Digitized quantitative multi-target common, biological detection

Abstract

And digitally quantitative multi-target sharing is realized. The present invention includes methods and devices for detecting and quantifying a plurality of biomolecules at a single molecule level using an integrated droplet microfluidic system. In one embodiment, the present invention provides real-time and digital detection of a plurality of biomolecules in a sample to quantify the plurality of biomolecules in an absolute and instant manner. In one embodiment, the present invention provides a method of diagnosing a disease comprising real-time and digital detection of a plurality of biomolecules in a sample using the method or device described herein.

Description

[0001] This application is a divisional application of a Chinese application, application number: 201910790298.3, application date: August 26, 2019. This application claims priority to a U.S. provisional application, application number: 62 / 723,455, application date: August 27, 2018. Field of the Invention

[0002] The present invention relates to methods and devices for detecting and quantifying a variety of biomolecules at the single molecule level using an integrated droplet microfluidics system. Background of the Invention

[0004] The background description of the present invention is only for the purpose of generally introducing the prior art of the present invention and shall not constitute any limitation to the present invention.

[0005] There are many biomarkers in human body fluids, such as in blood, urine, saliva or semen. Circulating biomarkers present in the blood include free DNA (cf-DNA, proteins, extracellular vesicles and circulating tumor cells. Accurate quantification of biomarkers is of great significance for the correct and reliable diagnosis, prognosis and progression monitoring of diseases or conditions, especially for early detection of diseases or prenatal screening in the form of non-invasive prenatal testing (NIPT). However, at the time of the present invention, the only widely used blood test for early prostate cancer detection is based on the measurement of the prostate specific antigen (PSA) marker, and the appropriate use of this test is still controversial. Measuring the number of biomarkers with high sensitivity and specificity is crucial to prevent misdiagnosis, because on the one hand too many healthy individuals will get false positive test results, resulting in unnecessary regular review procedures; on the other hand, false negative test results may lead to delayed treatment and adversely affect patients. In addition, from the perspective of scientific discovery, accurate quantification of biomarkers is important for understanding the association between biomarkers and corresponding diseases, and establishing reliable and robust associations for use as clinical indicators of diseases.

[0006] Many efforts have been made in academia and industry to quantify different types of biomarkers. Traditional methods for nucleic acid quantification include analysis of gel bands of some products, which can come from polymerase chain reaction (PCR), real-time PCR and some recent technologies such as next-generation sequencing (NGS) and other sequencing methods. Enzyme-linked immunosorbent assay (ELISA) methods have been widely used for protein quantification, and more recently protein mass spectrometry can also be used. For exosome quantification, methods such as nanoparticle tracking analysis (NTA) and ExoELISATM are widely adopted.

[0007] These methods involved above are widely used in laboratories or clinics. However, these methods cannot provide enough sensitivity for effective and sensitive diagnosis or cannot be absolutely quantified, and therefore cannot be used for diagnostic tests that require high-precision quantitative biomarkers to obtain effective results. In the present invention period, most of the traditional diagnostic methods that require quantitative markers use total volume determination to quantify markers, and can only give qualitative or semi-quantitative results, because the molecular information obtained is the average information of thousands of total volume determinations. Therefore, detection may not be very accurate. On the other hand, new methods based on NGS or mass spectrometry (MS) can achieve high throughput and accurate measurement of markers, but it is relatively time-consuming and expensive to implement. When these methods generate a large amount of unnecessary data to make later data analysis more complicated, this is a particularly realistic problem.

[0008] Another important issue is that most current diagnostic platforms cannot simultaneously detect and / or quantify multiple biomarkers of different types, such as DNA and protein markers. However, some diseases may require the co-detection of two or more markers for accurate diagnosis. For example, clinical studies have shown that the maximum sensitivity of plasma DNA testing ("liquid biopsy") is limited to localized cancers. The combined detection of genetic alterations and protein biomarkers can not only help identify the presence of relatively early cancers, but also help locate the organ of origin of these cancers. The accuracy of detection or diagnosis can be substantially improved because the test needs to co-detect multiple biomarkers and their hierarchical specific correlations in order to clinically determine the presence of a condition or disease.

[0009] In recent years, digital technologies such as digital PCR and digital ELISA technologies have emerged that use microfluidics to separate samples into thousands of separate aliquots of droplets. Thousands of PCR or ELISA reactions occur in independent spaces without interfering with each other, so that detection is reduced to the single molecule level and biomolecules can be absolutely and accurately quantified. This enables molecular diagnosis to have unprecedented accuracy while retaining the high specificity of PCR and ELISA tests. However, current digital platforms are limited to endpoint detection (i.e., detection after the reaction is completed) and a single type of reaction and detection (for example, digital PCR reactions and digital ELISA reactions cannot be integrated into one platform, such as performing PCR reactions and ELISA reactions in different droplets). Therefore, these current digital platforms cannot detect multiple biomolecules in real time.

