Volume fixing method, microfluidic volume fixing module, library building system and library building method

Abstract

The invention discloses a constant volume method, a microfluidic constant volume module, a library building system and a library building method, and belongs to the technical field of microfluidics. The constant volume method is applied to a microfluidic constant volume module, the microfluidic constant volume module comprises a first constant volume cavity, a constant volume feeding channel and an exhaust channel, and a feeding valve is arranged between the constant volume feeding channel and the first constant volume cavity; an exhaust valve is arranged between the exhaust channel and the first fixed containing cavity. The method comprises the steps that S1, the exhaust valve is closed, and the feeding valve is opened, so that fed materials enter the first constant-volume cavity through the constant-volume feeding channel; s2, after the feeding valve is closed, the exhaust valve is opened, so that gas in the first fixed containing cavity is exhausted through the exhaust channel; and S3, circularly executing the step S1 and the step S2 for set times until the first fixed containing cavity is filled with the fed material.

Description

Technical Field

[0001] The invention relates to the field of microfluidics, and in particular to a volume determination method, a microfluidics volume determination module, a library construction system, and a library construction method. Background Art

[0002] DNA sequencing library construction is a process of performing a series of treatments on the DNA samples to be sequenced to eventually form a fixed pattern. Among them, DNA sequencing refers to the analysis of the base sequence of a specific DNA fragment, that is, the arrangement of adenine (A), thymine (T), cytosine (C) and guanine (G). The emergence of rapid DNA sequencing methods has greatly promoted research and discovery in biology and medicine. Microfluidics technology is a technology that uses microchannels with a size of tens to hundreds of microns to process and manipulate tiny samples below picoliters. In the DNA sequencing library construction operation, microfluidics technology is used to perform library construction and other processing on the samples to be sequenced.

[0003] The microfluidic technology used in the prior art, especially the microfluidic technology in the DNA library construction process, has the defects of complex structure and cumbersome operation, which greatly limits the efficiency of DNA library construction and has room for further improvement. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defect of complicated volume treatment operation in the prior art, and provide a volume determination method, a microfluidic volume determination module, a library construction system, and a library construction method. The present invention solves the above technical problem through the following technical solutions:

[0005] In a first aspect, the present invention provides a constant volume method, which is applied to a microfluidic constant volume module, wherein the microfluidic constant volume module comprises a first constant volume chamber, a constant volume feed channel and a first exhaust channel, wherein a feed valve is provided between the constant volume feed channel and the first constant volume chamber, and the feed valve is used to control the on-off of the constant volume feed channel and the first constant volume chamber; an exhaust valve is provided between the first exhaust channel and the first constant volume chamber, and the exhaust valve is used to control the on-off of the first exhaust channel and the first constant volume chamber; the method comprises:

[0006] S1, closing the exhaust valve and opening the feed valve to allow the feed to enter the first constant volume chamber through the constant volume feed channel;

[0007] S2, closing the feed valve and then opening the exhaust valve, so that the gas in the first constant volume chamber is discharged from the first exhaust channel;

[0008] S3, cyclically executing step S1 and step S2 for a set number of times until the feed fills the first constant volume chamber.

[0009] In one embodiment, the constant volume feed channel includes a feed port, and a first feed valve is provided between the feed port and the first constant volume chamber, and the first feed valve is used to control the on-off between the feed port and the first constant volume chamber; the step S1 includes:

[0010] The exhaust valve is closed and the first feed valve is opened, and a set pressure is applied to the feed through a liquid adding device so that the feed enters the first constant volume chamber through the feed inlet.

[0011] In one embodiment, the constant volume module further includes a second constant volume chamber and a third constant volume chamber.

[0012] The two ends of the second constant volume chamber are respectively connected to the constant volume feed channel and the third constant volume chamber.

[0013] The second constant volume chamber is filled with a first feed material, and the third constant volume chamber is filled with a second feed material;

[0014] The feed valve comprises a second valve communicating with the second constant volume chamber and the constant volume feed channel, and a third valve communicating with the second constant volume chamber and the third constant volume chamber;

[0015] The step S1 comprises:

[0016] The exhaust valve is closed and the second valve and the third valve are opened, so that the second feed in the third constant volume chamber pushes the first feed in the second constant volume chamber and enters the first constant volume chamber through the constant volume feed channel.

[0017] In a second aspect, the present invention provides a microfluidic constant volume module, which is used to implement the constant volume method described in the first aspect; the constant volume module comprises:

[0018] The first constant volume chamber,

[0019] A constant volume feeding channel, wherein the constant volume feeding channel is connected to the first constant volume chamber,

[0020] a first exhaust channel, wherein the first exhaust channel is connected to the first constant volume chamber;

[0021] A feed valve is provided between the constant volume feed channel and the first constant volume chamber, and the feed valve is used to control the opening and closing of the first constant volume chamber and the constant volume feed channel;

[0022] An exhaust valve is provided between the first exhaust passage and the first constant volume chamber, and the exhaust valve is used to control the opening and closing of the first constant volume chamber and the first exhaust passage.

[0023] In one embodiment, the constant volume feed channel includes a first constant volume feed channel, the first constant volume feed channel is provided with a feed port, and the feed port is used to connect a liquid adding device;

[0024] The feed valve comprises a first valve disposed between the feed port and the first constant volume chamber, and the first valve is used to control the opening and closing of the feed port and the first constant volume chamber.

[0025] In one embodiment, the constant volume module further includes a second constant volume chamber and a third constant volume chamber, the two ends of the second constant volume chamber are respectively connected to the constant volume feed channel and the third constant volume chamber, the second constant volume chamber is filled with the first feed, and the third constant volume chamber is filled with the second feed;

[0026] The feed valve includes a second valve disposed between the second constant volume chamber and the constant volume feed channel, and a third valve disposed between the second constant volume chamber and the third constant volume chamber.

[0027] The second valve is used to control the opening and closing of the second constant volume chamber and the constant volume feeding channel.

[0028] The third valve is used to control the opening and closing of the second constant volume chamber and the third constant volume chamber.

[0029] In one embodiment, a second constant volume feed channel is further provided between the second constant volume chamber and the first constant volume chamber, the second constant volume feed channel is connected to the second constant volume chamber, and is used to connect a liquid adding device to fill the first feed into the second constant volume chamber;

[0030] A third constant volume feed channel is provided between the third constant volume chamber and the second constant volume chamber, the third constant volume feed channel is connected to the third constant volume chamber, and is used to connect a liquid adding device to fill the second feed into the third constant volume chamber;

[0031] A second exhaust passage is provided on a side of the third constant volume chamber away from the second constant volume chamber, and the second exhaust passage is connected to the second constant volume chamber through the third constant volume chamber.

[0032] A fourth valve is provided between the second exhaust channel and the third constant volume chamber, and the fourth valve is used to control the opening and closing of the second exhaust channel and the third constant volume chamber.

[0033] In one embodiment, at least one row of columns is disposed in the first constant volume cavity, and the interval between adjacent columns is less than or equal to a set distance.

[0034] In one embodiment, the microfluidic constant volume module includes a valve body layer, a spacer layer and a fluid layer which are stacked;

[0035] The first constant volume chamber is arranged in the fluid layer, the feed channel and the first exhaust channel penetrate the valve body layer and the spacer layer, and extend to the fluid layer;

[0036] The feed valve and the exhaust valve are arranged on the valve body layer, and include an inlet communicating with the outside of the valve body layer, and a valve body located inside the valve body layer;

[0037] The fluid layer is provided with a cut-off position, which is provided corresponding to the valve body and is used to fit with the spacer layer deformed under pressure to block the feed channel or the first exhaust channel.

[0038] In a third aspect, the present invention provides a library construction system, which includes the microfluidic constant volume module described in the second aspect.

[0039] In one embodiment, the system includes a PCR mixing and constant volume module and / or a sequencing adapter connection module;

[0040] The PCR mixing constant volume module and / or the sequencing adapter connection module comprises the microfluidic constant volume module according to any one of claims 4 to 9.

[0041] In one embodiment, the library construction system further includes a purification module, and the purification module includes:

[0042] A purification chamber, used for containing a sample to be purified;

[0043] A purification feed channel connected to the purification chamber, a fifth valve is provided between the purification feed channel and the purification chamber, and the fifth valve is used to control the on-off of the purification chamber and the purification feed channel;

[0044] The purification discharge channel is connected to the purification chamber, and a sixth valve is arranged between the purification discharge channel and the purification chamber. The sixth valve is used to control the on-off of the purification chamber and the purification discharge channel.

[0045] In one embodiment, the purification feed channel comprises a microbead feed channel, and a column is disposed in the purification chamber, and the column is used to block the microbeads entering the purification chamber.

[0046] In one embodiment, the purification feed channel comprises a lysate feed channel to inject lysate into the purification chamber; and / or

[0047] The purification feed channel comprises a microbead feed channel for injecting microbeads into the purification chamber; and / or

[0048] The purification feed channel includes a cleaning liquid channel to inject a cleaning liquid into the purification chamber; and / or

[0049] The purification feed channel includes an eluent channel for injecting eluent into the purification chamber.

[0050] In one embodiment, the purification module comprises a valve body layer, a spacer layer and a fluid layer arranged in a stacked manner;

[0051] The purification chamber is arranged in the fluid layer, and the purification feed channel and the purification discharge channel penetrate the valve body layer and the spacer layer and extend to the fluid layer;

[0052] The fifth valve and the sixth valve are arranged in the valve body layer, including an inlet connected to the outside of the valve body layer, and a valve body located inside the valve body layer; a stop position is arranged in the fluid layer, and the stop position is arranged corresponding to the valve body, and is used for being deformed under pressure to fit with the spacer layer to block the purification feed channel or the purification discharge channel.

[0053] In one embodiment, the library construction system further includes a purification module, and the purification module includes:

[0054] A sample container, used for containing a sample to be purified;

[0055] An extraction part, wherein the extraction part is provided with an extraction filter membrane for enriching and purifying the sample to be purified;

[0056] A suction component and a control valve, wherein the suction component is connected to the sample container and the extraction part through the control valve, and the suction component is used to cooperate with the control valve to suck the sample from the sample container and then pump it into the extraction part.

