveying oil; A droplet generating valve is connected to the oil channel, the outlet of the mixing device, and the second sample channel. The droplet generating valve includes a first droplet valve and a second droplet valve. By alternately opening the first droplet valve and the second droplet valve to spray the mixture and the second sample liquid into the oil in the oil channel, droplets of the mixture and droplets of the second sample liquid are alternately formed. A droplet output channel is used to output oil carrying droplets of the mixture and droplets of the second sample solution; A droplet storage array is connected to the droplet output channel. The droplet storage array includes multiple droplet storage units connected sequentially by the droplet channel. Each droplet storage unit has an upper cavity and a lower cavity that are interconnected and are used to fill a pair of droplets. The pair of droplets are a droplet of the mixture and a droplet of the second sample solution, respectively. The first droplet entering the droplet storage unit enters the lower cavity with lower pressure resistance. When the next droplet arrives at the droplet storage unit, the next droplet is loaded into the upper cavity. When both the upper and lower cavities of the droplet storage unit are occupied by droplets, subsequent droplets go to the next available droplet storage unit through the droplet channel.

2. The microfluidic device according to claim 1 further includes an inlet channel, wherein: The inlet channel is connected to the inlet of the annular channel via an inlet valve.

3. The microfluidic device according to claim 2 further includes a plurality of first sample channels, wherein: The first sample channel is used to input a first sample solution for mixing, and the plurality of first sample channels are connected to the inlet channel through a plurality of corresponding first sample valves.

4. The microfluidic device according to claim 3 further includes a peristaltic pump disposed on the inlet channel for controlling the amount of sample liquid pumped into the annular channel, wherein: The peristaltic pump is positioned between the plurality of first sample valves and the inlet valve.

5. The microfluidic device according to claim 4 further includes a first waste liquid channel and a buffer solution channel, wherein: The first waste liquid channel is connected to the inlet channel via a first waste liquid valve; The buffer channel is connected to the inlet channel via a buffer valve; The peristaltic pump is disposed between the buffer valve and the inlet valve, and the first waste liquid valve is disposed between the peristaltic pump and the inlet valve; The plurality of first sample valves are disposed between the buffer valve and the peristaltic pump.

6. The microfluidic device according to claim 5, further comprising: At least two isolation valves are disposed on the annular channel for isolating the annular channel into at least two segments, wherein at least one segment is connected to the inlet and outlet of the annular channel.

7. The microfluidic device according to claim 6 further includes an outlet channel and a second waste liquid channel, wherein: The outlet of the annular channel is connected to the outlet channel via an outlet valve; The outlet channel is connected to the droplet output channel via the droplet generating valve; The second waste liquid channel is connected to the outlet channel via a second waste liquid valve.

8. The microfluidic device according to claim 7 further includes a third waste liquid channel, wherein: The oil channel is connected to the droplet output channel via an oil valve; The second sample channel is connected to the droplet output channel via a droplet generation valve; The third waste liquid channel is connected to the droplet output channel via a third waste liquid valve.

9. The microfluidic device according to claim 8, further comprising a first control circuit for controlling the valve in the microfluidic device; When preparing a mixture of multiple first sample solutions, the first control circuit performs the following operations: Open the inlet valve and the first sample valve corresponding to the designated first sample solution among the multiple first sample solutions, and control the peristaltic pump to spray the designated first sample solution into the annular channel; Close the inlet valve and start the mixing valve to evenly distribute the designated first sample solution in the annular channel; Close the isolation valve to seal the designated first sample solution in the first section, open the inlet valve and the outlet valve to remove the designated first sample solution in the second section from the annular channel through the outlet; Open the inlet valve and the first sample valve corresponding to another first sample solution among the multiple first sample solutions, and control the peristaltic pump to spray the other first sample solution into the annular channel; Close the inlet valve and start the mixing valve to mix the multiple first sample solutions to obtain a mixture. The first control circuit controls the droplet generating valve to alternately generate droplets of the mixture and droplets of the second sample solution.