[0010] This requires improvements to existing equipment and detection technologies, hoping to provide more accurate and real-time detection results for process monitoring, and also hoping to detect multiple different types of multiple biomarkers at the same time. Summary of the invention

[0011] The present invention introduces for the first time a concept of using an integrated droplet digitalization platform of a microfluidic device to reduce the detection of multiple biomolecules to the single molecule level, and to perform multi-target co-detection, or / and to detect multiple biomolecules in a digital quantitative manner, thereby allowing effective and accurate testing or diagnosis.

[0012] In one aspect, the present invention provides methods and devices for detecting and quantifying a variety of biomolecules at the single molecule level using an integrated droplet microfluidics system.

[0013] In one embodiment, the present invention provides real-time and / or digital measurement of multiple biomolecules in a sample, thereby achieving absolute and simultaneous quantification of multiple biomolecules.

[0014] In some embodiments, an integrated droplet microfluidic generation and detection system for testing. The system includes four parts (from left to right): an inlet, a droplet generator, a droplet storage chamber, and an outlet. The inlet is used to introduce various liquids (such as oil, samples and reagents for reaction) into the droplet generator, wherein these liquids can be loaded separately through different inlets, or pre-mixed and loaded as a mixture through the same inlet. The sample containing biomolecules is prepared and divided into separate droplets by the droplet generator, and then the generated droplets are spread into a monolayer device in the droplet storage chamber. After the sample is divided, parallel in-situ reactions occur in each droplet, wherein the reaction is part of a reaction assay for analyzing the target biomolecule, and the reactions in different droplets can be the same or different. Once the reaction is completed, the signal indicating the presence of the target biomolecule can be detected by, for example, capturing an image of the droplets in the droplet storage chamber with a microscope camera. The image is then processed by a computer for counting and data analysis. The outlet is used to remove liquid or gas from the system. In one embodiment, the outlet is used to allow air or oil to be discharged. In another embodiment when the droplets need to be collected after the reaction, they can be collected from the droplet reservoir through the outlet.

[0015] In one embodiment, the method and apparatus include an integrated droplet microfluidics system capable of generating thousands of droplets, thereby partitioning a sample into thousands of individual droplets for subsequent reaction and analysis of each droplet.

[0016] In one embodiment, the present invention provides an integrated droplet microfluidics system for generating a large number of individual droplets from a sample.

[0017] In one embodiment, the integrated droplet microfluidics system is configured as a single microfluidics chip.

[0018] In one embodiment, the microfluidic system is composed of a droplet generator and a droplet reservoir. In one embodiment, a sample containing biomolecules is divided into individual droplets and passed through the droplet generator, and then the generated droplets are placed in the droplet reservoir in a single-layer configuration.

[0019] In one embodiment, the integrated droplet microfluidic system of the present invention includes multiple microfluidic channels for transferring fluids to or from various components of the system. In one embodiment, the droplet generator, droplet reservoir and / or outlet include one or more microfluidic channels that provide flow paths for fluids within these components. In one embodiment, one or more microfluidic channels are provided between different components of the integrated droplet microfluidic system (such as between the droplet generator and the droplet reservoir) for directing fluids from one component to another. In one embodiment, the precise type or configuration of microfluidic channels to be used (such as structure, length, spacing, number of branches and density) depends on the purpose of having the microfluidic channels and the desired flow resistance of the individual components.

[0020] In one embodiment, the present invention performs real-time and digital measurements of multiple droplets generated from a sample by quantifying and individually measuring the specific signal of each droplet.

[0021] In one embodiment, the integrated droplet microfluidic system of the present invention is combined with a motion temperature control system and a detection unit. By using the integrated droplet microfluidic system of the present invention, the present invention can guide thousands of droplets to perform multiple reactions, and can digitally detect different biomolecules in each of these droplets, thereby obtaining the absolute amount of target molecules in the sample for diagnosis or other analytical purposes. Since the measurement is real-time and single molecule, the present invention provides more useful and accurate information than existing endpoint digital measurement methods.

[0022] In one embodiment, the present invention provides a method for digitally quantifying and analyzing biological assays, comprising four steps: 1) sample preparation, 2) sample compartment, 3) reaction, and 4) Figure 1 Digital detection shown.

[0023] In one embodiment, the present invention provides a method for diagnosing a disease, comprising real-time and digital measurement of a plurality of biomolecules in a sample using the method or device described herein.

[0024] The present invention provides a droplet microfluidic system, comprising: a droplet generator and a storage device for storing a plurality of droplets, wherein each micropore of each storage device stores a droplet, wherein the droplet generator and the droplet storage device are an integrated structure, wherein the droplet generator and the droplet storage device are fluidically connected, and each micropore is fluidically connected.

[0025] In one example, the droplet generator includes an inlet, and the droplet reservoir includes an outlet.

[0026] In one embodiment, the droplet generator comprises a plurality of inlets, each inlet is used to receive a different reagent component, and one inlet is used to receive a sample.

[0027] In one embodiment, the droplet generator and the droplet reservoir are in fluid communication via a microfluidic channel.

[0028] In one embodiment, the microfluidic channel is made of one or more materials selected from silicon, glass, plastic and polydimethylsiloxane (PDMS).

[0029] In one embodiment, the diameter of the microfluidic channel is 1-2 times the diameter of the droplet.