[0057] In one embodiment, the library construction system further includes a PCR module, and the PCR module includes:

[0058] An inlet port, used for introducing a sample to be processed;

[0059] a denaturation zone pipeline, connected to the injection port;

[0060] a circulation zone pipeline, connected to the denaturation zone and including a plurality of groups of circulation unit pipelines; and

[0061] The sample outlet is connected to the circulation zone pipeline and is used to discharge the processed sample.

[0062] In one embodiment, the circulation unit pipeline includes a first pipeline and a second pipeline that are connected to each other, and the first pipeline is also connected to the denaturation zone pipeline; the processing temperature of the first pipeline is higher than the processing temperature of the second pipeline.

[0063] In one embodiment, the library construction system also includes a sample pool, which is used to receive samples discharged from the previous module. The sample pool includes: an inlet pipe, a sample container and an outlet pipe. A sealing plug is provided in the sample container, and the inlet pipe and the outlet pipe are arranged through the sealing plug.

[0064] In a fourth aspect, the present invention provides a library construction method, which is implemented using the library construction system provided in the third aspect.

[0065] In one embodiment, the method includes: a volume setting step performed according to the volume setting method described in the first aspect above.

[0066] In one embodiment, the method further includes a purification step implemented by a purification module in the library construction system, and the library construction method further includes: performing the volume determination step while performing the purification step.

[0067] In one embodiment, the purification step is a sample purification step, which is used to process the sample to be purified to obtain a purified sample.

[0068] The volume determination step is a PCR volume determination step, which is used to process the purified sample to obtain a PCR mixed sample;

[0069] or,

[0070] The purification step is a PCR purification step, which is used to purify the PCR mixed sample to obtain a PCR purified sample.

[0071] The volume determination step is a step of connecting sequencing adapters, which is used to perform adapter connection on the PCR purified sample.

[0072] The positive and progressive effects of the present invention are:

[0073] The constant volume method provided by the present invention realizes cyclic pressure relief by opening and closing the feed valve and the exhaust valve to complete the constant volume treatment of the feed. The overall operation is simple and easy to implement, which solves the problem of complex operation in the related technology. In addition, the microfluidic constant volume module based on it has a simple structure and is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 is a structural schematic diagram of a microfluidic constant volume module according to an exemplary embodiment;

[0075] Figure 2 is a flow chart of a volume determination method according to an exemplary embodiment;

[0076] Figure 3 is a structural diagram of a microfluidic constant volume module according to another exemplary embodiment;

[0077] Figure 4 is a structural diagram of a microfluidic constant volume module according to another exemplary embodiment;

[0078] Figure 5 is a schematic diagram of a microfluidic constant volume module according to another exemplary embodiment;

[0079] Fig. 6A and Figure 6B is a schematic diagram of a microfluidic stop valve in different states according to an exemplary embodiment;

[0080] Fig. 7A is a schematic diagram of a microfluidic constant volume module according to another exemplary embodiment;

[0081] Figure 7B is a schematic diagram of a microfluidic constant volume module according to another exemplary embodiment;

[0082] Figure 8 is a schematic diagram of a library building system according to an exemplary embodiment;

[0083] Fig. 9 is a schematic diagram of a PCR module according to an exemplary embodiment;

[0084] Fig.10 is a schematic diagram of a first purification module according to an exemplary embodiment;

[0085] Fig.11 is a schematic diagram of a second purification module according to an exemplary embodiment;

[0086] Fig.12 is a schematic diagram of a purification module according to an exemplary embodiment;

[0087] Fig.13 is a schematic diagram of a purification module according to an exemplary embodiment. DETAILED DESCRIPTION

[0088] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0089] Example 1

[0090] Embodiment 1 of the present invention provides a volume determination method, which is applied to a microfluidic volume determination module. Before describing the volume determination method, the structure of the microfluidic volume determination module is first introduced.

[0091] Figure 1 FIG. 1 is a schematic diagram of the structure of a microfluidic constant volume module according to an exemplary embodiment. Figure 1As shown, the microfluidic constant volume module includes a first constant volume chamber 111, a constant volume feed channel 120 and a first exhaust channel 130a. A feed valve 121 is provided between the constant volume feed channel 120 and the first constant volume chamber 111, and the feed valve 121 is used to control the on-off of the constant volume feed channel 120 and the first constant volume chamber 111. An exhaust valve 131 is provided between the first exhaust channel 130a and the first constant volume chamber 111, and the exhaust valve 131 is used to control the on-off of the first exhaust channel 130a and the first constant volume chamber 111.

[0092] Figure 2 FIG. 1 is a flow chart of a method for determining volume according to an exemplary embodiment. Figure 2 As shown, the volume determination method provided in Example 1 of the present invention includes:

[0093] Step S1, close the exhaust valve and open the feed valve to allow the feed to enter the first constant volume chamber through the constant volume feed channel.

[0094] Combination Figure 1 In step S1, the exhaust valve 131 is closed and the feed valve 121 is opened, and the feed is added to the first constant volume chamber 111 through the feed channel 120 by using the liquid adding device. In this process, as the feed continuously enters the first constant volume chamber 111, the air pressure in the first constant volume chamber 111 gradually increases, forming an air pressure difference with the environment outside the microfluidic constant volume module.

[0095] Step S2, after closing the feed valve, open the exhaust valve to allow the gas in the first constant volume chamber to be discharged from the first exhaust channel.

[0096] Combination Figure 1 In step S2, the feed valve 121 is closed and the exhaust valve 131 is opened. At this time, under the action of pressure, the gas in the first constant volume chamber 111 is discharged through the first exhaust channel 130a.

[0097] Step S3, cyclically executing steps S1 and S2 for a set number of times until the feed fills the first constant volume chamber.

[0098] Repeat step S1 and step S2 to achieve material filling by using the pressure difference between the inside and outside of the first constant volume chamber 111 until the first constant volume chamber 111 is full. Since the volume of the first constant volume chamber 111 is fixed, the constant volume process is completed by performing step S1 and step S2 multiple times.

[0099] In a specific example, the volume of the first constant volume chamber 111 is Tμl. In step S1, the pressure applied to the feed is xpsi, and the feed will quickly flow into the first constant volume chamber 111. Volume.

[0100] In step S2, the feed valve 121 is closed and the exhaust valve 131 is opened to discharge the gas in the first constant volume chamber 111 until the air pressure in the first constant volume chamber 111 reaches equilibrium with the external air pressure. Then, step S1 and step S2 are cyclically executed until the first constant volume chamber 111 is filled with feed.

[0101] The constant volume method provided in Example 1 of the present invention is implemented based on a microfluidic constant volume module. The overall method realizes cyclic pressure relief by opening and closing the feed valve 121 and the exhaust valve 131, evacuates the gas in the first constant volume chamber 111, realizes precise liquid control, and completes the constant volume processing of the feed. The overall operation is simple and easy to implement, which solves the problem of complex operation in the related art.

[0102] In the embodiment of the present invention, there are multiple ways to add feed to the first constant volume chamber 111 through the feed channel 121, which are described in detail below with reference to the accompanying drawings.

[0103] Figure 3 is a structural diagram of a microfluidic constant volume module according to another exemplary embodiment, such as Figure 3 As shown, the constant volume feed channel 120 includes a feed port 122, and a first feed valve 121a is provided between the feed port 122 and the first constant volume chamber 111. The first feed valve 121a is used to control the on-off between the feed port 122 and the first constant volume chamber 111. At this time, step S1 includes: closing the exhaust valve 131 and opening the first feed valve 121a, applying a set pressure to the feed through a liquid adding device, so that the feed enters the first constant volume chamber 111 through the feed port 122. In this example, liquid is added to the first constant volume chamber 111 directly through the feed port 122 using a liquid adding device.

[0104] Figure 4 is a structural diagram of a microfluidic constant volume module according to another exemplary embodiment, such as Figure 4 As shown, the constant volume module also includes a second constant volume chamber 112 and a third constant volume chamber 113. The two ends of the second constant volume chamber 112 are respectively connected to the constant volume feed channel 120 and the third constant volume chamber 113. The second constant volume chamber 112 is filled with the first feed, and the third constant volume chamber 113 is filled with the second feed. The feed valve 121 includes a second valve 121b connecting the second constant volume chamber 112 and the constant volume feed channel 120, and a third valve 121c connecting the second constant volume chamber 112 and the third constant volume chamber 113.

[0105] Combination Figure 4 In the microfluidic constant volume module shown, step S1 includes: closing the exhaust valve 131 and opening the second valve 121b and the third valve 121c, so that the second feed in the third constant volume chamber 113 pushes the first feed in the second constant volume chamber 112 and enters the first constant volume chamber 111 through the constant volume feed channel 120.

[0106] In this example, the pressure difference between the inside and outside of the constant volume chamber is used to achieve the mixing and constant volume of different feeds. Among them, the second constant volume chamber 112 is also connected to the second exhaust channel and the feed channel for filling the first feed, and the third constant volume chamber 113 is also connected to the third exhaust channel and the feed channel for filling the second feed. In this way, by cycling the above steps S1 and S2, the constant volume of the first feed and the second feed is completed.

[0107] In summary, the volume determination method provided in Example 1 of the present invention uses a microfluidic positioning module to solve the defects of being complicated and difficult to operate in the related art. The overall solution is easy to implement, simple to operate, and can achieve precise liquid control, thereby improving the efficiency of the volume determination method and the overall process including the volume determination method.

[0108] Example 2

[0109] Embodiment 2 of the present invention provides a microfluidic volume control module, which is used to implement the volume control method provided in the above embodiment 1. Figure 1 The constant volume module includes a first constant volume chamber 111, a constant volume feed channel 120 and a first exhaust channel 130a. The constant volume feed channel 120 is connected to the first constant volume chamber 111. In addition, a feed valve 121 is provided between the constant volume feed channel 120 and the first constant volume chamber 111, and the feed valve 121 is used to control the on-off of the first constant volume chamber 111 and the constant volume feed channel 120. The first exhaust channel 130a is connected to the first constant volume chamber 111, and an exhaust valve 131 is provided between the first exhaust channel 130a and the first constant volume chamber 111. The exhaust valve 131 is used to control the on-off of the first constant volume chamber 111 and the first exhaust channel 130a.

