Reaction system and kit based on digital microfluidic technology platform

Abstract

The invention provides a reaction system and a kit based on a digital microfluidic technology platform. The reaction system comprises: a water phase, wherein the water phase does not contain a surfactant; and the continuous phase comprises silicone oil, and the silicone oil contains a non-ionic surface active agent. When the reaction system is applied to a digital micro-fluidic platform, the reagent consumption cost can be saved, and the influence of addition of an exogenous surfactant on a complex biochemical reaction is reduced. Specifically, the method is applied to the field of sequencing, and the problems of low sequencing data volume and even sequencing failure caused by surfactant residues are solved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and more specifically to a reaction system and a kit based on a digital microfluidic technology platform, and more specifically to a reaction system, a library construction method, a sequencing method and a kit based on a microfluidic technology platform. Background Art

[0002] Digital microfluidics (DMF) is an emerging microfluidic technology that uses precise manipulation of droplets in the microliter to nanoliter range to achieve complex laboratory analysis. The core is to discretize the liquid flow, and the droplets act as separate reaction containers. These droplets are individually controlled through the electrowetting phenomenon on a special medium (electro-wetting on dielectric, referred to as EWOD). Digital microfluidics has important application value in biology, chemistry, medicine and other fields, especially in trace liquid analysis, biochemical reactions, digital PCR and other aspects. It is simple to operate and highly automated, which helps to avoid cross-contamination and loss of analytes in droplets.

[0003] The core and difficulty of digital microfluidics is to use electrical signals on the chip to precisely manipulate the movement of trace amounts of liquid. The principle is to change the voltage of the chip electrode to change the hydrophilicity of its surface accordingly, so that the contact angle of the droplet on the adjacent electrode surface is different, so that the droplet has a difference in surface tension in different directions, so as to manipulate the droplet to produce directional movement, splitting, merging and other phenomena. In actual operation, the droplet is usually first distributed from the reservoir in the closed area, and according to the application requirements, the multiple droplets in the closed area are independently and in real time controlled through other basic operations of the droplet to complete the pretreatment of the sample, and then driven to the open area to complete the post-processing and analysis detection. If necessary, the droplet can also be returned to the closed area according to the application requirements, and the required droplet operation and analysis can be performed on it, or the droplet can be directly collected in the open area. To achieve the free reciprocating movement of the droplet in the closed-open interval, the initial contact angle between the droplet in the reservoir and the electrode must be in a suitable range. Therefore, the current mainstream solution is to add a certain concentration of surfactant to the droplet to reduce the surface tension of the droplet. Usually, the HLB range is between 8 and 16 for O / W type emulsifiers, and the preferred non-ionic surfactant is the polyoxyethylene sorbitan fatty acid ester series.

[0004] Since polyoxyethylene sorbitan fatty acid esters have more hydrophilic groups, namely polyoxyethylene groups, they are highly hydrophilic and can easily enter into biochemical reagents in the aqueous phase. However, such non-ionic surfactants are equivalent to exogenous interferents for most biochemical reactions, especially molecular biological reactions, and will significantly inhibit or even cause the failure of biochemical reactions, directly resulting in the inability to develop many important applications based on digital microfluidics technology. In addition, polyoxyethylene sorbitan fatty acid esters may be unstable under high-pressure sterilization, especially in buffer solutions containing metal cations. They are sensitive to heat and their molecular structure will change under long-term exposure to light. Aqueous solutions will also undergo auto-oxidation during storage. These characteristics result in the stability and shelf life of biochemical reagents currently used in digital microfluidics failing to meet actual needs.

[0005] As an emerging technology, digital microfluidics has broad application prospects, but its biochemical system still needs to be further optimized to avoid interfering with biochemical reactions and affecting the quality of sequencing library construction. Summary of the invention

[0006] This application is made by the inventor based on the discovery of the following problems:

[0007] The existing digital microfluidic aqueous biochemical system is difficult to accurately prepare (lower content) and has complex operation due to the additional addition of surfactants (such as Tween, etc.); in addition, adding Tween to biochemical reagents will also have unknown effects on biochemical reactions. When the Tween content is too high, it will seriously affect the progress of the biochemical reaction. For example, Tween 20 with a mass concentration of more than 1% will seriously affect the progress of the biochemical reaction.

[0008] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the inventors have found through a large number of experiments that in the process of constructing a sequencing library for a microfluidic device, no surfactant is added to the aqueous phase (sequencing reagents, such as enzymes, primers and buffers, etc.), and a non-ionic polysiloxane surfactant is added to the continuous phase (silicone oil). This type of non-ionic surfactant can be miscible with silicone oil in any ratio, and it is easy to form stable oil-in-water droplets, which can make the separation and pipetting of biochemical reagents in the aqueous phase undisturbed, avoiding the problem of low sequencing data or sequencing failure caused by residual surfactant.