[0030] In one embodiment, the droplet generator is a surface tension-based droplet generator.

[0031] In one embodiment, the droplet generator includes a cross-flow structure that allows the continuous phase and the dispersed phase to intersect at a specific angle θ.

[0032] In one embodiment, the droplet generator comprises a stepped emulsification structure.

[0033] In one embodiment, when the sample is a cell or an exosome, the one inlet is used to receive the cell or exosome sample, and the other one or more inlets are used to receive a lysis reagent or an oily substance.

[0034] In one embodiment, the droplet generator includes a droplet sorting and separation unit.

[0035] In one embodiment, the height of the micropores of the droplet storage is 1-1.5 times the diameter of the droplet.

[0036] In one embodiment, the droplet reservoir includes a plurality of rows of anchoring structures for anchoring droplets to predetermined positions in the droplet reservoir.

[0037] In one embodiment, the anchoring structures take the form of struts such as columns arranged in a manner to capture individual droplets.

[0038] In one embodiment, the anchors are in the form of grooves that capture individual droplets via surface tension.

[0039] In one embodiment, the droplet contains no more than one copy or one target component to be analyzed in a subsequent step.

[0040] In one embodiment, the target molecules are nucleic acids and proteins.

[0041] In one embodiment, the droplet contains a single cell or a single exosome. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 An embodiment of the present invention is shown for implementing a digital quantitative bioassay comprising four steps: 1) sample preparation, 2) sample discretization (or compartmentalization of the sample), 3) reaction, and 4) digital detection.

[0043] Figure 2 An embodiment of the present invention is shown comprising an integrated droplet microfluidics system for digitally quantified biological assays.

[0044] Figure 3 An example of droplet generation in a specific embodiment of the present invention is shown.

[0045] Figure 4 An example of droplet generation in another embodiment of the present invention is shown.

[0046] Figure 5 An embodiment of an anchoring structure within a droplet reservoir is shown that captures a single droplet at a predetermined location within the droplet reservoir.

[0047] Figure 6 Another embodiment of an anchoring structure within a droplet reservoir is shown.

[0048] Figure 7 An embodiment of a droplet generation device having a flow focusing structure connected downstream to a droplet reservoir is shown.

[0049] Figure 8 A fluorescence image of the droplet obtained by a CCD camera is shown.

[0050] Fig. 9 An embodiment of an integrated droplet microfluidics system of the present invention is shown for digital quantification of RNA from single exosomes.

[0051] Fig.10 The process of digitally quantifying RNA from individual exosomes is shown.

[0052] Fig.11 Two images of fluorescent signals are shown, obtained from droplets containing amplified DNA molecules in an endpoint digital detection of target DNA molecules using the present invention. Detailed description

[0054] Unless otherwise specified in the present invention, the terms of the present invention are interpreted according to the ordinary meaning.

[0055] Detection

[0056] Detection means to test or examine the presence of a substance or material. The substance or material may be, for example but not limited to, a chemical substance, an organic compound, an inorganic compound, a metabolite, a drug or a drug metabolite, an organic tissue or a metabolite of an organic tissue, a nucleic acid, a protein or a polymer. In addition, detection may also mean the amount of the substance or material tested, and assay may also mean immunoassay, chemical assay, enzyme assay, nucleic acid assay, etc.

[0057] The present invention provides methods and devices for detecting and quantifying multiple biomolecules at the single molecule level using an integrated droplet microfluidic system. In one embodiment, the method and device include an integrated droplet microfluidic system capable of generating thousands of droplets, thereby dividing a sample into thousands of individual droplets for subsequent reactions and analysis of each droplet.

[0058] In one embodiment, the present invention performs real-time and digital measurements of multiple droplets generated from a sample by quantifying and individually measuring the specific signal of each droplet.

[0059] In one embodiment, the present invention provides a method for diagnosing a disease, comprising real-time and digital measurement of a plurality of biomolecules in a sample using the methods and devices described herein.

[0060] As shown herein, the present invention introduces for the first time a concept of co-detecting multiple biomolecules at the single-molecule level using a digital platform in a microfluidic setting and quantifying these multiple biomolecules in real time, thereby allowing for efficient and accurate diagnosis.

[0061] The integrated droplet microfluidic system of the present invention is combined with a motion temperature control system and a detection unit. By using the integrated droplet microfluidic system of the present invention, the present invention can guide thousands of droplets to perform multiple reactions, and can digitally detect different biomolecules in each of these droplets, thereby obtaining the absolute amount of target molecules in the sample for diagnosis or other analytical purposes. Since the measurement is real-time and single molecule, the present invention provides more useful and accurate information than existing endpoint digital measurement methods.

[0062] In one embodiment, the present invention provides a method and apparatus for digitally quantifying and analyzing biological assays, comprising four steps: 1) sample preparation, 2) sample compartment, 3) reaction, and 4) Figure 1 Digital detection shown.