[0110] The microfluidic constant volume module supports the constant volume method provided in Example 1. When used, the feed valve 121 and the exhaust valve 131 are opened and closed according to the constant volume method to achieve feed constant volume. The module has a simple structure and is easy to use, which solves the defects of complex hardware module structure and cumbersome operation used in microfluidic technology in related technologies.

[0111] In one example, see Figure 3 The constant volume feed channel 120 includes a first constant volume feed channel 120a, and the first constant volume feed channel 120a is provided with a feed port 122, and the feed port 122 is used to connect the liquid adding device. The feed valve 121 includes a first feed valve 121a provided between the feed port 122 and the first constant volume chamber 111, and the first feed valve 121a is used to control the on-off of the feed port 122 and the first constant volume chamber 111. In this way, when the first feed valve 121a is opened, the feed is directly added to the feed port 122 through the liquid adding device.

[0112] In one example, Figure 4is a schematic diagram of a constant volume module according to another exemplary embodiment, Figure 4 As shown, the constant volume module also includes a second constant volume chamber 112 and a third constant volume chamber 113, and the two ends of the second constant volume chamber 112 are connected to the constant volume feed channel 120 and the third constant volume chamber 113 respectively. In addition, the second constant volume chamber 112 is filled with the first feed, and the third constant volume chamber 113 is filled with the second feed. The feed valve 121 includes a second valve 121b arranged between the second constant volume chamber 112 and the constant volume feed channel 120, and a third valve 121c arranged between the second constant volume chamber 112 and the third constant volume chamber 113. The second valve 121b is used to control the on-off of the second constant volume chamber 112 and the constant volume feed channel 120, and the third valve 121c is used to control the on-off of the second constant volume chamber 112 and the third constant volume chamber 113.

[0113] In this way, by opening the second valve 121b and the third valve 121c, the first feed in the second constant volume chamber 112 and the second feed in the third constant volume chamber 113 enter the first constant volume chamber 111, achieving mixed feed constant volume of the first feed and the second feed.

[0114] Figure 5 is a schematic diagram of a constant volume module according to another exemplary embodiment, Figure 5 As shown, a second constant volume feed channel 120b is further provided between the second constant volume chamber 112 and the first constant volume chamber 111, and the second constant volume feed channel 120b is connected to the second constant volume chamber 112 and is used to connect the liquid adding device to fill the first feed into the second constant volume chamber 112. A first control valve 121e is provided on the second constant volume feed channel 120b.

[0115] A third constant volume feed channel 120c is provided between the third constant volume chamber 113 and the second constant volume chamber 112, and the third constant volume feed channel 120c is connected to the third constant volume chamber 113, and is used to connect a liquid adding device to fill the second feed into the third constant volume chamber 113. A second control valve 121f is provided on the third constant volume feed channel 120c. In addition, a second exhaust channel 130b is provided on the side of the third constant volume chamber 113 away from the second constant volume chamber 112, and the second exhaust channel 130b is connected to the second constant volume chamber 112 through the third constant volume chamber 113. A fourth valve 121d is provided between the second exhaust channel 130b and the third constant volume chamber 113, and the fourth valve 121d is used to control the opening and closing of the second exhaust channel 130b and the third constant volume chamber 113.

[0116] In this way, in the process of realizing the first feed constant volume through the second constant volume chamber 112, the second control valve 121f is turned off to block the third constant volume feed channel 120c, and the second exhaust channel 130b is used as the exhaust channel. In the process of realizing the second feed constant volume through the third constant volume chamber 113, the first control valve 121e is turned off to block the second constant volume feed passage 120b. And the second exhaust channel 130b is also used as the exhaust channel. In other words, the second constant volume chamber 112 and the third constant volume chamber 113 share the second exhaust channel 130b. In this way, the number of exhaust channels is reduced, thereby simplifying the structure of the microfluidic constant volume module and reducing the module hardware manufacturing cost.

[0117] In one example, at least one row of columns 140 is provided in the first constant volume chamber 111, and the interval between adjacent columns 140 is less than or equal to the set distance. Through the blocking effect of the columns 140 and the surface tension of the liquid, the first feed and the second feed can be evenly mixed after entering the first constant volume chamber 111. Among them, the arrangement and number of the columns 140 are not specifically limited. For example, two rows of columns 140 are provided in the first constant volume chamber 111, and each row of columns 140 is arranged in an arc shape.

[0118] In one example, the feed valve and the exhaust valve of the microfluidic constant volume module are microfluidic stop valves. Fig. 6A and Figure 6B is a schematic diagram of the structure of a microfluidic stop valve in different states according to an exemplary embodiment. Fig. 6A and Figure 6B As shown, the overall microfluidic constant volume module includes a stacked valve body layer 210, a spacer layer 220 and a fluid layer 230. Among them, the constant volume chamber (the first constant volume chamber 111 is taken as an example in the figure), the feed channel (the first constant volume feed channel 120a is taken as an example in the figure), and the exhaust channel (the first exhaust channel 130a is taken as an example in the figure) are arranged in the fluid layer 230. In addition, the feed channel and the exhaust channel penetrate the valve body layer 210 and the spacer layer 220, and extend to the fluid layer 230. On the valve body layer 210, the feed channel is formed with a feed port, and the exhaust channel is formed with an exhaust port.

[0119] The feed valve and the exhaust valve are arranged in the valve body layer 210. Taking the feed valve 121 in the figure as an example, the feed valve 121 includes an inlet 1211 connected to the outside of the valve body layer 210, and a valve body 1212 located inside the valve body layer 210. A stop position 231 is also arranged in the fluid layer 230, and the stop position 231 is arranged corresponding to the valve body 1212. The spacer layer 220 is located between the valve body layer 210 and the fluid layer 230. At the valve body 1212 and the stop position 231, the spacer layer 220 is used to be deformed by the fluid in the valve body 1212 until it fits with the stop position 231 (such as Figure 6B As shown), the feed channel or the exhaust channel is blocked.

[0120] When the microfluidic stop valve is in use, the working fluid is passed through the conduit into the inlet 1211 on the valve body layer 210 and enters the valve body 1212. By applying pressure to the working fluid, the spacer layer 220 at the valve body 1212 is compressed and deformed, and the spacer layer 220 gradually approaches the fluid layer 230 until it fits with the stop position 231 (such as Figure 6B When the pressure on the working fluid is removed, the spacer layer 220 of the valve body 1212 recovers its deformation, and the cut-off position 231 of the fluid layer recovers the flow of the fluid in the fluid channel in the fluid layer 230 (as shown in FIG. Fig. 6A shown).

[0121] When manufacturing the integral microfluidic stop valve, the valve body layer 210 is made into a template by negative photoresist soft lithography, and then the valve body 1212 is made by casting and demolding polydimethylsiloxane (PDMS). The spacer layer 220 is formed by spin coating PDMS on a silicon wafer and then heating and curing. The fluid layer 230 is made into its cavity area (including a constant volume cavity, a fluid channel, and an exhaust channel) by negative photoresist soft lithography, and then positive photoresist soft lithography is used and then continued to heat to soften the positive photoresist to form a stop position 231 with an arc-shaped cross-sectional feature. In addition, optionally, the valve body layer 210 is preferentially made, and a punch is used to punch out the feed port, exhaust port, and valve body inlet on the valve body layer 210. It is then bonded to the spacer layer 220, and a punch is used to punch out the opening on the spacer layer 220, and finally aligned and bonded to the fluid layer 230.

[0122] In summary, the microfluidic constant volume module provided in Example 2 of the present invention can realize constant volume of feed by controlling the feed valve and the exhaust valve according to the constant volume method provided in Example 1. The overall microfluidic constant volume module has a simple structure and is easy to implement.

[0123] In this embodiment 2, the following specific examples are provided to illustrate the structure of the microfluidic constant volume module.

[0124] Fig. 7A is a schematic diagram of a microfluidic constant volume module according to another exemplary embodiment. Fig. 7A The microfluidic constant volume module shown is a PCR constant volume module, which is used for PCR constant volume. Taking the PCR constant volume module for processing DNA samples as an example, the function of the PCR constant volume module is to mix and constant volume the purified DNA sample with the PCR Mix for achieving the purpose of PCR amplification, so as to prepare for the subsequent PCR amplification operation.

[0125] like Fig. 7AAs shown, in this example, the feed specifically includes PCR Mix, the constant volume feed channel specifically includes the PCR Mix feed channel 321, the first constant volume chamber specifically includes the PCR Mix constant volume chamber 320, the first exhaust channel specifically includes the PCR Mix exhaust channel 323, the feed valve specifically includes the third stop valve 322, and the exhaust valve specifically includes the fourth stop valve 324.

[0126] The PCR Mix constant volume chamber 320 is connected to a PCR Mix feed channel 321. A third stop valve 322 is provided between the inlet of the PCR Mix feed channel 321 and the PCR Mix constant volume chamber 320. The third stop valve 322 has an inlet located on the valve body layer, and the inlet is used to apply pressure to the working liquid to block the PCR Mix feed channel 321. The PCR Mix constant volume chamber 320 is also connected to a PCRMix exhaust channel 323. A fourth stop valve 324 is provided between the outlet of the PCR Mix exhaust channel 323 and the PCR Mix constant volume chamber 320. The fourth stop valve 324 has an inlet located on the valve body layer, and the inlet is used to apply pressure to the working liquid to block the PCRMix exhaust channel 323.

[0127] In this example, the PCR constant volume module also includes an eluent holding chamber 310. The eluent holding chamber 310 is connected to an eluent feed channel 311. A first stop valve 312 is arranged between the inlet of the eluent feed channel 311 and the eluent holding chamber 310, and the first stop valve 312 has an inlet located on the valve body layer, and the inlet is used to pass the working liquid to block the eluent feed channel 311. The eluent holding chamber 310 is also connected to an eluent exhaust channel 313. A second stop valve 314 is arranged between the outlet of the eluent exhaust channel 313 and the eluent holding chamber 310, and the second stop valve 314 has an inlet located on the valve body layer, and the inlet is used to pressurize the working liquid to block the eluent exhaust channel 313.