[0009] In the first aspect of the present application, the present application proposes a reaction system based on a microfluidic technology platform. According to an embodiment of the present application, the reaction system comprises: an aqueous phase, wherein the aqueous phase does not contain a surfactant; and a continuous phase, wherein the continuous phase comprises silicone oil, wherein the silicone oil contains a nonionic surfactant.

[0010] According to the embodiments of the present application, the reaction system applied to the digital microfluidic platform can save reagent consumption costs and reduce the impact of the addition of exogenous surfactants on complex biochemical reactions.

[0011] According to an embodiment of the present application, the above reaction system further includes at least one of the following additional features:

[0012] According to an embodiment of the present application, the nonionic surfactant has the same main chain structure as the silicone oil. According to an embodiment of the present application, the nonionic surfactant having the same main chain structure as the silicone oil can be better soluble in the continuous phase.

[0013] According to an embodiment of the present application, the nonionic surfactant includes a nonionic polysiloxane surfactant. According to an embodiment of the present application, the inventors finally found through a large number of screenings that nonionic polysiloxane surfactants have better compatibility with silicone oils, and that the results are better after being used to construct a sequencing library and sequencing.

[0014] According to the embodiments of the present application, the HLB value of the nonionic polysiloxane surfactant is not greater than 10. According to the embodiments of the present application, the HLB value is greater than 10 and is easily soluble in the aqueous phase. In order to be better compatible with the continuous phase (silicone oil), the inventors screened a surfactant with an HLB value of no more than 10.

[0015] According to an embodiment of the present application, the HLB value of the nonionic polysiloxane surfactant is no more than 5.

[0016] According to an embodiment of the present application, the HLB value of the nonionic polysiloxane surfactant is less than 3 and not less than 1.

[0017] According to an embodiment of the present application, the nonionic polysiloxane surfactant has a structure as shown in formula (I), wherein one or more -CH3 at one end of the siloxane in the structure as shown in formula (I) is replaced by one or more nonionic organic polar groups.

[0018]

[0019] According to an embodiment of the present application, the nonionic organic polar group includes polyether, alkanolamide, ester, glycoside, fatty alcohol and polyol. Preferably, the nonionic organic polar group is polyether, ester, fatty alcohol and polyol.

[0020] According to an embodiment of the present application, the modification degree of the nonionic polysiloxane surfactant is not less than 3.

[0021] It should be noted that the degree of modification refers to the number of modified groups or functional groups in the compound molecule. According to an embodiment of the present application, the degree of modification of the nonionic polysiloxane surfactant includes 3, 4, 5, 6, 7, 8, 9, and 10.

[0022] According to an embodiment of the present application, the nonionic polysiloxane surfactant includes at least one of cetyl PEG / PPG-10 / 1 polydimethylsiloxane and Silwet L-77.

[0023] According to an embodiment of the present application, the mass fraction of the nonionic surfactant in the continuous phase is 0.001%-0.1%.

[0024] According to an embodiment of the present application, the mass fraction of the nonionic surfactant in the continuous phase is 0.001%.

[0025] According to an embodiment of the present application, the mass fraction of the nonionic surfactant in the continuous phase is 0.005%.

[0026] According to an embodiment of the present application, the mass fraction of the nonionic surfactant in the continuous phase is 0.01%.

[0027] In the second aspect of the present application, the present application proposes a library construction method based on a microfluidic technology platform. According to an embodiment of the present application, the method comprises: placing the reaction system described in the first aspect of the present application in a microfluidic device to construct a sequencing library.

[0028] According to the embodiments of the present application, the biochemical reagent formula of the aqueous phase in the digital microfluidic sequencing is consistent with the original system, ensuring that the performance of the digital microfluidic biochemical system is not affected, and the automation of complex biochemical processes (such as NGS library construction) can be realized, expanding the application scenarios of digital microfluidics. In addition, it also makes the preparation of biochemical reagents in the aqueous phase easier, avoiding the cumbersome steps of introducing different surfactant addition amounts of multiple types of reagents, greatly simplifying the preparation process of digital microfluidic biochemical reagents, and promoting the large-scale application of digital microfluidic platforms. More importantly, compared with the Span series of surfactants, silicone oil non-ionic surfactants are extremely soluble in silicone oil, and can be completely dissolved in silicone oil without the help of magnetic heating stirring and ultrasonic and other complex treatments, eliminating the deviation between batches of the continuous phase (oil phase) introduced by the latter due to complex operation.