10. The microfluidic device according to claim 1: The droplet storage array also includes electrode channels for generating an electric field at the droplet storage unit to promote the merging of the pair of droplets; The microfluidic device also includes a second control circuit for applying square wave pulses or sine wave pulses to the electrode channels.

11. A method for manufacturing a microfluidic device, the microfluidic device comprising a mixing device, a second sample channel, an oil channel, a droplet generating valve, and a droplet output channel, wherein the mixing device is used to mix multiple first sample liquids, the mixing device comprising an annular channel and a mixing valve, the annular channel comprising an inlet and an outlet, the inlet being used to input the first sample liquids, the outlet being used to output the mixture of the first sample liquids, and the mixing valve being used to control the fluid flow in the annular channel; The second sample channel is used to transport the second sample liquid; the oil channel is used to transport oil; the droplet generating valve is connected to the oil channel, the outlet of the mixing device and the second sample channel; the droplet generating valve includes a first droplet valve and a second droplet valve, and by alternately opening the first droplet valve and the second droplet valve to spray the mixture and the second sample liquid into the oil in the oil channel respectively, droplets of the mixture and droplets of the second sample liquid are alternately formed; A droplet output channel is used to output oil carrying droplets of the mixture and droplets of the second sample solution; A droplet storage array, connected to the droplet output channel, includes multiple droplet storage units sequentially connected by the droplet channel. Each droplet storage unit has an upper cavity and a lower cavity that are interconnected, for filling a pair of droplets. The pair of droplets are a droplet of the mixture and a droplet of the second sample solution, respectively, and are stored in the upper cavity and the lower cavity. The first droplet entering the droplet storage unit enters the lower cavity with lower pressure resistance. When the next droplet arrives at the droplet storage unit, the next droplet is loaded into the upper cavity. When both the upper and lower cavities of the droplet storage unit are occupied by droplets, subsequent droplets go to the next available droplet storage unit through the droplet channel. The method includes: On a first wafer, a fluid channel layer mold is fabricated using a first photoresist, the fluid channel layer including the annular channel; A fluid channel layer is formed on the first wafer using the fluid channel layer mold; On the second wafer, a control channel layer mold is fabricated using a second photoresist; A control channel layer is formed on the second wafer using the control channel layer mold. The fluid channel layer is peeled off from the first wafer; The fluid channel layer and the control channel layer are aligned and bonded, and the second wafer is removed.

12. The method of claim 11, further comprising: The droplet storage array and electrode channels of the microfluidic device were 3D printed on the first wafer; The fluid channel layer further includes any one or more of the following: inlet channel, first sample channel, buffer solution channel, first waste liquid channel, outlet channel, second sample channel, second waste liquid channel, oil channel, third waste liquid channel, droplet output channel, droplet channel, first electrode channel, and second electrode channel of the microfluidic device.

13. A method of using a microfluidic device, the microfluidic device comprising a mixing device, a second sample channel, an oil channel, a droplet generating valve, an outlet channel, and a droplet output channel, wherein the mixing device is used to mix multiple first sample liquids, the mixing device comprising an annular channel and a mixing valve, the annular channel comprising an inlet and an outlet, the inlet being used to input the first sample liquids, the outlet being used to output the mixture of the first sample liquids, the outlet of the annular channel being connected to the outlet channel via an outlet valve, the mixing valve being used to control the fluid flow in the annular channel; the second sample channel being used to transport a second sample liquid; the oil channel being used to transport oil; the droplet generating valve being connected to the oil channel, the outlet of the mixing device, and the second sample channel; the droplet generating valve comprising a first droplet valve and a second droplet valve, wherein by alternately opening the first droplet valve and the second droplet valve to spray the mixture and the second sample liquid into the oil in the oil channel respectively, droplets of the mixture and droplets of the second sample liquid are alternately formed; A droplet output channel is used to output oil carrying droplets of the mixture and droplets of the second sample solution; A droplet storage array, connected to the droplet output channel, includes multiple droplet storage units sequentially connected by the droplet channel. Each droplet storage unit has an upper cavity and a lower cavity that are interconnected, for filling a pair of droplets. The pair of droplets are a droplet of the mixture and a droplet of the second sample solution, respectively, and are stored in the upper cavity and the lower cavity. The first droplet entering the droplet storage unit enters the lower cavity with lower pressure resistance. When the next droplet arrives at the droplet storage unit, the next droplet is loaded into the upper cavity. When both the upper and lower cavities of the droplet storage unit are occupied by droplets, subsequent droplets go to the next available droplet storage unit through the droplet channel. The method includes: The sample solution to be mixed is introduced into the annular channel through the inlet; The flow of sample solution in the annular channel is controlled by the mixing valve to generate a mixture of the sample solution; The mixture is delivered to the droplet generating valve through the outlet of the annular channel; Droplets of the mixture are generated through the droplet generating valve.