[0128] The eluent holding chamber 310 is used to hold the eluent output by the upper-level device of the PCR constant volume module. In addition, the eluent holding chamber 310 is also used to cooperate with the upper-level device of the PCR constant volume module to discharge the gas in the device. Specifically, the upper-level device is connected to the eluent feed channel 311, which is used to pass the eluent into the feed channel 311. During the feeding process, the second stop valve 314 is opened so that the gas passed by the upper-level device passes through the eluent holding chamber 310 and is discharged by the eluent exhaust channel 313. At the same time, the eluent enters and is stored in the eluent holding chamber 310.

[0129] In addition, the eluent holding chamber 310 is also connected to the PCR Mix constant volume chamber 320. The PCR constant volume module also includes a fifth stop valve 340 disposed between the eluent holding chamber 310 and the PCR Mix constant volume chamber 320. The fifth stop valve 340 has an inlet located on the valve body layer, and the inlet is used to pressurize the working liquid to block the communication between the PCR Mix constant volume chamber 320 and the eluent holding chamber 310.

[0130] Furthermore, by closing the fifth stop valve 340, the PCR constant volume module upper process and the PCR Mix constant volume can be performed independently without interfering with each other. Specifically, when the upper process of PCR purification is performed, the PCR Mix feed channel 321 and the PCR Mix exhaust channel 323 are used to achieve constant volume of the PCR Mix phase.

[0131] In this example, the PCR constant volume module also includes a first mixing constant volume chamber 330. The first mixing constant volume chamber 330 is connected to a mixing exhaust channel 331. A seventh stop valve 360 ​​is arranged between the outlet of the mixing exhaust channel 331 and the first mixing constant volume chamber 330, and the seventh stop valve 360 ​​has an inlet located on the valve body layer, and the inlet is used to pass the working fluid to block the mixing exhaust channel 331.

[0132] The first mixing constant volume chamber 330 is also connected to the PCR Mix constant volume chamber 320, and an eighth stop valve 350 is arranged between the two. The eighth stop valve 350 has an inlet located on the valve body layer, and the inlet is used to introduce working fluid to achieve communication or blocking between the PCR Mix constant volume chamber 320 and the first mixing constant volume chamber 330.

[0133] When the PCR constant volume module is in use, the eluent holding chamber 310 is used to hold the eluent output by the upper module by controlling the first stop valve 312 and the second stop valve 314, and the PCR Mix constant volume chamber 320 is used to realize the PCR Mix constant volume by controlling the third stop valve 322 and the fourth stop valve 324. After the PCR Mix constant volume is completed, the fifth stop valve 340 and the eighth stop valve 350 are opened, so that the eluent in the eluent holding chamber 310 and the PCR Mix in the PCR Mix constant volume chamber 320 enter the first mixing constant volume chamber 330. And specifically through the cyclic pressure relief method provided in Example 1, the constant volume of the mixed liquid is completed. Among them, a column is also provided in the first mixing constant volume chamber 330, and the column is used to ensure that different liquids are evenly mixed.

[0134] In the PCR constant volume module, a first buffer feed channel 361 and a second buffer feed channel 362 connected to the first mixing constant volume chamber 330 are also included. Optionally, a ninth stop valve 371 is provided between the first buffer feed channel 361 and the first mixing constant volume chamber 330, and the communication between the first buffer feed channel 361 and the first mixing constant volume chamber 330 is controlled by the ninth stop valve 371. A tenth stop valve 372 is provided between the second buffer feed channel 362 and the first mixing constant volume chamber 330, and the communication between the second buffer feed channel 362 and the first mixing constant volume chamber 330 is controlled by the tenth stop valve 372. Optionally, the first buffer feed channel 361 and the second buffer feed channel 362 are located on one side of the first mixing constant volume chamber 330 close to the PCRMix constant volume chamber 320, and are specifically located between the eighth stop valve 350 and the first mixing constant volume chamber 330. In this way, by turning off the eighth stop valve 350, it is avoided that the PCR Mix phase enters the buffer channel when the PCR Mix phase is constant volume. It should be noted that the first buffer solution and the second buffer solution are different.

[0135] The first mixing constant volume chamber 330 is also connected to a drainage channel 363. After the mixing and constant volume is completed, the first buffer solution (such as a spacing buffer) is first introduced through the first buffer solution feed channel 361, and then the second buffer solution (such as a binding buffer) is introduced through the second buffer solution channel 362 to discharge the mixed liquid in the first mixing constant volume chamber 330 from the drainage channel 363. In addition, a stop valve is provided between the outlet of the drainage channel 363 and the first mixing constant volume chamber 330, which is used to control the connection or blocking of the outlets of the first mixing constant volume chamber 330 and the drainage channel 363. In particular, when mixing and constant volume is performed, the outlets of the first mixing constant volume chamber 330 and the drainage channel 363 are blocked by the stop valve, and together with the shut-off eighth stop valve 350, the volume accuracy of the mixing constant volume chamber is ensured.

[0136] In summary, before the microfluidic constant volume module works, the conduit filled with the shut-off valve working fluid is connected to the inlet of the shut-off valve, and the conduits filled with various reagents are connected to the inlets of various feed channels.

[0137] After starting the work, determine the fluid channel that needs to be cut off, and apply pressure to the working fluid through the corresponding stop valve inlet to discharge the gas between the inlet and the cut-off position in the fluid channel, and then continue to apply pressure to the working fluid to achieve the blocking of the fluid channel.

[0138] When the microfluidic constant volume module is used to perform the PCR constant volume process, the constant volume method provided in Example 1 can be used. The overall method realizes the constant volume process by controlling the opening and closing of different stop valves, which is simple to operate and easy to implement. In addition, the microfluidic constant volume module has a reasonable structure and solves the problem of complex microfluidic structure in related technologies.

[0139] Figure 7B is a schematic diagram of a microfluidic constant volume module according to another exemplary embodiment. Figure 7B The microfluidic constant volume module shown is used for the sequencing adapter connection process. Taking the microfluidic constant volume module for DNA sequencing adapter connection as an example, the purpose of the sequencing adapter connection process is to mix the purified DNA sample with the various phase reagents of the sequencing adapter to a fixed volume, so as to connect the sequencing adapter to the DNA sample to be sequenced, and realize the subsequent sequencing steps.

[0140] like Figure 7B As shown, in this example, the first feed specifically includes the enzyme phase, and the second feed specifically includes the enzyme buffer. The first constant volume chamber specifically includes the second mixed constant volume chamber 450, the second constant volume chamber specifically includes the enzyme phase constant volume chamber 440, and the third constant volume chamber specifically includes the enzyme buffer constant volume chamber 430. The first exhaust channel specifically includes the mixed exhaust channel 452, and the exhaust valve specifically includes the twenty-second stop valve 453. The second constant volume feed channel specifically includes the enzyme phase feed channel 442, and the first control valve specifically includes the twentieth stop valve 443. The third valve specifically includes the nineteenth stop valve 441. The second exhaust channel specifically includes the general exhaust channel 422, and the fourth valve specifically includes the fourteenth stop valve 424. The third constant volume feed channel specifically includes the enzyme buffer feed channel 431, and the first control valve specifically includes the sixteenth stop valve 432.

[0141] Specific to Figure 7B In the microfluidic constant volume module shown, the module also includes a purification eluent holding chamber 410. The purification eluent holding chamber 410 is used to hold the output liquid of the previous process of the sequencing connector connection process, specifically the eluent purified by PCR. The purification eluent holding chamber 410 is connected to the purification eluent feed channel 411 and the purification eluent constant volume exhaust channel 412. The purification eluent feed channel 411 is located on the side of the purification eluent holding chamber 410 close to the upper-level process module, and the purification eluent constant volume exhaust channel 412 is located on the side of the purification eluent holding chamber 410 away from the upper-level process module.

[0142] An eleventh stop valve 413 is provided between the entrance of the purified eluent holding chamber 410 and the purified eluent holding chamber 410, and the eleventh stop valve 413 has an entrance located on the valve body layer. The purified eluent feed channel 411 is blocked by applying pressure to the working liquid at the entrance, and the purified eluent feed channel 411 is kept unobstructed by removing the working liquid pressure. A twelfth stop valve 414 is provided on the purified eluent constant volume exhaust channel 412, and the twelfth stop valve 414 has an entrance located on the valve body layer. The purified eluent constant volume exhaust channel 412 is blocked by applying pressure to the working liquid at the entrance, and the purified eluent constant volume exhaust channel 412 is kept unobstructed by removing the working liquid pressure.

[0143] When the purified eluent holding chamber 410 is in use, the eleventh stop valve 413 and the twelfth stop valve 414 cooperate with each other, and according to the constant volume method provided in Example 1, the feeding process of the purified eluent is realized by utilizing the principle of cyclic pressure relief. Before the feeding is completed, the purified eluent holding chamber 410 and the purified eluent constant volume exhaust channel 412 are utilized to exhaust the gas in the previous stage device.

[0144] Specific to Figure 7B In the microfluidic constant volume module shown, the module also includes a joint complex constant volume chamber 420. The joint complex constant volume chamber 420 is used to accommodate the joint complex phase. The joint complex constant volume chamber 420 is connected to the joint complex feed channel 421 and the universal exhaust channel 422. Among them, the joint complex feed channel 421 is located on the side of the joint complex constant volume chamber 420 close to the purified eluent holding chamber 410, and the universal exhaust channel 422 is located on the side of the joint complex constant volume chamber 420 away from the purified eluent holding chamber 410.

[0145] A thirteenth stop valve 423 is provided between the inlet of the joint compound feed channel 421 and the joint compound constant volume chamber 420. The thirteenth stop valve 423 has an inlet located on the valve body layer. The joint compound feed channel 421 is blocked by applying pressure to the working fluid at the inlet, and the composite feed channel 421 is kept unobstructed by removing the pressure on the working fluid.

[0146] A fourteenth stop valve 424 is provided between the outlet of the common exhaust channel 422 and the joint compound constant volume chamber 420. The fourteenth stop valve 424 has an inlet located on the valve body layer, and the common exhaust channel 422 is blocked by applying pressure to the working fluid through the inlet, and the common exhaust channel 422 is kept unobstructed by removing the working fluid pressure.