[0029] According to the embodiments of the present application, the above-mentioned library construction method based on the microfluidic technology platform may also include the following technical features:

[0030] According to an embodiment of the present application, the sequencing library construction is achieved in the following manner: adding the aqueous phase in the reaction system to the microfluidic chip, the aqueous phase including microfluidic library construction reagents and samples to be tested, and the aqueous phase does not contain a surfactant; covering the microfluidic chip with the aqueous phase added with the continuous phase in the reaction system, the continuous phase including silicone oil, and the silicone oil containing a non-ionic surfactant; placing the microfluidic chip covered with the continuous phase in a microfluidic device for sequencing library construction.

[0031] In the third aspect of the present application, the present application proposes a sequencing method. According to an embodiment of the present application, the method comprises: preparing a sequencing library based on the method described in the second aspect of the present application; and sequencing the sequencing library on a sequencing platform.

[0032] According to the embodiments of the present application, the sequencing method can ensure sequencing stability and sequencing quality. The sequencing method is based on a fully automatic high-throughput sequencing detection system and can achieve full automation of detection and analysis.

[0033] According to an embodiment of the present application, the sequencing platform includes at least one selected from Illumina's HiSeq, NextSeq, MiSeq, NovaSeq platforms, BGISEQ, MGISEQ and DNBSEQ platforms, Thermo Fisher or LifeTechnologies' Ion Torrent platform, PacBio's REVIO system, ONSO system and SEQUEL IIe system.

[0034] It should be noted that, in this article, there is no particular limitation on the sequencing platform, and it can be any common second-generation or third-generation sequencing platform.

[0035] In the fourth aspect of the present application, the present application proposes a library construction kit for use in a microfluidic technology platform. According to an embodiment of the present application, the kit includes: an aqueous phase, which is a microfluidic library construction reagent, and the aqueous phase does not contain a surfactant; a continuous phase, and the continuous phase includes silicone oil, and the silicone oil contains a non-ionic surfactant. The use of the aforementioned kit can significantly improve the efficiency of library construction, and no surfactant is added to the library construction reagent, which greatly ensures the stability of the reagent and realizes a high-throughput, high-efficiency and low-cost library construction process.

[0036] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0038] Figure 1 is a schematic diagram of the microfluidic chip according to Example 2 of the present application;

[0039] Figure 2 It is the result of pathogenic microorganism identification and analysis based on the sequencing data described in Example 3 of the present application;

[0040] Figure 3 It is a comparison of the fragment analysis results of the products generated by different blocking solutions described in Comparative Example 1 of the present application;

[0041] Figure 4 This is the performance verification result of different blocking solutions in multiplex PCR according to Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0042] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0043] In the present application, the nonionic polysiloxane surfactant can be applied to various digital microfluidic platforms. The digital microfluidic platform includes a microfluidic chip and a microfluidic device, which are detachably connected. Silicone oil is widely used as a continuous phase in droplet microfluidics due to its good biological inertness. When the microfluidic platform generates droplets, it is not only related to the physical properties (hydrophobicity) of the chip channel, but also to the surface tension difference between the two phases used to form the droplets. Surfactants are amphiphilic molecules that have the effect of reducing surface tension. Adding surfactants is conducive to the generation of droplets on the microfluidic platform.

[0044] Prior art related to this application

[0045] Digital microfluidic devices, which have been widely studied and applied, generally adopt a double-substrate structure similar to a "sandwich", in which a fluid-fluid dispersion (mainly an oil-in-water emulsion) is produced in the middle, that is, silicone oil or mineral oil as a continuous phase and aqueous phase droplets as a dispersed phase, with different concentrations of non-ionic surfactants added to both phases.

[0046] Commonly used non-ionic surfactants include polyoxyethylene sorbitan fatty acid ester (trade name Tween) and sorbitan fatty acid ester (trade name Span) series. Tween series belongs to W / O type emulsifier, which is easily soluble in water phase. In the prior art, a certain concentration of polyoxyethylene sorbitan monolaurate (trade name Tween20) is usually added to the water phase. In silicone oil as a continuous phase, the lower HLB value of Span series surfactant makes it insoluble in water phase but soluble in oil phase. In the prior art, a certain concentration of sorbitan trioleate (trade name Span85) is often added. The interaction between the two reduces the surface tension of the droplets in the closed area, so that the biochemical reaction reagents can be accurately separated and pipetted on the microfluidic chip through the switch of the electrode.