14. The method according to claim 13, wherein, The microfluidic device further includes an inlet channel and multiple first sample channels. The inlet channel is connected to the inlet of the annular channel via an inlet valve. The first sample channels are used to input a first sample solution for mixing. The multiple first sample channels are connected to the inlet channel via corresponding multiple first sample valves. The annular channel is provided with at least two isolation valves for isolating the annular channel into at least two segments, wherein at least one segment is connected to the inlet and outlet of the annular channel. Inputting the sample solution to be mixed into the annular channel through the inlet includes: Open the inlet valve and the first sample valve corresponding to the designated first sample solution, and input the designated first sample solution into the annular channel; Close the inlet valve and start the mixing valve to evenly distribute the designated first sample solution in the annular channel; Close the isolation valve to seal the designated first sample solution in the first section, open the inlet valve and the outlet valve to remove the designated first sample solution in the second section from the annular channel through the outlet; Open the inlet valve and the first sample valve corresponding to another first sample solution among the multiple first sample solutions, and input the other first sample solution into the annular channel.

15. The method according to claim 14, wherein, A peristaltic pump is installed on the inlet channel, positioned between the plurality of first sample valves and the inlet valve, to control the amount of sample liquid pumped into the annular channel. The step of inputting the designated first sample solution into the annular channel includes controlling the peristaltic pump to spray the designated first sample solution into the annular channel; The input of the other first sample solution into the annular channel includes controlling the peristaltic pump to spray the other first sample solution into the annular channel.

16. The method according to claim 15, wherein, The microfluidic device further includes a buffer channel connected to the inlet channel via a buffer valve. The peristaltic pump is disposed between the buffer valve and the inlet valve, and the plurality of first sample valves are disposed between the buffer valve and the peristaltic pump. The step of clearing the designated first sample solution in the second segment from the annular channel through the outlet includes: opening the buffer valve, the inlet valve, and the outlet valve; spraying buffer solution into the annular channel through the peristaltic pump; rinsing the second segment with the buffer solution; thereby clearing the designated first sample solution in the second segment from the annular channel through the outlet.

17. The method according to claim 16, wherein, The microfluidic device further includes a first waste liquid channel, which is connected to the inlet channel via a first waste liquid valve. The first waste liquid valve is disposed between the peristaltic pump and the inlet valve. The method further includes: After the other first sample solution is introduced into the annular channel, the inlet valve is closed, the buffer valve and the first waste liquid valve are opened, the inlet channel is flushed with the buffer solution, and the waste liquid generated flows out from the first waste liquid channel.

18. The method according to claim 14, wherein, The step of controlling the flow of sample solution in the annular channel through the mixing valve to generate the mixture of sample solution includes: Open the isolation valve and start the mixing valve to mix the sample solution in the annular channel to generate the sample solution mixture.

19. The method according to claim 13, wherein, The step of conveying the mixture to the droplet generating valve through the outlet of the annular channel includes: A buffer solution is introduced into the annular channel, which pushes the mixture out of the annular channel to the droplet generating valve.

20. The method according to claim 13, wherein, The microfluidic device further includes an outlet channel, the outlet of the annular channel is connected to the outlet channel via an outlet valve, the outlet channel is connected to the droplet output channel via the droplet generating valve, and the oil channel is connected to the droplet output channel via an oil valve. The generation of droplets of the mixture via the droplet generating valve includes: Open the oil valve and control the opening time of the droplet generation valve to spray the mixture into the oil to generate droplets of the mixture.