[0147] In addition, a fifteenth stop valve 415 is provided between the joint complex constant volume chamber 420 and the purified eluent holding chamber 410. The fifteenth stop valve 415 blocks the connection between the joint complex constant volume chamber 420 and the purified eluent holding chamber 410. When performing constant volume of the joint complex phase, the connection between the joint complex constant volume chamber 420 and the purified eluent holding chamber 410 is blocked, so that the joint complex only passes into the joint complex constant volume chamber 420. Using the thirteenth stop valve 423 and the fourteenth stop valve 424, according to the constant volume method provided in Example 1, the joint complex phase constant volume is completed by adopting a cyclic pressure relief method.

[0148] The enzyme buffer constant volume chamber 430 is used to accommodate enzyme buffer and is connected to the enzyme buffer feed channel 431. Optionally, the enzyme buffer feed channel 431 is located on the side of the enzyme buffer constant volume chamber 430 away from the joint complex constant volume chamber 420. The enzyme buffer feed channel 431 is used to pass enzyme buffer into the enzyme buffer constant volume chamber 430. A sixteenth stop valve 432 is provided between the inlet of the enzyme buffer feed channel 431 and the enzyme buffer constant volume chamber 430. The sixteenth stop valve 432 forms an inlet on the valve body layer, and the inlet is used to pressurize the working fluid to block the enzyme buffer feed channel 431.

[0149] And, the enzyme buffer constant volume chamber 430 is also connected to the general exhaust channel 422, and the fourteenth stop valve 424 is also used to block the communication between the outlet of the general exhaust channel 422 and the enzyme buffer constant volume chamber 430. In addition, a seventeenth stop valve 433 is provided between the fourteenth stop valve 424 and the enzyme buffer constant volume chamber 430, and an eighteenth stop valve 425 is provided between the fourteenth stop valve 424 and the joint compound constant volume chamber 420. In this way, when constant volume is performed through the enzyme buffer constant volume chamber 430, the eighteenth stop valve 425 is turned off to prevent gas from entering the joint compound constant volume chamber 420. When constant volume is performed through the joint compound constant volume chamber 420, the seventeenth stop valve 433 is turned off to prevent gas from entering the enzyme buffer constant volume chamber 430.

[0150] In this way, when the enzyme buffer constant volume chamber 430 is constant volume according to the constant volume method provided in Example 1, the universal exhaust channel 422 is used as an exhaust passage for pressure relief. Accordingly, there is no need to set an exhaust channel separately for the enzyme buffer constant volume chamber 430, reducing the structural complexity of the microfluidic constant volume module.

[0151] The enzyme phase constant volume chamber 440 is used to accommodate the enzyme phase and is connected to the enzyme buffer constant volume chamber 430. A nineteenth stop valve 441 is provided between the enzyme buffer constant volume chamber 430 and the enzyme phase constant volume chamber 440. The nineteenth stop valve 441 forms an inlet on the valve body layer, which is used to apply pressure to the working fluid to block the communication between the enzyme buffer constant volume chamber 430 and the enzyme phase constant volume chamber 440.

[0152] The enzyme phase constant volume chamber 440 is also connected to the enzyme phase feed channel 442, which is located on the side of the enzyme phase constant volume chamber 440 away from the enzyme buffer constant volume chamber 430. A twentieth stop valve 443 is provided between the enzyme phase feed channel 442 and the enzyme phase constant volume chamber 440, and the twentieth stop valve 443 forms an inlet on the valve body layer, and a positive pressure is applied to the working fluid through the inlet to block the enzyme phase feed channel 442.

[0153] When the enzyme phase constant volume is performed by the constant volume method provided in Example 1 using the enzyme phase constant volume chamber 440, the enzyme phase is introduced through the enzyme phase feed channel 442. At this time, the 19th stop valve 441 is kept closed. When the pressure is released, the 20th stop valve 443 is turned off, and the 19th stop valve 441, the 17th stop valve 433 and the 14th stop valve 424 are opened, so that the gas is discharged from the general exhaust channel 422 through the enzyme buffer constant volume chamber 430. In this way, the exhaust channel 422 is used for exhaust pressure relief by controlling the stop valve, thereby there is no need to set a separate exhaust channel for the enzyme phase constant volume chamber 440, which further reduces the structural complexity of the microfluidic constant volume module.

[0154] Due to the provision of a universal exhaust channel 422, the microfluidic constant volume module, when in use, preferentially performs enzyme phase constant volume, and then performs enzyme phase buffer constant volume. Accordingly, the enzyme phase overflowing into the enzyme buffer constant volume chamber 430 during enzyme phase constant volume is discharged. In addition, when the purified eluent holding chamber 410 is utilized to perform purified eluent constant volume, the joint complex constant volume chamber 420 can be utilized synchronously to perform joint complex constant volume.

[0155] Finally, the structure of the second mixing constant volume chamber 450 is described. The second mixing constant volume chamber 450 is used to mix the components that have been fixed in volume in the previous sequence, specifically including the eluate, the linker complex, the enzyme phase buffer and the enzyme phase of the mixed PCR purification. The second mixing constant volume chamber 450 is provided with a column 140, and the columns 140 are used to achieve uniform mixing of the components. The distribution form of the columns 140 is not specifically limited. Figure 7B Only two rows of arc-shaped columns 140 are taken as an example.

[0156] The second mixing constant volume chamber 450 is connected to the enzyme phase constant volume chamber 440, and a twenty-first stop valve 451 is provided between the second mixing constant volume chamber 450 and the enzyme phase constant volume chamber 440, and the twenty-first stop valve 451 is used to block the communication between the second mixing constant volume chamber 450 and the enzyme phase constant volume chamber 440. In addition, the second mixing constant volume chamber 450 is also connected to a mixed exhaust passage 452, and the mixed exhaust passage 452 is located on the side of the second mixing constant volume chamber 450 away from the enzyme phase constant volume chamber 440. A twenty-second stop valve 453 is provided between the outlet of the mixed exhaust passage 452 and the second mixing constant volume chamber 450, and the twenty-second stop valve 453 is used to block the mixed exhaust passage 452.

[0157] Keep the twenty-first stop valve 451 closed before mixing and volume setting, and when the volume setting of each component is completed. Open the twenty-first stop valve 451, the nineteenth stop valve 441, the seventeenth stop valve 433, and the fifteenth stop valve 415 to allow the PCR purification eluate, the linker complex enzyme phase buffer and the enzyme phase to enter the second mixing and volume setting chamber 450. In addition, the pressure is released by opening the twenty-second stop valve 453 using the mixing exhaust channel 452. Accordingly, according to the volume setting method provided in Example 1, the mixing and volume setting of the second mixing and volume setting chamber 450 is achieved by adopting a cyclic pressure relief method.

[0158] In addition, the second mixing constant volume chamber 450 is also connected to a liquid outlet channel 454, and a twenty-third stop valve 455 is provided between the outlet of the liquid outlet channel 454 and the twenty-second stop valve 453. The twenty-third stop valve 455 is closed during the constant volume period of the second mixing constant volume chamber 450, and after the mixing constant volume is completed, the twenty-third stop valve 455 is opened to discharge the mixture from the liquid outlet channel 454.

[0159] In addition, a buffer feed channel 456 is provided between the twenty-first stop valve 451 and the second mixing constant volume chamber 450. A twenty-third stop valve 457 is provided between the inlet of the buffer feed channel 456 and the second mixing constant volume chamber 450, and the twenty-third stop valve 457 is used to block the buffer feed channel 456. By opening the twenty-third stop valve 457, a buffer is introduced through the buffer feed channel 456, and the buffer (e.g., Binding Buffer) is used to completely discharge the mixed components in the second mixing constant volume chamber 450.

[0160] In summary, before the microfluidic constant volume module works, the conduit filled with the shutoff valve working fluid is connected to the inlet of the shutoff valve, and the conduits filled with various reagents are connected to the inlets of various feed channels. After starting the work, the fluid channel that needs to be shut off is determined, and the working fluid is pressurized through the corresponding shutoff valve inlet to achieve the blocking of the fluid channel.

[0161] When the microfluidic constant volume module is used to perform the sequencing adapter connection and mixing process, the constant volume method provided in Example 1 can be used. The overall method realizes the constant volume process by controlling the opening and closing of different stop valves, which is simple to operate and easy to implement. In addition, the microfluidic constant volume module has a reasonable structure and solves the problem of complex microfluidic structure in related technologies.

[0162] Example 3

[0163] Embodiment 3 of the present invention provides a database building system. Figure 8 FIG. 1 is a schematic diagram of a library building system according to an exemplary embodiment. Figure 8 As shown, the library construction system includes the microfluidic constant volume module provided in the above-mentioned embodiment 2. In particular Fig. 7AThe PCR constant volume module 300 shown, and / or, Figure 7B The sequencing adapter connection module 400 shown. It should be noted that, Figure 8 The library construction system shown also includes Fig. 7A The PCR constant volume module shown and Figure 7B The sequencing adapter ligation module is shown.

[0164] The database construction system provided in the embodiment of the present invention can implement the volume determination method provided in Example 1 by adopting the volume determination module provided in Example 2. Based on the easy-to-operate feature of the volume determination method provided in Example 1, the operation difficulty of the overall database construction system is reduced, and the efficiency of the database construction process is improved.

[0165] In addition to the microfluidic constant volume module, the library construction system provided in Example 3 of the present invention further includes at least one of a PCR module, a first purification module (eg, a lysate purification module or a PCR purification module), and a second purification module. Furthermore, different modules are connected by conduits.

[0166] In one example, the library construction system further includes a PCR module 500. Taking the library construction system for DNA library construction as an example, the PCR module 500 is used to complete the amplification of the DNA sample and increase the amount of the sample to be sequenced. Fig. 9 FIG. 1 is a schematic diagram of a PCR module according to an exemplary embodiment. Fig. 9 As shown, the PCR module 500 includes: an injection port 510, a denaturation zone pipeline 520, a circulation zone pipeline 530 and a sample outlet 540.

[0167] The injection port 510 is used to introduce the sample to be processed. The denaturation zone pipeline 520 is connected to the injection port 510, and is used to perform denaturation treatment on the sample to be processed entering through the injection port 510. The circulation zone pipeline 530 is connected to the denaturation zone pipeline 520, and includes a plurality of groups of circulation unit pipelines 531. The circulation zone pipeline 530 realizes cyclic amplification through the plurality of groups of circulation unit pipelines 531. The sample outlet 540 is connected to the circulation zone pipeline 530, and is used to discharge the processed sample.