[0047] The Tween series surfactants used in the prior art are W / O type emulsifiers, and the HLB value is generally greater than 10. Adding Tween series surfactants to the aqueous phase is equivalent to adding additional exogenous substances compared to the original reaction system, which will have a certain negative impact on most biochemical reactions, especially biochemical reactions of sensitive molecules in the system (such as DNA enzyme interruption, DNA fragment ligation reaction, reverse transcription reaction, PCR amplification, cDNA second-chain synthesis and DNA rolling circle amplification, etc.), resulting in a large deviation in the results and even reaction failure. Taking the DNBSEQ sequencing platform manufactured by BGI as an example, Tween series surfactants will cause abnormal loading of DNB nanospheres and even prevent the sequencing reaction from proceeding smoothly. In addition, since low concentrations of Tween need to be added to small volumes of reaction reagents, this not only makes the operation complicated, but also requires multiple operations for multiple reagents in one reaction, and the accuracy is difficult to guarantee. The Span series of surfactants present in silicone oil will also have unknown effects on biochemical reactions. For example, when the Span85 content exceeds 0.07%, it will seriously affect the uniformity of the magnetic beads, causing the magnetic beads to aggregate and precipitate on the digital microfluidic chip, resulting in library construction failure.

[0048] Reaction system based on microfluidic technology platform

[0049] In one aspect of the present application, the present application proposes a reaction system based on a microfluidic technology platform. According to an embodiment of the present application, the reaction system comprises: an aqueous phase, wherein the aqueous phase does not contain a surfactant; and a continuous phase, wherein the continuous phase comprises silicone oil, wherein the silicone oil contains a nonionic surfactant.

[0050] According to the embodiments of the present application, the reaction system described in the present application can be widely used in PCR, vector construction, sequencing library construction, and sequencing processes. There is no need to add surfactants to the aqueous phase, thereby eliminating the interference of surfactants on the reaction system; and reducing the cost of adding surfactants.

[0051] Library construction method based on microfluidic technology platform

[0052] In one aspect of the present application, the present application proposes a library construction method based on a microfluidic technology platform. According to an embodiment of the present application, the method comprises: placing the reaction system described in the first aspect of the present application in a microfluidic device to construct a sequencing library.

[0053] According to the embodiments of the present application, the sequencing library construction method solves the problem that the existing microfluidic biochemical system has complex biochemical reagent preparation steps and large differences in accuracy due to the addition of a small amount of non-ionic surfactant, which leads to the inhibition of important molecular biological and biochemical reactions and seriously interferes with the loading of DNB nanospheres on the sequencing chip (such as the MGI DNBSEQ sequencing platform), making digital microfluidics limited to some simple applications and not suitable for complex biochemical processes (such as NGS sequencing library construction, etc.).

[0054] It should be noted that after screening a series of surfactants, the inventors found that the nonionic polysiloxane surfactant having the structure shown in the main chain of formula (I) can be miscible with the continuous phase (silicone oil) in any ratio, and it is easy to form stable oil-in-water droplets, which can make the biochemical reagents in the aqueous phase be normally separated and pipetted, thereby ensuring the stability of the library construction reagents and a higher sequencing data volume (the success rate of the library construction point is more than 97%, such as the MGI sequencing platform after the library is built, MGISEQ-200FCL sequencing> 500M). The inventors also found that by replacing one or more nonionic organic polar groups on -CH3 in the structure shown in formula (I), the HLB value (lipophile-hydrophile balance value) of the surface activity can be reduced, so that the surfactant can be better soluble in the continuous phase.

[0055] Specifically, for ease of understanding, the technical solution of the present application is described exemplarily below:

[0056] According to an embodiment of the present application, a certain volume of sealing liquid (continuous phase) is evenly filled into a microfluidic chip with a parallel double-substrate structure through a funnel, and then the prepared nucleic acid samples and biochemical reagents are directly added to the reagent storage area on the chip (the temperature of the storage area can be controlled by a water cooling module, for example, it can be controlled at 4 degrees Celsius), the microfluidic chip is placed on a digital microfluidic device, and the run button in the program is clicked. The reaction reagents can move and mix under the control of the electrodes, and the bottom of the digital microfluidic chip, that is, the temperature control module and the magnetic control module on the microfluidic device are used to realize various biochemical reactions and magnetic bead purification of nucleic acids.

[0057] The sample DNA on the microfluidic chip first enters the reaction zone under the control of the electrode, and then the corresponding reaction reagent enters. In the reaction zone, DNA fragments are sheared, end repaired, adapters are connected, magnetic beads are purified and PCR amplified, and the PCR products are thermally denatured to obtain single-stranded circular DNA, which is then subjected to rolling circle amplification to obtain DNA nanoballs. In addition, after the DNB nanoballs are generated, the products can be quantified using the fluorescence quantification module in the digital microfluidic device.

[0058] Sequencing methods

[0059] In another aspect of the present application, the present application proposes a sequencing method. According to an embodiment of the present application, the method comprises: preparing a sequencing library based on the method described in the first aspect of the present application; and sequencing the sequencing library on a sequencing platform.