[0063] Figure 2An embodiment of an integrated droplet microfluidics system for digital quantitative bioassay is shown. The system includes four parts (from left to right): an inlet, a droplet generator, a droplet reservoir, and an outlet. The inlet is used to introduce various liquids (such as oil, samples for reactions, and reagents) into the droplet generator, wherein these liquids can be loaded separately through different inlets (such as Figure 2 As shown), or premixed and loaded as a mixture through the same inlet. A sample containing biomolecules is prepared and divided into individual droplets by a droplet generator, and the generated droplets are then spread into a monolayer device in a droplet reservoir. After the sample is divided, parallel in situ reactions occur in each droplet, where the reaction is part of a reaction assay for analyzing the target biomolecule, and the reactions in different droplets can be the same or different. Once the reaction is complete, a signal indicating the presence of the target biomolecule can be detected by, for example, capturing an image of the droplet with a microscope camera in the droplet reservoir. The image is then processed by a computer for digital counting and data analysis. The outlet is used to remove liquid or gas from the system. In one embodiment, the outlet is used to allow air or oil to drain. In another embodiment, when the droplets need to be collected after the reaction, they can be collected from the droplet reservoir through the outlet.

[0064] Sample preparation

[0065] The present invention can be used for various types of samples containing target biomolecules. In one embodiment, the sample is a liquid sample obtained directly from a living organism, including but not limited to humans, animals, plants, fungi, microorganisms such as bacteria and viruses. In one embodiment, the sample is a solution containing target biomolecules, wherein the solution can be obtained by another separate process of treating target biomolecules. For example, this independent process is a process of separating, purifying or concentrating target biomolecules in a sample containing target biomolecules, or a pretreatment process, that is, pretreating a sample containing target biomolecules at a certain temperature or pressure by certain enzymes or reagents. The sample here can also include biological liquids. The sample or the initial state of the sample can be liquid, solid or semi-solid, and the solid or semi-solid sample can be converted into a liquid sample by any appropriate method, such as mixing, mashing, macerating, incubating, dissolving and enzymolysis, etc., and then the sample is processed into droplets. During the dropletization process, before the process, the sample can be processed in some ways, including filtering, dissolving, etc., and other reagents can also be mixed to improve some properties of the sample, so as to facilitate the formation of micro-droplets in the later stage or provide better results in the detection. Samples can be taken from the human body, animals, plants, and nature. Samples taken from the human body can be, for example, liquid samples such as blood, serum, urine, cerebrospinal fluid, sweat, lymph, saliva, and gastric juice; solid or semi-solid samples such as feces, hair, keratin, tartar, and nails. Samples taken from plants can be, for example, solid samples such as roots, stems, and leaves; liquid or semi-solid samples such as tissue fluid and cell fluid prepared from roots, stems, and leaves. Samples taken from nature can be, for example, liquid samples such as rainwater, river water, seawater, and groundwater; solid or semi-solid samples such as soil, rocks, ores, and petroleum.

[0066] In one embodiment, samples include, but are not limited to, blood, plasma, serum, tissue, urine, saliva, feces, materials that can be used for smears, and excreta such as tears, sputum, nasal mucus, vaginal secretions, and penile excreta. In one embodiment, samples include, but are not limited to, bacteria, viruses, bacterial cultures, viral cultures, cell cultures, cell suspensions, and adherent cells. In another embodiment, samples include, but are not limited to, food, feed, and plants.

[0067] In one embodiment, the sample can be obtained directly from a subject. In one embodiment, the sample is one or more biological molecules obtained from a subject, including immediate or final products that can be obtained from various molecular tests such as polymerase chain reaction (PCR).

[0068] In one embodiment, the present invention includes the step of providing or preparing a sample, which contains a target biomolecule to be detected or quantified. These target molecules or analytes are of different types, and the types here refer to the presence of different types of analytes in the same sample, such as different biomolecules or different biomarkers, such as nucleic acids, proteins, polysaccharides and small molecule peptides in a cell, and the types of these different markers or analytes are different. The present invention can simultaneously test multiple different types of analytes on a carrier, such as a chip, such as simultaneous detection of proteins and nucleic acids, such as simultaneous detection of nucleic acids and proteins associated with a certain disease, such as nucleic acid mutations and protein molecule levels of a certain cancer or tumor. The carrier here can refer to the microfluidic chip of the present invention, or it can be a device that combines a droplet generator and a droplet storage in an integrated structure, such as a single small hole in a droplet storage, each droplet performs a different test or a different reaction in a droplet, such as a nucleic acid binding reaction, a nucleic acid amplification reaction or a protein reaction, and the reaction here is a binding reaction, an amplification reaction or any other reaction. Here, the so-called different types can also refer to the same classification, but the specific subdivisions also have different meanings. For example, if they are all nucleic acid detection, the DNA level and RNA level can be detected at the same time. Here, the DNA level and RNA level can be considered as different types of detection. For another example, if they are all protein levels, but the biological surface markers of different proteins can also be considered as different types of detection, such as a variety of protein molecules associated with a certain disease, these protein molecules have different polypeptide sequences, different protein three-dimensional spatial structures, and one or more of different sites. Although they are all called protein levels, the protein markers are different, and the detection device or detection method of the present invention can also be used to perform simultaneous detection of different types of protein molecules, and provide the content of multiple substances for a specific disease at the same time, and these multiple substances are all from the same sample, which reduces multiple sampling or reduces the amount of sample used, and can issue multiple test results at the same time, such as disease diagnosis.