[0168] Optionally, the circulation unit pipeline 531 includes a first pipeline 531a and a second pipeline 531b that are connected. Fig. 9 As shown, the circulation unit pipeline 531 first extends in a set direction, then bends 180° and extends in a direction opposite to the set direction. In this way, the processing path of the sample is greatly improved through a plurality of groups of circulation unit pipelines 531, and the amplification effect is optimized. Among them, in the circulation unit pipeline 531 connected to the deformation zone pipeline 520, the first pipeline 531a is specifically connected to the deformation zone pipeline 520. In addition, the processing temperature of the first pipeline 531a is higher than the processing temperature of the second pipeline 531b. See Fig. 9In the orientation shown, the second pipeline 531b is distributed on both sides of the first pipeline 531a. Accordingly, the sample that has been subjected to high temperature treatment in the denaturation zone pipeline 520 preferentially enters the high temperature portion of the circulation unit pipeline 531 (i.e., the first pipeline 531a), and then enters the low temperature portion of the circulation unit pipeline 531 (i.e., the second pipeline 531b). Moreover, in the two connected circulation unit pipelines 531, the first pipeline 531a is connected. That is, in the previous circulation unit pipeline 531, the sample passes through the first pipeline 531a and enters the first pipeline 531a of the next circulation unit pipeline 531. Among them, the processing temperature range of the first pipeline 531a is 90°C-98°C, and the processing temperature range of the second pipeline 531b is 50°C-60°C.

[0169] Optionally, the circulation unit pipeline 531 further includes a third pipeline, the processing temperature of the third pipeline is lower than the processing temperature of the first pipeline 531a, and higher than the processing temperature of the second pipeline 531b. For example, the processing temperature of the third pipeline is 70°C-80°C. Accordingly, the processing temperature distribution of the circulation unit pipeline 531 is changed in a gradient to optimize the amplification effect.

[0170] Optionally, the circulation zone pipeline 530 further includes an extension zone pipeline 532, and the extension zone pipeline 532 is connected to the second pipeline 531b in the circulation unit pipeline 531 near the sample outlet 540. The extension zone pipeline 532 is used to receive the sample passing through all the circulation unit pipelines 531 and perform low-temperature base pairing on the sample. In addition, the extension zone pipeline 532 is also connected to the sample outlet 540 to discharge the sample that has undergone low-temperature base pairing out of the sample outlet 540.

[0171] Optionally, in order to accurately obtain the temperature of different areas on the PCR module, temperature measurement points are set on the module to install (for example, by drilling and embedding) temperature sensors. Fig. 9 As shown, a first temperature measuring point 551 is set in the area where the first pipeline 531a is located, a second temperature measuring point 552 and a third temperature measuring point 553 are set in the area where the second pipeline 531b is located, and temperature sensors are installed at the temperature measuring points.

[0172] Continue to see Figure 8 , the library construction system also includes a first purification module. Specifically, the first purification module includes a lysate purification module 600A and a PCR purification module 600B. Taking the library construction system for DNA library construction as an example, the lysate purification module 600A is used to extract the DNA sequence in the incoming lysate and remove other impurities in the lysate to complete the subsequent PCR amplification step. The PCR purification module 600B is used to extract the DNA sequence in the sample after PCR treatment and remove other impurities in the sample after PCR treatment to complete the subsequent sequencing adapter connection step.

[0173] In the embodiment of the present invention, the lysate purification module 600A and the PCR purification module 600B adopt the same design in structure, and the first purification module will be uniformly introduced below. Fig.10 FIG. 1 is a schematic diagram of a first purification module according to an exemplary embodiment. Fig.10 As shown, the first purification module includes a purification chamber 610 , a purification feed channel 620 , and a purification outlet channel 630 .

[0174] The purification chamber 610 is used to accommodate various components to purify the lysate. The purification feed channel 620 is connected to the purification chamber 610. In addition, a fifth valve is provided between the purification feed channel 620 and the purification chamber 610, and the fifth valve is used to control the on-off of the purification chamber 610 and the purification feed channel 620. The purification discharge channel 630 is connected to the purification chamber 610, and a sixth valve is provided between the purification discharge channel 630 and the purification chamber 610, and the sixth valve is used to control the on-off of the purification chamber 610 and the purification discharge channel 630.

[0175] The fifth valve is opened to connect the purification feed channel 620, so that different feeds enter the purification chamber 610. The sixth valve is opened to connect the purification discharge channel 630, so that the purified sample is discharged from the purification chamber 610.

[0176] Optionally, the fifth valve and the sixth valve adopt the valve body structure as shown in FIG6. That is, the overall purification module 600 includes a valve body layer, a spacer layer and a fluid layer that are stacked. At this time, the purification chamber 610, the purification feed channel 620 and the purification discharge channel 630 are all arranged in the fluid layer. In addition, the purification feed channel 620 and the purification discharge channel 630 penetrate the valve body layer and the spacer layer and extend to the fluid layer.

[0177] In one example, the purification feed channel 620 includes a microbead feed channel 621, which is used to inject microbeads into the purification chamber 610. In addition, a column 611 is provided in the purification chamber 610, and the column 611 is used to bind the microbeads. Optionally, the column 611 is provided near the connection between the purification chamber 610 and the purification discharge channel 630. Accordingly, the microbeads entering the purification chamber 610 through the microbead feed channel 621 are blocked in front of the column 611.

[0178] The fifth valve includes a twenty-fourth stop valve 641, which is disposed between the inlet of the microbead feeding channel 621 and the purification chamber 610. The working fluid is pressurized through the inlet formed at the valve body layer of the twenty-fourth stop valve 641 to block the microbead feeding channel 621.

[0179] In one example, the purification feed channel 620 includes a sample feed channel 622. In the lysate purification module 600A, the sample feed channel 622 is used to pass the lysate into the purification chamber 610. In the PCR purification module 600B, the sample feed channel 622 is used to pass the sample processed by PCR into the purification chamber 610.

[0180] The fifth valve includes a twenty-fifth stop valve 642 disposed between the inlet of the sample feed channel 622 and the purification chamber 610. The twenty-fifth stop valve 642 is used to block the sample feed channel 622, and the twenty-fifth stop valve 624 is also used to block the inlet of the sample feed channel 622 and the inlet of the microbead feed channel 621. Accordingly, the feeding effect of the sample feed channel 622 and the microbead feed channel 621 is guaranteed, so that the feed enters the purification chamber 610 through the feed channel, and does not flow to other feed channels, or discharge from the first purification module.

[0181] In one example, the purification feed channel 620 includes a cleaning liquid channel 623, which is used to inject cleaning liquid into the purification chamber 610. The cleaning liquid is used to clean the microbeads in the purification chamber 610, and the lysate remaining in the purification chamber 610 and the waste liquid of washing the microbeads are discharged from the purification chamber 610.

[0182] The fifth valve includes a twenty-sixth stop valve 643, which is disposed between the inlet of the cleaning liquid channel 623 and the purification chamber 610. The working liquid is pressurized through the inlet formed in the valve body layer of the twenty-sixth stop valve 643 to block the cleaning liquid channel 623. The twenty-sixth stop valve 643 also blocks the communication between the inlet of the cleaning liquid channel 623 and the inlet of the sample feeding channel 622 and the inlet of the microbead feeding channel 621, thereby ensuring that the cleaning liquid enters the purification chamber 610.

[0183] In one example, the purification feed channel 620 includes an eluent channel 624, and the eluent channel 624 is used to inject an eluent into the purification chamber 610. The DNA sample connected to the microbeads is eluted by the eluent to obtain a purified sample.

[0184] The fifth valve includes a twenty-seventh stop valve 644, which is disposed between the entrance of the eluent channel 624 and the purification chamber 610. The twenty-seventh stop valve 644 applies pressure to the working liquid through the entrance formed in the valve body layer to block the eluent channel 624. The twenty-seventh stop valve 644 also blocks the mutual communication between the eluent channel 624 and the entrances of the sample feed channel 622, the microbead feed channel 621, and the cleaning liquid channel 623, thereby ensuring that the eluent enters the purification chamber 610.

[0185] The purification discharge channel 630 includes a sample discharge channel 631 and a waste liquid discharge channel 632 which are connected to the purification chamber 610. The sample discharge channel 631 is used to discharge the sample eluted by the eluent, and the waste liquid discharge channel 632 is used to discharge waste liquid such as the cleaning liquid and the microbead liquid phase.

[0186] The sixth valve includes a twenty-eighth stop valve 651 and a twenty-ninth stop valve 652. The twenty-eighth stop valve 651 is disposed between the outlet of the sample discharge channel 631 and the purification chamber 610, and is used to block the sample discharge channel 631. The twenty-ninth stop valve 652 is disposed between the outlet of the waste liquid discharge channel 632 and the purification chamber 610, and is used to block the waste liquid discharge channel 632. When in use, the twenty-eighth stop valve 651 and the twenty-ninth stop valve 652 make the two discharge channels independent of each other.

[0187] In summary, when the first purification module is in use, the twenty-fourth stop valve 641 is opened, and the microbead phase (e.g., magnetic bead phase) is introduced into the purification chamber 610 through the main feed channel 621. Under the blocking effect of the column in the purification chamber 610, the microbeads in the microbead phase are accumulated in the purification chamber 610. At the same time, the twenty-ninth stop valve 652 is kept open and the twenty-eighth stop valve 651 is closed, so that the waste liquid is discharged from the waste liquid discharge channel 632. Afterwards, the twenty-fourth stop valve 641 is closed, the twenty-fifth stop valve 642 is opened, and the lysate is introduced into the purification chamber 610 through the sample feed channel 622. The microbeads in the purification chamber 610 capture the DNA sequence in the lysate, and the waste liquid is discharged through the waste liquid discharge channel 632. Afterwards, the twenty-fifth stop valve 642 is closed, the twenty-sixth stop valve 643 is opened, and the cleaning liquid 622 is introduced into the purification chamber 610 through the cleaning liquid channel 622. The microbeads are cleaned by the cleaning liquid, and the lysate remaining in the purification chamber 610 is discharged. After cleaning is completed, close the twenty-ninth stop valve 652 and open the twenty-eighth stop valve 651, and at the same time close the twenty-sixth stop valve 643 and open the twenty-seventh stop valve 644, introduce the eluent into the purification chamber 610 through the eluent channel 624, and discharge the purified sample from the sample discharge channel 631.