[0060] According to an embodiment of the present application, the sequencing is performed based on a sequencing library constructed by the aforementioned method, which solves the problem of low data volume or even sequencing failure caused by surfactant residues.

[0061] It should be noted that the sequencing can be performed by a sequencing platform. According to the embodiments of the present application, the sequencing platform that can be used to perform the above sequencing method can be selected from but not limited to Illumina's HiSeq / Miseq / Nextseq / Novaseq sequencing platforms, Thermo Fisher / Life Technologies' Ion Torrent platforms, BGISEQ and MGISEQ / DNBSEQ platforms of BGI, and single-molecule sequencing platforms; the sequencing method can be selected from single-end sequencing or double-end sequencing.

[0062] Reagent test kit

[0063] In another aspect of the present application, the present application proposes a library construction kit for use in a microfluidic technology platform. According to an embodiment of the present application, the kit includes: an aqueous phase, which is a microfluidic library construction reagent and does not contain a surfactant; and a continuous phase, which includes silicone oil and contains a nonionic surfactant. According to an embodiment of the present application, the kit can improve the efficiency of library construction, and since the sequencing reagent does not contain a surfactant, the stability of the aqueous phase reagent is further guaranteed.

[0064] Beneficial Effects

[0065] According to the embodiments of the present application, a surfactant is added to the silicone oil, which avoids the cumbersome operation of adding a surfactant to a variety of biochemical reagents. Previously, it was necessary to add surfactants to the 10 or so reagents used for library construction. Now, it is only necessary to add surfactants to the silicone oil to meet the liquid separation and pipetting requirements of all library construction reagents. In addition, the surfactant is added to the silicone oil instead of the biochemical reagent, which reduces the impact on the physicochemical properties and biochemical reactions of the biochemical reagents, greatly improves the success rate of library construction, and the sequencing data is also increased from the original 350M to 500M. In addition, this method combines a microfluidic device to build a library, which greatly reduces the consumption of biochemical reagents. Only 6ng DNA is needed to complete the entire process. The use of related biochemical reagents is reduced by about 5 times compared to manual library construction. For example, FEA (Fluorescent EndAdapter, fluorescent end adapter) reagents only need 1μL, which greatly saves the cost of library construction. Further reduce manual operation time. Manual library construction often takes up to 8 hours of manual operation. With microfluidic library construction, manual operation time is only about 15 minutes, and the rest of the work can be completed on the microfluidic device. The library construction time on the microfluidic device is only 3 hours, and DNB nanospheres for subsequent sequencing can be obtained. In addition, while reducing manual operation time, it also reduces some errors caused by manual operation. The error of microfluidic library construction is much smaller than that of manual library construction.

[0066] It should be noted that the features and technical effects described in this article for different aspects can be used as reference for each other and will not be repeated here.

[0067] The present application is illustrated below by way of examples, but this should not be construed as limiting the scope of the subject matter of the present application to the following examples. All technologies implemented based on the above content of the present application belong to the scope of the present application. The compounds or reagents used in the following examples can be purchased from commercial sources or prepared by conventional methods known to those skilled in the art; the experimental instruments used can be purchased from commercial sources.

[0068] It should be noted that, for the convenience of description, in the following embodiments, silicone oil containing nonionic surfactant is collectively referred to as sealing liquid.

[0069] Example 1: Preparation of blocking solution

[0070] This example is a blocking solution preparation, and the reagents used are shown in Table 1:

[0071] Table 1:

[0072] Component name brand Part Number Dimethicone Myrelle GEL-DMS-T05 Cetyl PEG / PPG-10 / 1 Polydimethylsiloxane SILTECH SilubeT308-16-TT Silwet L-77 Yisheng Biology 41008ES10

[0073] 1. Prepare a blocking solution containing 0.01% cetyl PEG / PPG-10 / 1 polydimethylsiloxane (blocking solution 1)

[0074] 1.1 Take a suitable preparation bottle and weigh it with a balance (precision of 0.1 mg) (tare weight), and use the tare preparation bottle to weigh 10g of polydimethylsiloxane;

[0075] 1.2 Use a pipette to draw 1 μL of cetyl PEG / PPG-10 / 1 polydimethylsiloxane and add it all to 10 mL of silicone oil to prepare a blocking solution. Shake it up and down 5-10 times.

[0076] Note: Cetyl PEG / PPG-10 / 1 polydimethylsiloxane has a high viscosity. It needs to be aspirated slowly when using a pipette. When adding, only insert the tip of the pipette into the silicone oil, add it slowly, and blow back and forth several times to ensure that the liquid in the pipette tip is completely added to the silicone oil.