[0069] In one embodiment, the size of the sample is 1 microliter to 500 microliters. A sample volume that is too large may take longer to generate droplets. A sample volume that is too small may make it difficult to load the sample into the droplet generator.

[0070] In one embodiment, the viscosity of the sample is no greater than 0.5 Ns / m 2 In one embodiment, the sample does not contain solvent components, such as dimethyl sulfoxide (DMSO), which may cause droplet merging.

[0071] Integrated droplet microfluidics system

[0072] In some specific ways, the present invention provides an integrated detection system or detection device, which includes a droplet generating device and a droplet storage device. The so-called integration refers to two components connected as an integral structure to form an integral structure, which can simultaneously complete different functions, but the completion of these functions may have a certain order, so that they are integrated together, reducing the cumbersome operation steps of non-integrated operations. For example, in traditional technology, when using a microfluidic system for detection, liquid generation requires a separate droplet generation system to complete, and then after the droplets are generated, another device is used to store these droplets. The stored droplets are generally multiple droplets mixed together for storage, and then when a reaction is required, the storage container is directly used for reaction or transferred to a separate reaction device for reaction, such as incubation, cyclic heating amplification, etc. After the reaction is completed, when the detection is performed, it is also necessary to separate the multiple droplets separately, and then read the test results one by one, such as using a flow cytometer for detection. This not only has cumbersome operation steps and many steps, but also brings a lot of uncertainty to the final test results. For example, there are many operating steps, and different personnel may have batch differences in operation, which increases the risk factors that affect the final test results; for example, frequent transfer of droplets causes droplet loss and fusion; for example, mixing multiple droplets together for reaction may cause problems such as uneven heat transfer, and special attention should be paid to the control of incubation temperature; for example, multi-step transfer may also cause cross-contamination, false positives, and affect the accuracy of the experiment, which is particularly important in clinical testing. The present invention combines the functional components for forming droplets with reactions and detections. It only needs to prepare samples, and then all steps can be completed in one step, which greatly simplifies the operating steps and reduces the adverse effects of different steps on the final detection. In addition, the droplet storage device does not mix multiple droplets together, but individual droplets exist independently. For example, a hole only includes one liquid, and droplets exist independently, which ensures the independence of the reactions between droplets. At the same time, during reaction and detection, they will not interfere with each other.

[0073] Therefore, in a specific embodiment, the present invention provides a detection system, which includes a liquid generation system and a storage system, and the storage system is used to store droplets generated by droplets. In some embodiments, the droplet generation system is capable of generating multiple individual tiny droplets, and the storage system includes multiple storage units, such as storage holes and cavities, and each individual hole or cavity stores a droplet. In some embodiments, the droplet generation system and the storage system are liquid-connected through a microfluidic channel. The storage system here includes an inlet and an outlet for the liquid, and includes multiple storage holes, cavities, holes, etc. between the inlet and the outlet. It can be understood that the storage system here can be used directly to carry out a certain reaction, for example Figure 2 The systems in the Figure 2 On the right side of the image, the storage system includes multiple microporous structures, each of which stores a droplet. When these liquids include substances necessary for the reaction, such as when performing nucleic acid amplification, there are primers, enzymes necessary for amplification, etc., which can be directly allowed to the storage system for reaction. At this time, the storage system is a reaction container, which includes multiple separate holes, each of which includes a separate droplet. There is a separate and independent reaction in each droplet, and each droplet will have an independent final reaction result. After the reaction is completed, the storage system can be directly tested for the reaction results, and the results of multiple independent reactions (multiple independent droplets) can be tested simultaneously at one time. Compared with traditional multi-step tests, the operation is simple, and multiple different or identical test results can be obtained.

[0074] Therefore, the generation of individual droplets can also be used for simultaneous detection of different types of analytes, such as the detection of nucleic acids and proteins, which will be described in detail later.

[0075] In a specific embodiment, for example Figure 2 As shown, the integrated droplet microfluidic system of the present invention for digital quantification of biological assays includes an inlet, a droplet generator, a droplet reservoir and an outlet.

[0076] In one embodiment, the present invention provides an integrated droplet microfluidics system for generating a large number of individual droplets from a sample.

[0077] In one embodiment, the integrated droplet microfluidics system is configured as a single microfluidics chip.

[0078] In one embodiment, the microfluidic system is composed of a droplet generator and a droplet reservoir. In one embodiment, a sample containing biomolecules is divided into individual droplets and passed through the droplet generator, and then the generated droplets are placed in the droplet reservoir in a single-layer configuration.

[0079] Here, the microfluidic chip can also be a single-layer configuration of the droplet reservoir.