[0188] Continue to see Figure 8 The library construction system further includes a second purification module 700. The second purification module 700 is located after the sequencing adapter connection process and is used to extract the DNA sequence in the connection sequencing product and remove other impurities in the connection sequencing product.

[0189] In one example, the second purification module 700 adopts a similar structural design to the first purification module. Specifically, Fig.11 FIG. 1 is a schematic diagram of a second purification module according to an exemplary embodiment. Fig.11As shown, the second purification module 700 also includes a purification chamber 610, a purification feed channel 620, a purification discharge channel 630, a fifth valve and a sixth valve. Different from the first purification module, the second purification module 700 also includes the following structures:

[0190] The purification feed channel 620 also includes a first sequencing buffer feed channel 625. The first sequencing buffer feed channel 625 is used to introduce a sequencing buffer, and the sequencing buffer is used to discharge the eluent from the purification chamber 610 to participate in the subsequent process of DNA sequencing library construction.

[0191] The fifth valve also includes a 30th stop valve 645, which is disposed between the inlet of the sequencing buffer feed channel 625 and the purification chamber 610. The 30th stop valve 645 not only blocks the sequencing buffer feed channel 625, but also blocks the connection with the inlets of the microbead feed channel 621, the sample feed channel 622, the cleaning liquid channel 623, and the eluent channel 624. In this way, the sequencing buffer can be passed into the purification chamber 610 through the first sequencing buffer feed channel 625.

[0192] The purification chamber 610 is also connected to a second sequencing buffer feed channel 633, which is connected to the purification outlet channel 630 and is used to introduce the sequencing buffer and push the sequencing buffer and the eluent to the next process module. A thirty-first stop valve 653 is provided on the second sequencing buffer feed channel 633 to block the second sequencing buffer feed channel 633. Optionally, compared with the waste liquid outlet channel 632, the second sequencing buffer feed channel 633 is provided near the outlet of the sample outlet channel 631.

[0193] In one example, the first purification module and the second purification module 700 use extraction filter membranes to achieve DNA sample enrichment. Fig.12 FIG. 1 is a schematic diagram of a purification module according to an exemplary embodiment. Fig.12 As shown, in this example, the purification is achieved by using an extraction filter membrane 800 sealed in a cavity instead of microbeads and a purification chamber.

[0194] In one example, the first purification module and the second purification module 700 are implemented in another manner, and are collectively described as purification modules below. Fig.13 FIG. 1 is a schematic diagram of a purification module according to an exemplary embodiment. Fig.13 As shown, the purification module includes: a sample container 710, an extraction part 720, a suction component 730 and a control valve.

[0195] The sample container 710 is used to hold the sample to be purified. An extraction filter membrane is provided in the extraction part 720, and the extraction filter membrane is used to enrich and purify the sample to be purified. Optionally, the extraction membrane is provided in the extraction part 720 in the form of a threaded joint. The suction assembly 730 is connected to the sample container 710 and the extraction part 720 through a control valve, and is used to cooperate with the control valve to suck the sample from the sample container 710 and pump it into the extraction part 720.

[0196] Specifically, the control valve includes a first solenoid valve 741 connected to the sample container 710, and the first solenoid valve 741 is also connected to the suction assembly 730. When the first solenoid valve 741 is in a powered-on state, the first solenoid valve 741 connects the suction assembly 730 and the sample container 710. At this time, the suction assembly 730 performs a liquid extraction operation to extract a sample. In addition, a one-way valve 750 is also provided between the suction assembly 730 and the extraction part 720, and the one-way valve 750 ensures that the liquid pumped out of the suction assembly 730 smoothly enters the extraction part 720, thereby preventing the liquid in the extraction part 720 from flowing back.

[0197] The purification module further includes a cleaning liquid container 760 for containing cleaning liquid. The control valve further includes a second solenoid valve 742 connected to the cleaning liquid container 760, and the second solenoid valve 742 is also connected to the suction assembly 730. After the sample is pumped into the extraction part 720, the first solenoid valve 741 is closed, the second solenoid valve 742 is opened, and the suction assembly 730 extracts the cleaning liquid. After the liquid extraction operation, the second solenoid valve 742 is closed, and the cleaning liquid is pumped into the extraction part 720 through the suction assembly 730.

[0198] The purification module further includes an air pump 770 connected to the extraction part 720, and the control valve further includes a third electromagnetic valve 743 connected to the air pump 770. After the cleaning liquid is pumped in, only the third electromagnetic valve 743 is opened, and the air pump 770 is started to pass airflow to the extraction part 720 to dry the extraction filter membrane.

[0199] The purification module 700 further includes an eluent container 780 connected to the suction assembly 730, and the control valve further includes a fourth solenoid valve 744 connected to the eluent container 780 and the one-way valve 750. After the drying is completed, the fourth solenoid valve 744 is opened to suck the eluent through the suction assembly 730. Thereafter, the fourth solenoid valve 744 is closed and the sucked eluent is injected into the extraction part 720 to elute the DNA sequence enriched on the extraction membrane in the extraction part 720.

[0200] The purification module further includes a waste liquid container 790 connected to the extraction part 720, and the control valve further includes a fifth solenoid valve 745 connected to the waste liquid container 790 and the extraction part 720. During the extraction, cleaning and drying process, the fifth solenoid valve 745 is kept open so that the waste liquid and waste gas are passed into the waste liquid container 790. During the elution process, the fifth solenoid valve 745 is kept closed so that the eluent enters the next process module.

[0201] Continue to see Figure 8 In Example 3 of the present invention, the library building system further includes a sample pool 900. The sample pool 900 is used to receive samples discharged from the upper module. The sample pool 900 includes: an inlet pipe 910, a sample container 920 and an outlet pipe 930. A sealing plug 921 is provided in the sample container 920, and the inlet pipe 910 and the outlet pipe 930 are provided through the sealing plug 921. Different modules are connected as a whole through the sample pool 900 to improve the overall library building efficiency.

[0202] In summary, the database construction system provided in the embodiment of the present invention can implement the volume determination method provided in Example 1 by adopting the volume determination module provided in Example 2. Based on the easy-to-operate feature of the volume determination method provided in Example 1, the operation difficulty of the overall database construction system is reduced, and the efficiency of the database construction process is improved.

[0203] Example 4

[0204] Embodiment 4 of the present invention provides a library construction method, which is implemented by using the library construction system provided in the above embodiment 3. Figure 8 The library construction method includes: using a first purification module to perform a sample purification step, using a microfluidic constant volume module to perform a PCR constant volume step, using a PCR module to perform a PCR step, using the first purification module to perform a PCR purification step, using a microfluidic constant volume module to perform a sequencing adapter connection step, and using a second purification module to perform an adapter purification step.

[0205] In one example, the library construction method includes the following steps:

[0206] Step 1: Perform a first purification treatment on the sample using a microfluidic purification module to obtain a first purified sample.

[0207] Step 2: Perform PCR volume-fixing treatment on the first purified sample using a microfluidic volume-fixing module to obtain a mixed sample.

[0208] Step 3: Use a PCR module to perform PCR on the mixed sample to obtain a PCR sample.

[0209] Step 4: Perform a second purification process on the PCR sample using a microfluidic purification module to obtain a second purified sample.

[0210] Step 5: Use a microfluidic constant volume module to connect the second purified sample to the sequencing connector to obtain a connector sample.

[0211] Step 6: Use a microfluidic purification module to purify the connector sample to obtain a library construction sample.

[0212] In combination with a specific example, the sample to be sequenced in the overall method is a bacterial or cell lysate, which is mixed with Binding Buffer and then introduced into the library construction system provided in Example 3.

[0213] Step 1: Purify the incoming lysate through the lysate purification module to obtain a first purified sample. Specifically, after the DNA sample is enriched, the excess sample is washed with a cleaning solution, and the DNA sequence connected to the magnetic beads is eluted with an elution solution.

[0214] Step 2: Perform PCR volume determination on the first purified sample through the PCR volume determination module to obtain a mixed sample. Specifically, the eluent and PCR mix are mixed and volume determined, and then the spacer buffer and the binding buffer are used to push the mixed sample to the PCR module in the microfluidic device.

[0215] Step 3: Perform PCR amplification on the mixed sample through a PCR module to obtain a PCR sample.

[0216] Step 4: Purify the PCR sample through the PCR purification module to obtain a second purified sample. After step 3, the PCR sample is mixed with the Binding Buffer in the sample chamber, and then passed into the PCR purification module. After enrichment, the excess sample is washed with a cleaning solution, and the DNA sequence connected to the magnetic beads is eluted with an elution solution to obtain a second purified sample.

[0217] Step 5: Mix the second purified sample with various reagents required for sequencing adapter connection through the sequencing adapter connection module, make up to volume, and incubate until the DNA sample is connected to the sequencing adapter. After the adapter reaction is completed, a adapter sample is obtained.

[0218] Step 6: The adapter sample is purified by the second purification module to obtain a library sample. Optionally, after step 5, the adapter sample is mixed with the Binding Buffer in the sample chamber, and then passed into the second purification module. After enrichment, the excess sample is washed with a cleaning solution, and the DNA sequence connected to the magnetic beads is eluted with an elution solution. After that, it is mixed with the sequencing buffer in the sample chamber, and finally passed into the sequencing chip for sequencing.

[0219] The volume determination method is implemented according to the volume determination method provided in Example 1. In addition, the volume determination step is implemented using the volume determination module provided in Example 2. At this time, by using the volume determination module in Example 2, the volume determination step can be performed while performing the purification step, thereby improving the library construction efficiency.

[0220] Specifically, the above purification step is a sample purification step, which is used to process the sample to be purified to obtain a purified sample. The volume determination step that can be performed simultaneously is a PCR volume determination step, which is used to process the purified sample to obtain a PCR mixed sample.

[0221] Alternatively, the purification step is a PCR purification step, which is used to purify the PCR mixed sample to obtain a PCR purified sample. The volume determination step that can be performed simultaneously is a sequencing adapter ligation step, which is used to perform adapter ligation on the PCR purified sample.