[0077] 1.3 Turn on the ultrasonic cleaning machine, put the mixed sealing solution into the ultrasonic cleaning machine, and use the ultrasonic cleaning machine to ultrasonicate for 10 minutes, with an ultrasonic power of 100%, and the ultrasonic temperature does not need to be controlled;

[0078] 1.4 After the timing is over, take out the sealing liquid from the ultrasonic cleaning, wipe the outside of the bottle dry, put it on the magnetic stirrer, set the speed to 300r / min, set the timing to 10min, and start stirring;

[0079] 1.5 Dispense the blocking solution in step 1.4 into glass tubes equipped with rotors, 3.1 mL per tube, put on the lid without sealing it tightly, then evacuate the tube for 80 minutes, press the tube lid tightly and evacuate and seal it with an aluminum foil bag.

[0080] 2. Prepare blocking solution containing 0.001% Silwet L-77 (blocking solution 2)

[0081] 2.1 Take a suitable preparation bottle and weigh it with a balance (precision of 0.1 mg) (tare weight), and use the tare preparation bottle to weigh 10g of polydimethylsiloxane;

[0082] 2.2 Use a pipette to draw 0.1 μL of Silwet L-77 and add it all to the weighed silicone oil to prepare the blocking solution. Shake up and down 5-10 times;

[0083] Note: The viscosity of Silwet L-77 is slightly higher than that of conventional reagents. When using a pipette, it needs to be aspirated slowly. When adding, only insert the tip of the pipette into the silicone oil, add it slowly, and blow back and forth several times to ensure that the liquid in the pipette tip is completely added to the silicone oil.

[0084] 2.3 Turn on the ultrasonic cleaning machine, put the mixed sealing solution into the ultrasonic cleaning machine, and use the ultrasonic cleaning machine to ultrasonicate for 10 minutes, with an ultrasonic power of 100%, and the ultrasonic temperature does not need to be controlled;

[0085] 2.4 After the timing is over, take out the sealing liquid from the ultrasonic cleaning, wipe the outside of the bottle dry, put it on the magnetic stirrer, set the speed to 300r / min, set the timing to 10min, and start stirring;

[0086] 2.5 Dispense the above blocking solution into glass tubes equipped with rotors, 3.1 mL per tube, put on the lid without tightening it, then evacuate the tube for 80 minutes, press the tube lid tightly and evacuate and seal it with an aluminum foil bag.

[0087] Example 2: Construction of a fully automated enzyme digestion DNA library based on digital fluidics using blocking solution 1

[0088] The digital microfluidic platform used in the construction of the fully automatic enzyme digestion DNA library in this embodiment is the DNBelab-D4RS digital sample preparation system (Cat. No.: 900-000369-00) manufactured by MGI, and the reagent kit used is the MGIEasy enzyme digestion DNA library preparation reagent kit (Cat. No.: 1000006988) manufactured by MGI.

[0089] In this example, human gDNA was used as a template, with an initial input of 50 ng. After the entire sample preparation card was completely filled with 3.1 ml of the blocking solution 1 prepared in Example 1, enzyme cleavage, end repair and dA tail addition, adapter ligation, PCR amplification, one-step preparation of DNB and DNB quantitative components, and magnetic bead purification components were prepared according to the kit instructions. Then all reagents and samples were added to the microfluidic chip filled with the blocking solution ( Figure 1 ), click the corresponding program to start automatic database building.

[0090] Finally, the DNB concentrations of the four samples with known concentrations (DNBSEQ platform-specific library) were all above 10 ng / μL (Table 2), meeting the quality control standards.

[0091] Table 2:

[0092]

[0093] The four libraries constructed above were mixed together and sequenced using MGISEQ-200 with the sequencing mode of SE100. The sequencing results are shown in Table 3, which met the FCL single-slide effective reads quantity quality control standard.

[0094] Table 3:

[0095]

[0096] By performing human resequencing analysis on the above-mentioned offline data, as shown in Table 4, the result indicators all meet the requirements.

[0097] Table 4:

[0098]

[0099]

[0100] Example 3: Fully automated mNGS library construction based on digital fluidics using blocking solution 2

[0101] The digital microfluidic platform used in the construction of the fully automatic enzyme-cut DNA library in this embodiment is the DNBelab-D4RS digital sample preparation system (Cat. No.: 900-000369-00) manufactured by MGI, and the reagent kit used is the DNBelab-D4RS enzyme-cut DNA library preparation reagent kit V2.0 (Cat. No.: 940-001198-00) manufactured by MGI.

[0102] In this example, human and different proportions of Meta DNA were mixed as templates, with an initial input of 6 ng. After the entire sample preparation card was completely filled with 3.1 ml of the blocking solution 2 prepared in Example 1, enzyme cutting, end repair, dA tail addition, adapter ligation, PCR amplification, one-step preparation of DNB and DNB quantitative components, and magnetic bead purification components were prepared according to the kit instructions. Then, all reagents and samples were added to the microfluidic chip filled with the blocking solution ( Figure 1 ), click the corresponding program to start automatic database building.