[0080] The inlet is used to introduce various liquids (such as oil, samples and reagents for carrying out reactions) into the droplet generator. Figure 2As shown, oil, sample and reagents 1 to N (i.e. various reagents required for subsequent reactions, which may be the same or different) are introduced into the droplet generator through different inlets. In one embodiment, when the sample and reagents do not produce a chemical reaction, they can be pre-mixed and loaded into the droplet generator through one inlet as a mixture. In another embodiment, when the sample and one or more reagents react, these reagents and samples cannot be introduced into the droplets as a mixture, but can be loaded into the droplet generator through different inlets and then divided into or formed into droplets at the connection of the droplet generator.

[0081] For example, when performing digital PCR for hot-start amplification of quantified DNA in droplets, the sample containing the target nucleotide and the reagents for hot-start amplification (including primers, polymerase, and other buffers) can be pre-mixed and loaded into the same inlet where the droplets are generated, because the reaction will only start when the temperature rises to the working temperature of the enzyme. However, if the enzyme in the reagent can catalyze any substrate of the sample at room temperature, the reagent containing the enzyme and the sample need to be loaded into the droplet generator through different inlets and then mixed in each droplet at the connection of the droplet generator.

[0082] Figure 3 and Figure 4 Two embodiments of droplet generators using the present invention are shown. Figure 3 In the process, the sample and reagents to be subjected to the subsequent reaction are premixed, and the resulting mixture is placed in a droplet generator for producing droplets. Figure 4 In the experiment, the lysis buffer and cells or exosomes are pre-mixed, which will cause the cells or exosomes to lyse prematurely, and the expression at the single cell level cannot be observed. Therefore, the original sample containing cells or exosomes and the lysis buffer are loaded into the droplet generator through different inlets, so that they will not contact before being encapsulated into the droplets. As for the reaction generated after encapsulation, it is also generated in independent droplets, and the droplets will not affect each other.

[0083] The droplet generator of the present invention can be any structure or system that can divide or form a liquid sample into a large number of droplets. In one embodiment, the droplet generator includes but is not limited to flow focusing, T-junction and emulsification reagents, etc.

[0084] The droplet reservoir of the present invention can be any module that can hold droplets generated by a droplet generator. In one embodiment, the design of the droplet reservoir of the present invention depends on the total number and volume of droplets required for a particular assay.

[0085] In one embodiment, the generated droplets are expanded in a droplet reservoir in a monolayer configuration. After the sample is partitioned, parallel reactions are carried out in separate droplets in the droplet reservoir, followed by detection and analysis of target biomolecules.

[0086] In one embodiment, the outlet is used to remove liquid or gas from the integrated system of the present invention. In one embodiment, the outlet is used to exhaust air or oil. In another embodiment, when droplets need to be collected after the reaction, they can be collected from the droplet storage chamber through the outlet, and the collected droplets are used for other reactions or other purposes.

[0087] In one embodiment, the integrated droplet microfluidic system of the present invention further comprises a unit for sorting droplets and separating. For example, a sorting junction comprising electrodes in an alternating current (AC) electric field is included in the present integrated droplet microfluidic system to select specific droplets, for example, containing a specific type of target molecule or observing a fluorescent signal from a droplet having a specific property as described herein (Baret, Jean-Christophe, 2009).

[0088] In one embodiment, the integrated droplet microfluidic system of the present invention includes multiple microfluidic channels for transferring fluids to or from various components of the system. In one embodiment, the droplet generator, droplet reservoir and / or outlet include one or more microfluidic channels that provide flow paths for fluids within these components. In one embodiment, one or more microfluidic channels are provided between different components of the integrated droplet microfluidic system (such as between the droplet generator and the droplet reservoir) for directing fluids from one component to another. In one embodiment, the precise type or configuration of microfluidic channels to be used (such as structure, length, spacing, number of branches and density) depends on the purpose of having the microfluidic channels and the desired flow resistance of the individual components.

[0089] In one embodiment, the microfluidic channel is made of a material selected from silicon, glass, plastic and polydimethylsiloxane (PDMS).

[0090] In one embodiment, the same type or configuration of microfluidic channels can be used in various components of the integrated droplet microfluidic system of the present invention. In another embodiment, various types or configurations of microfluidic channels can be used in various components of the integrated droplet microfluidic system of the present invention.

[0091] In one embodiment, the droplet generator of the present invention includes two microfluidic channels for transporting oil and one or more microfluidic channels for transporting sample fluid and / or reagents. In one embodiment, the actual configuration depends on the type of emulsion selected and the number of inlets required.

[0092] In one embodiment, a microfluidic channel is used to connect the droplet generator and the droplet reservoir. In one embodiment, the diameter of the microfluidic channel is 1-2 times the diameter of the droplet. The larger diameter microfluidic channel helps stabilize the droplets as they pass through the channel, and compressing the fluid flow in the channel will also help stabilize the droplets.

[0093] In one embodiment, the droplet reservoir does not have any microfluidic channels, and the generated droplets will self-assemble to spread on the flat surface within the chamber. In the case where the droplet reservoir has a well, the droplets will spread within the chamber and then be guided into the well by surface tension.

[0094] In one embodiment, the outlet of the present invention comprises a microfluidic channel having a diameter of several hundred microns.

[0095] In one embodiment, the microfluidic channel is rectangular (eg, has a rectangular cross-section). In another embodiment, the microfluidic channel has a circular cross-section.