21. The method according to claim 20, wherein, The second sample channel is connected to the droplet output channel via a droplet generation valve, and the method further includes: Open the oil valve, input the second sample liquid into the droplet generating valve from the second sample channel, control the opening time of the droplet generating valve, and spray the second sample liquid into the oil to generate droplets of the second sample liquid.

22. The method according to claim 21, wherein, The method further includes: Droplets of the mixture and droplets of the second sample solution are generated alternately; A pair of droplets are sequentially transported to the droplet storage unit through the droplet output channel and stored in the upper and lower chambers respectively. The pair of droplets are droplets of the mixed solution and droplets of the second sample solution.

23. The method according to claim 22, wherein, In the working state, the upper cavity is located above the lower cavity.

24. The method according to claim 22, wherein, The droplet storage array further includes electrode channels, and the method further includes: An electric field is generated at the droplet storage unit through the electrode channel to promote the merging of the pair of droplets.

25. The method according to claim 24, wherein, The generation of an electric field at the droplet storage unit through the electrode channel includes: A square wave pulse or a sine wave pulse is applied to the electrode channel to generate an electric field at the droplet storage unit through the electrode channel.

26. A method for measuring enzyme properties using a microfluidic device, the microfluidic device comprising a mixing device, a second sample channel, an oil channel, a droplet generation valve, and a droplet output channel, wherein the mixing device is used to mix multiple first sample solutions, the mixing device comprising an annular channel and a mixing valve, the annular channel comprising an inlet and an outlet, the inlet being used to input the first sample solutions, the outlet being used to output the mixture of the first sample solutions, and the mixing valve being used to control the fluid flow in the annular channel; The second sample channel is used to transport the second sample liquid; the oil channel is used to transport oil; the droplet generating valve is connected to the oil channel, the outlet of the mixing device and the second sample channel; the droplet generating valve includes a first droplet valve and a second droplet valve, and by alternately opening the first droplet valve and the second droplet valve to spray the mixture and the second sample liquid into the oil in the oil channel respectively, droplets of the mixture and droplets of the second sample liquid are alternately formed; A droplet output channel is used to output oil carrying droplets of the mixture and droplets of the second sample solution; A droplet storage array, connected to the droplet output channel, includes multiple droplet storage units sequentially connected by the droplet channel. Each droplet storage unit has an upper cavity and a lower cavity that are interconnected, for filling a pair of droplets. The pair of droplets are a droplet of the mixture and a droplet of the second sample solution, respectively, and are stored in the upper cavity and the lower cavity. The first droplet entering the droplet storage unit enters the lower cavity with lower pressure resistance. When the next droplet arrives at the droplet storage unit, the next droplet is loaded into the upper cavity. When both the upper and lower cavities of the droplet storage unit are occupied by droplets, subsequent droplets go to the next available droplet storage unit through the droplet channel. The method includes: The various first sample solutions are mixed using a microfluidic device to generate a substrate mixture, which is used to react with an enzyme to test the properties of the enzyme. The substrate mixture is output to a droplet generating valve, through which substrate droplets are generated.

27. The method according to claim 26, wherein, The substrate includes any one or more of the following: fluorescent substrate, non-fluorescent substrate, and inhibitor; The microfluidic device further includes a second sample channel connected to the droplet generation valve, and the method further includes: outputting an enzyme solution to the droplet generation valve through the second sample channel to generate enzyme droplets from the droplet generation valve; The substrate mixture and the enzyme solution are alternately output to the droplet generation valve to alternately generate substrate droplets and enzyme droplets.

28. The method according to claim 27, wherein, The method further includes: A pair of droplets generated by the droplet generating valve are sequentially transported to the droplet storage unit and stored in the upper and lower chambers, respectively. The pair of droplets are a substrate droplet and an enzyme droplet.

29. The method according to claim 28, wherein, In the working state, the upper cavity is located above the lower cavity.

30. The method according to claim 29, wherein, The droplet storage array further includes electrode channels, and the method further includes: An electric field is generated at the droplet storage unit through the electrode channel to promote the merging of the pair of droplets.

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