[0103] Finally, the DNB concentrations of the four samples with known concentrations (DNBSEQ platform-specific library) were all above 10 ng / μL (Table 5), meeting the quality control standards.

[0104] Table 5:

[0105]

[0106] The four libraries constructed above were mixed together and sequenced using DNBSEQ-G99 with the sequencing mode of SE100. The sequencing results are shown in Table 6, which met the quality control standard of the number of effective reads per slide.

[0107]

[0108] This example uses the MGI PFIV5.0 process to analyze pathogenic microorganisms in the offline data, and the result indicators meet the requirements ( Figure 3 ).

[0109] Comparative Example 1: Compatibility test of the new system using blocking solution 1 or 2 and the traditional system using the old blocking solution for biochemical reactions

[0110] In this embodiment, the old blocking solution specifically refers to silicone oil with 0.01%-0.04% span85 added thereto, and the aqueous phase reagent matched therewith is added with 0.05%-0.15% Tween-20.

[0111] The digital microfluidic platform used in the construction of the fully automatic enzyme digestion DNA library in this example is the DNBelab-D4RS digital sample preparation system (Cat. No.: 900-000369-00) manufactured by MGI, and the kits used are the MGI MGIEasy Fast PCR-FREE enzyme digestion library preparation reagent kit (Cat. No.: 940-000021-00) and the ATOPlex RNA multiplex PCR library construction kit set V3.1 (Cat. No.: 940-000127-00).

[0112] In this example, human standard NA12878 was used as a template for enzyme digestion and shearing, and the initial input amount was 150 ng. After the entire sample preparation card was completely filled with 3.1 ml of each of the blocking solutions 1 and 2 prepared in Example 1 and the old blocking solution, the enzyme digestion and shearing, end repair and dA tail addition, and magnetic bead purification components were prepared according to the instructions of the kit. Then, all reagents and samples were added to the microfluidic chip containing the blocking solution, and the corresponding program was clicked to start the running process. After the reaction was completed, the product was taken for fragment range analysis ( Figure 3 ).

[0113] In this embodiment, the multiplex PCR amplification reaction uses the Jingliang COVID-19 standard as a template, with an initial input of 100 copies. After the entire sample preparation card is completely filled with 3.1 ml of each of blocking solution 1 and 2 (prepared in Example 1) and the old blocking solution (Comparative Example 1), the RT-PCR reaction solution, primer pool, and magnetic bead purification component are prepared according to the instructions of the kit, and then all reagents and samples are added to the microfluidic chip containing the blocking solution ( Figure 1 ), click the corresponding program to start the process, and after the reaction is completed, take the product for quantitative analysis ( Figure 4 ).

[0114] The results showed that compared with the traditional system using the old blocking solution, the new system using blocking solutions 1 and 2 made the enzyme-cutting bands more uniformly diffuse, and there was no large fragment aggregation due to incomplete cutting; compared with the traditional system, the efficiency of the multiplex PCR reaction of the new system was increased by 10%, indicating that the new system would be more compatible with important biochemical reactions and could significantly improve the efficiency of biochemical reactions.

[0115] Comparative Example 2: Comparison of sequencing quality of MGISEQ-200 using the new system with blocking solution 1 or 2 and the traditional system with the old blocking solution

[0116] In this embodiment, the old blocking solution specifically refers to silicone oil with 0.01%-0.04% span85 added thereto, and the aqueous phase reagent matched therewith is added with 0.05%-0.15% Tween-20.

[0117] The digital microfluidic platform used in the fully automatic enzyme cleavage and multiplex PCR amplification reaction in this embodiment is the DNBelab-D4RS digital sample preparation system (Cat. No.: 900-000369-00) manufactured by MGI, and the kit used is the DNBelab-D4RS enzyme cleavage DNA library preparation kit set V2.0 (Cat. No.: 940-001198-00) manufactured by MGI.

[0118] In this example, human standard NA12878 was used as a template, and the initial input amount was 10 ng. After the entire sample preparation card was completely filled with the blocking solutions 1 and 2 prepared in Example 1 and the old blocking solution totaling 3.1 ml, the sample was thawed on ice for enzyme digestion, end repair and addition of dA tail, adapter connection, PCR amplification, and one-step preparation of DNB and DNB quantitative components according to the kit instructions. Subsequently, all reagents and samples were added to the microfluidic chip containing the blocking solution, and the corresponding program was clicked to start the automatic library construction. After the library construction was completed, DNBs were collected, and the sequencing operation was performed with reference to the MGISEQ-200RS High-throughput (Fast) Sequencing Reagent Set Instructions for Use.