[0096] Discretization - droplet generation and storage

[0097] In one embodiment, the present invention includes a droplet generator of any structure or system that can divide a liquid sample into a large number of droplets (such as thousands or millions of droplets). In one embodiment, the droplet generator includes but is not limited to structures of flow focusing, cross-flow, co-flow, step emulsification reagents and microchannel emulsification. P. Zhu and L. Wang (2017) describe some techniques for generating droplets, the contents of which are fully cited by reference.

[0098] In one embodiment, the droplet generator of the present invention is a droplet generating device based on the shear force principle, which uses shear pressure to squeeze the fluid into small droplets. In one embodiment, the shear droplets based on the droplet generating device include but are not limited to devices including cross-flow structures, co-flow structures and flow focusing structures.

[0099] In one embodiment, the droplet generator of the present invention is a surface tension-based droplet generation device, wherein surface tension is the main driving force in the droplet breakup process. In one embodiment, the surface tension-based droplet generation device includes but is not limited to those comprising a T-structure combined with a step emulsifier and a microchannel emulsification structure.

[0100] In one embodiment, the droplet generator of the present invention comprises the droplet generating structure described in WO2016189383A1, the content of which is fully incorporated into the content of the present invention by reference.

[0101] In one embodiment, methods capable of generating droplets can be used in the present invention to generate droplets, including but not limited to high shear stirring, ultrasonic emulsification, high pressure homogenization and membrane emulsification.

[0102] In one embodiment, the droplet generator of the present invention includes a fluid focusing structure that compresses the fluid to enhance the focusing effect. In one embodiment, the flow focusing structure is a 2D planar flow focusing structure. Figure 4 An embodiment of a droplet generator is shown, which includes a flow focusing structure and a droplet reservoir coupled to the droplet generator downstream thereof. Figure 4 In the process, the sample in the central channel is sheared by the fluid from the side channel and split into small droplets, which are then sucked into the droplet reservoir due to capillary forces.

[0103] In one embodiment, the droplet generator of the present invention comprises a cross-flow structure that allows the continuous phase and the dispersed phase to intersect at a specific angle θ. In one embodiment, the droplet generator of the present invention comprises a T-type, Y-type, double T-type, K-type or V-type junction structure.

[0104] In one embodiment, the droplet generator of the present invention comprises a co-flow structure in which the dispersed fluid lines are sheared by the surrounding flowing continuous phase. In one embodiment, the co-flow structure is a 2D planar co-flow structure.

[0105] In one embodiment, the droplet generator of the present invention comprises a stepped emulsification structure. In one embodiment, the droplet generator of the present invention comprises a stepped emulsification structure combined with a horizontal or vertical T-shaped structure.

[0106] In one embodiment, the droplet generator of the present invention comprises a microchannel emulsification structure.

[0107] In one embodiment, the droplet generator of the present invention includes the droplet generating structure described in WO2016189383A1, the content of which is fully cited as the content of the present invention.

[0108] In one embodiment, the components or parts of the droplet generator responsible for droplet generation (such as sample discretization) have a hydrophobic surface. This can be achieved by coating the surface of the component or part with a chemical surface that couples with hydrophobic groups. In one embodiment, a surfactant such as Span 80, Tween 20 or Abil EM 90 is added to the oil phase or the water phase to avoid droplet coalescence or prevent molecules such as enzymes, DNA or RNA from adhering to solid surfaces or water-oil interfaces.

[0109] In one embodiment, the droplets generated are emulsified droplets, and are not limited to a particular type of emulsion. In one embodiment, emulsions include, but are not limited to, oil-in-water, water-in-oil, and water-oil-water double emulsions.

[0110] In one embodiment, oil and surfactant are used to generate droplets. In one embodiment, the ratio of surfactant to oil is 1-5% (weight percentage). In one embodiment, oil is used to generate droplets including but not limited to mineral oil, silicone oil, fluorinated oil, hexadecane and vegetable oil. In one embodiment, the surfactant used is but not limited to Triton 80, Tween 20 / 80, ABIL EM 90 and phospholipids. Baret and Jean-Christophe (2012) describe that surfactants can be used in droplet microfluidics, which content is fully incorporated into the present invention by reference.

[0111] The droplet reservoir of the present invention can be any module that can hold or retain droplets, including but not limited to droplets generated by a droplet generator. In one embodiment, the design of the droplet reservoir of the present invention depends on the total number and volume of droplets required for the reaction or measurement to be performed in the subsequent step.

[0112] In one embodiment, the size of the droplet reservoir of the present invention is larger than the total volume of the droplets generated. In one embodiment, the height of the droplet reservoir of the present invention is 1-1.5 times the diameter of the droplets generated. Figure 3 As shown, the height of the droplet reservoir is 1-1.5 times the diameter of the droplet; the droplet is stretched in the droplet reservoir in a monolayer configuration.

[0113] In one embodiment, the droplet storage chamber of the present invention is connected to the droplet generator of the present invention, so that the droplets generated are sucked into the droplet storage chamber by capillary action. In one embodiment, the droplets are accumulated and dispersed in the droplet storage chamber in a special way. In one embodiment, the droplets are loosely or randomly accumulated and dispersed in the droplet storage chamber.