[0222] In summary, Example 4 of the present invention provides a library construction method, which is implemented based on the library construction system provided in Example 3. The overall method is highly efficient, easy to control and operate, and effectively solves the defects of the library construction method in the related art that it is difficult to operate and inefficient.

[0223] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that this is only for illustration and the protection scope of the present invention is defined by the appended claims. Those skilled in the art may make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but these changes and modifications all fall within the protection scope of the present invention.

Claims

1. A method for determining volume, characterized in that: The constant volume method is applied to a microfluidic constant volume module, the microfluidic constant volume module comprises a first constant volume chamber, a constant volume feed channel and a first exhaust channel, a feed valve is arranged between the constant volume feed channel and the first constant volume chamber, the feed valve is used to control the on-off of the constant volume feed channel and the first constant volume chamber; an exhaust valve is arranged between the first exhaust channel and the first constant volume chamber, the exhaust valve is used to control the on-off of the first exhaust channel and the first constant volume chamber; the method comprises: S1, closing the exhaust valve and opening the feed valve to allow the feed to enter the first constant volume chamber through the constant volume feed channel; S2, closing the feed valve and then opening the exhaust valve, so that the gas in the first constant volume chamber is discharged from the first exhaust channel; S3, cyclically executing step S1 and step S2 for a set number of times until the feed fills the first constant volume chamber.

2. The volume determination method according to claim 1, characterized in that: The constant volume feed channel includes a feed port, and a first feed valve is provided between the feed port and the first constant volume chamber, and the first feed valve is used to control the on-off between the feed port and the first constant volume chamber; the step S1 includes: The exhaust valve is closed and the first feed valve is opened, and a set pressure is applied to the feed through a liquid adding device so that the feed enters the first constant volume chamber through the feed inlet.

3. The volume determination method according to claim 1, characterized in that: The constant volume module further includes a second constant volume chamber and a third constant volume chamber. The two ends of the second constant volume chamber are respectively connected to the constant volume feed channel and the third constant volume chamber. The second constant volume chamber is filled with a first feed material, and the third constant volume chamber is filled with a second feed material; The feed valve comprises a second valve communicating with the second constant volume chamber and the constant volume feed channel, and a third valve communicating with the second constant volume chamber and the third constant volume chamber; The step S1 comprises: The exhaust valve is closed and the second valve and the third valve are opened, so that the second feed in the third constant volume chamber pushes the first feed in the second constant volume chamber and enters the first constant volume chamber through the constant volume feed channel.

4. A microfluidic constant volume module, characterized in that: The module is used to implement the volume determination method described in any one of claims 1 to 3; the volume determination module includes: The first constant volume chamber, A constant volume feeding channel, wherein the constant volume feeding channel is connected to the first constant volume chamber, a first exhaust channel, wherein the first exhaust channel is connected to the first constant volume chamber; A feed valve is provided between the constant volume feed channel and the first constant volume chamber, and the feed valve is used to control the opening and closing of the first constant volume chamber and the constant volume feed channel; An exhaust valve is provided between the first exhaust passage and the first constant volume chamber, and the exhaust valve is used to control the opening and closing of the first constant volume chamber and the first exhaust passage.

5. The microfluidic constant volume module according to claim 4, characterized in that: The constant volume feed channel comprises a first constant volume feed channel, wherein the first constant volume feed channel is provided with a feed port, and the feed port is used to connect a liquid adding device; The feed valve comprises a first valve disposed between the feed port and the first constant volume chamber, and the first valve is used to control the opening and closing of the feed port and the first constant volume chamber.

6. The microfluidic constant volume module according to claim 4, characterized in that: The constant volume module further includes a second constant volume chamber and a third constant volume chamber, the two ends of the second constant volume chamber are respectively connected to the constant volume feed channel and the third constant volume chamber, the second constant volume chamber is filled with the first feed, and the third constant volume chamber is filled with the second feed; The feed valve includes a second valve disposed between the second constant volume chamber and the constant volume feed channel, and a third valve disposed between the second constant volume chamber and the third constant volume chamber. The second valve is used to control the opening and closing of the second constant volume chamber and the constant volume feeding channel. The third valve is used to control the opening and closing of the second constant volume chamber and the third constant volume chamber.

7. The microfluidic constant volume module according to claim 6, characterized in that: A second constant volume feed channel is further provided between the second constant volume chamber and the first constant volume chamber, the second constant volume feed channel is connected to the second constant volume chamber and is used to connect a liquid adding device to fill the first feed into the second constant volume chamber; A third constant volume feed channel is provided between the third constant volume chamber and the second constant volume chamber, the third constant volume feed channel is connected to the third constant volume chamber, and is used to connect a liquid adding device to fill the second feed into the third constant volume chamber; A second exhaust passage is provided on a side of the third constant volume chamber away from the second constant volume chamber, and the second exhaust passage is connected to the second constant volume chamber through the third constant volume chamber. A fourth valve is provided between the second exhaust channel and the third constant volume chamber, and the fourth valve is used to control the opening and closing of the second exhaust channel and the third constant volume chamber.

8. The microfluidic constant volume module according to claim 4, characterized in that: At least one row of columns is arranged in the first constant volume cavity, and the interval between adjacent columns is less than or equal to a set distance.

9. The microfluidic constant volume module according to claim 4, characterized in that: The microfluidic constant volume module comprises a valve body layer, a spacer layer and a fluid layer which are stacked; The first constant volume chamber is arranged in the fluid layer, the feed channel and the first exhaust channel penetrate the valve body layer and the spacer layer, and extend to the fluid layer; The feed valve and the exhaust valve are arranged on the valve body layer, and include an inlet communicating with the outside of the valve body layer, and a valve body located inside the valve body layer; The fluid layer is provided with a cut-off position, which is provided corresponding to the valve body and is used to fit with the spacer layer deformed under pressure to block the feed channel or the first exhaust channel.

10. A database building system, characterized in that: The library construction system comprises the microfluidic constant volume module according to any one of claims 4 to 9.

11. The library building system according to claim 10, characterized in that: The system includes a PCR mixing and constant volume module and / or a sequencing adapter connection module; The PCR mixing constant volume module and / or the sequencing adapter connection module comprises the microfluidic constant volume module according to any one of claims 4 to 9.

12. The library building system according to claim 10, characterized in that: The library construction system also includes a purification module, which includes: A purification chamber, used for containing a sample to be purified; A purification feed channel connected to the purification chamber, a fifth valve is provided between the purification feed channel and the purification chamber, and the fifth valve is used to control the on-off of the purification chamber and the purification feed channel; The purification discharge channel is connected to the purification chamber, and a sixth valve is arranged between the purification discharge channel and the purification chamber. The sixth valve is used to control the on-off of the purification chamber and the purification discharge channel.

13. The library building system according to claim 12, characterized in that: The purification feed channel comprises a microbead feed channel. A column is arranged in the purification cavity, and the column is used to block the microbeads entering the purification cavity.

14. The library building system according to claim 12, characterized in that: The purification feed channel comprises a lysate feed channel for injecting lysate into the purification chamber; and / or The purification feed channel comprises a microbead feed channel for injecting microbeads into the purification chamber; and / or The purification feed channel includes a cleaning liquid channel to inject a cleaning liquid into the purification chamber; and / or The purification feed channel includes an eluent channel for injecting eluent into the purification chamber.

15. The library building system according to claim 12, characterized in that: The purification module comprises a valve body layer, a spacer layer and a fluid layer which are stacked; The purification chamber is arranged in the fluid layer, and the purification feed channel and the purification discharge channel penetrate the valve body layer and the spacer layer and extend to the fluid layer; The fifth valve and the sixth valve are arranged on the valve body layer, and include an inlet communicating with the outside of the valve body layer, and a valve body located inside the valve body layer; The fluid layer is provided with a cut-off position, which is provided corresponding to the valve body and is used for deforming under pressure to fit with the spacing layer so as to block the purification feed channel or the purification discharge channel.

16. The library building system according to claim 10, characterized in that: The library construction system also includes a purification module, which includes: A sample container, used for containing a sample to be purified; An extraction part, wherein the extraction part is provided with an extraction filter membrane for enriching and purifying the sample to be purified; A suction component and a control valve, wherein the suction component is connected to the sample container and the extraction part through the control valve, and the suction component is used to cooperate with the control valve to suck the sample from the sample container and then pump it into the extraction part.

17. The library building system according to claim 10, characterized in that: The library construction system further includes a PCR module, which includes: An inlet port, used for introducing a sample to be processed; a denaturation zone pipeline, connected to the injection port; a circulation zone pipeline, connected to the denaturation zone and including a plurality of groups of circulation unit pipelines; and The sample outlet is connected to the circulation zone pipeline and is used to discharge the processed sample.

18. The library building system according to claim 17, characterized in that: The circulation unit pipeline includes a first pipeline and a second pipeline that are connected to each other. The first pipeline is also connected to the denaturation zone pipeline. The processing temperature of the first pipeline is higher than the processing temperature of the second pipeline.

19. The library building system according to any one of claims 12 to 18, characterized in that: The library building system also includes a sample pool, which is used to receive samples discharged from the upper module. The sample pool includes: an inlet pipe, a sample container and an outlet pipe. A sealing plug is provided in the sample container, and the inlet pipe and the outlet pipe are provided through the sealing plug.

20. A library construction method, characterized in that: The method is implemented by using the library building system described in any one of claims 10 to 19.

21. The library construction method according to claim 20, characterized in that: The method comprises: a volume determination step performed according to the volume determination method according to any one of claims 1 to 3.

22. The library construction method according to claim 21, characterized in that: The method further includes a purification step implemented by using a purification module in the library construction system, and the library construction method further includes: performing the volume determination step while performing the purification step.

23. The library construction method according to claim 22, characterized in that: The purification step is a sample purification step, which is used to process the sample to be purified to obtain a purified sample. The volume determination step is a PCR volume determination step, which is used to process the purified sample to obtain a PCR mixed sample; or, The purification step is a PCR purification step, which is used to purify the PCR mixed sample to obtain a PCR purified sample. The volume determination step is a step of connecting sequencing adapters, which is used to perform adapter connection on the PCR purified sample.

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