[0119] The results are shown in Table 7. Each blocking solution produced 3 runs and 12 lanes of DNB. The library construction success rate standard was DNB concentration>20ng / μL, the success rate of blocking solutions 1 and 2 was 100%, and the success rate of the old blocking solution was 25%; the MGISEQ-200RS qualification standard after sequencing was FCL Total Reads≥500M, the qualification rate of blocking solutions 1 and 2 was 100%, and the success rate of the old blocking solution was 0%. The amount of sequencing data of blocking solutions 1 and 2 increased by more than 80% compared with the old blocking solution, which fully demonstrated the good compatibility of the new system for sequencing.

[0120] Table 7:

[0121]

[0122]

[0123] In summary, the library construction method based on the digital microfluidic technology platform of the present application has achieved the successful application of complex biochemical processes (such as the construction of the second-generation sequencing library on the digital microfluidic platform). The library sequencing results obtained are no different from those of manual operations, but have obvious advantages in the use of biochemical reagents in a single reaction and automated operations, and are of great significance in the field of digital microfluidic technology.

[0124] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0125] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A reaction system based on a microfluidic technology platform, characterized in that: include: an aqueous phase, wherein the aqueous phase does not contain a surfactant; and The continuous phase comprises silicone oil, and the silicone oil contains a nonionic surfactant.

2. The reaction system according to claim 1, characterized in that The nonionic surfactant has the same main chain structure as the silicone oil.

3. The reaction system according to claim 2, characterized in that The nonionic surfactant includes a nonionic polysiloxane surfactant.

4. The reaction system according to claim 3, characterized in that: The HLB value of the nonionic polysiloxane surfactant is not greater than 10; Optionally, the HLB value of the nonionic polysiloxane surfactant is not greater than 5; Preferably, the HLB value of the nonionic polysiloxane surfactant is less than 3; More preferably, the HLB value of the nonionic polysiloxane surfactant is less than 3 and not less than 1.

5. The reaction system according to claim 3, characterized in that: The nonionic polysiloxane surfactant has a structure as shown in formula (I), wherein one or more -CH3 at one end of the siloxane in the structure as shown in formula (I) is replaced by one or more nonionic organic polar groups. Optionally, the nonionic organic polar groups include polyethers, alkanolamides, esters, glycosides, fatty alcohols and polyols; preferably, the nonionic organic polar groups are polyethers, esters, fatty alcohols and polyols.

6. The reaction system according to claim 5, characterized in that The modification degree of the nonionic polysiloxane surfactant is not less than 3.

7. The reaction system according to claim 3, characterized in that: The nonionic silicone surfactant includes at least one of cetyl PEG / PPG-10 / 1 polydimethylsiloxane and Silwet L-77.

8. The reaction system according to claim 1, characterized in that: The mass fraction of the nonionic surfactant in the continuous phase is 0.001%-0.1%; Preferably, the mass fraction ratio of the nonionic surfactant in the continuous phase is 0.001%; Preferably, the mass fraction ratio of the nonionic surfactant in the continuous phase is 0.005%; More preferably, the mass fraction of the nonionic surfactant in the continuous phase is 0.01%.

9. A library construction method based on a microfluidic technology platform, characterized in that: include: The reaction system according to any one of claims 1 to 8 is placed in a microfluidic device to construct a sequencing library.

10. The method according to claim 9, characterized in that The sequencing library construction is achieved in the following manner: Adding the aqueous phase in the reaction system into the microfluidic chip, wherein the aqueous phase includes microfluidic library preparation reagents and samples to be tested, and the aqueous phase does not contain a surfactant; Covering the microfluidic chip with the aqueous phase with a continuous phase in the reaction system, wherein the continuous phase includes silicone oil, and the silicone oil contains a nonionic surfactant; The microfluidic chip covered with the continuous phase is placed in a microfluidic device to construct a sequencing library.

11. A sequencing method, characterized in that: include: Prepare a sequencing library based on the method of claim 9 or 10; Sequencing the sequencing library on a sequencing platform; Optionally, the sequencing platform includes at least one selected from Illumina's HiSeq, NextSeq, MiSeq, NovaSeq platforms, BGISEQ, MGISEQ and DNBSEQ platforms of BGI, IonTorrent platforms of Thermo Fisher or Life Technologies, REVIO system, ONSO system and SEQUEL IIe system of PacBio.

12. A library construction kit for use in a microfluidic technology platform, characterized in that: include: Aqueous phase, which is a microfluidic library preparation reagent and does not contain a surfactant; The continuous phase comprises silicone oil, and the silicone oil contains a nonionic surfactant.

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