[0114] In one embodiment, the droplets are dispersed in a specific or predetermined manner, and the droplet reservoir includes multiple rows of anchoring structures for anchoring the droplets to predetermined positions in the droplet reservoir. In one embodiment, the anchoring structures are in the form of pillars such as columns arranged in a manner to capture a single droplet ( Figure 5 As droplets pass through the droplet reservoir, they will be trapped in the spaces between the pillars. In one embodiment, the anchors are combined with grooves to capture individual droplets by surface tension ( Figure 6 In one embodiment, the droplet reservoir of the present invention comprises an anchoring structure or equivalents described in the art, such as Abbyad (2010) and Huebner (2008), which are incorporated herein by reference in their entirety.

[0115] In one embodiment, the droplets are randomly stacked without anchoring structures within the droplet reservoir.

[0116] Characteristics of droplets

[0117] In one embodiment, the number, size (eg, diameter), volume, and type of emulsion of droplets produced or used in the present invention depends on the processing and analysis required following use of the present invention.

[0118] In one embodiment, the number of droplets produced ranges from a few hundred to several million.

[0119] In one embodiment, the droplets produced range in size from about 5 microns to about 200 microns.

[0120] In one embodiment, the volume of the droplets produced ranges from about 0.65 fL (femtoliters) to about 4 nL (nanoliters).

[0121] In one embodiment, the droplets generated are of uniform diameter. In one embodiment, the droplets generated have a uniform diameter with a coefficient of variation of less than 5%. In another embodiment, droplets of different diameters are generated by adjusting the loading pressure.

[0122] In general, the size and volume of droplets processed and analyzed in a particular assay are fairly consistent at the same time, thereby minimizing the effects on the results due to droplet size and volume differences.

[0123] In one embodiment, each droplet generated by the present invention contains no more than one copy or one target molecule (such as a cell, exosome, or a type of biomolecule) to be analyzed in a subsequent step. In one embodiment, the number of droplets to be generated and the volume of sample introduced for generating the droplets are adjusted in a manner such that each droplet generated can contain no more than one target molecule. The digital method of distributing target molecules to a large number of droplets theoretically follows the Poisson distribution principle (Majumdar, 2015). Therefore, the target molecule can be quantified by counting the number of droplets containing one or more copies of the target molecule. In order to achieve absolute quantification, each droplet should contain no more than one copy of the target molecule. In general, if the ratio of the number of droplets to the number of target molecules is no greater than 10, according to the Poisson distribution principle, more than 99% of the droplets will include no more than one copy of the target molecule, while if the ratio is 3, the percentage will be 96%. For example, when using the present invention to digitally quantify exosomes, 10 times more antibody-conjugated beads than the expected number of exosomes are used to ensure that each bead captures no more than one target exosome for absolute quantification. Alternatively, if each droplet is not guaranteed to have a single copy of the target molecule (i.e., some droplets may contain more than one copy of the target molecule), Poisson statistics is used to calculate the absolute number of target molecules (Majumdar, 2015).

[0124] The "copy" mentioned in the present invention does not only refer to the copy of nucleic acid in the usual sense, but refers to the number of target molecules. For example, a protein molecule in a sample can become a copy, a nucleic acid molecule can become a copy of nucleic acid, and a polypeptide chain molecule can be called a copy of polypeptide. Therefore, the unit quantifier of copy here can be a unit measured by number. Alternatively, a cell can also be called a copy of a cell, which actually means one cell. When there are two cells, it can be called 2 copies of a cell. Although each cell is different, it only refers to two cells, which only means quantity. Therefore, when using copies to measure a specific marker substance or analyzed substance (nucleic acid, DNA or RNA), it only indicates the number of marker substances, and does not indicate whether these marker substances or target molecules are the same type of substances. For example, 1 copy of DNA or RNA only indicates the number of DNAs present, and does not necessarily mean that when talking about 2 copies of DNA, it does not mean that the two DNAs belong to the same DNA. DNA DNA can be the same or different. For example, one sample contains the DNA of virus A and the other contains the DNA of virus B. In this case, we can say that there are two copies of DNA. For example, a droplet includes one copy of the DNA of virus A, or two copies of the virus DNA. In this case, the two virus DNAs can be two DNAs of virus A, or two DNAs of virus B, or one DNA of virus A and one DNA of virus B.

[0125] In one embodiment, the size and number of droplets generated by the droplet generator of the present invention are sufficient to accurately quantify the target molecules in the sample to achieve a high dynamic range. Generally, for digital analysis techniques, which are used to distinguish target molecules to be detected, the dynamic range of detection (such as the range of the number of target molecules accurately detected by digital analysis techniques) is determined by two main parameters: the size and total number of droplets, which are limited by the distinguishing ability of the droplet generating device. For example, it is reported that the dynamic range of a typical digital PCR is 0-10 6 , which means that if the target nucleic acid molecule exceeds 10 6 Due to the limitation of copies / μL, typical dPCR cannot determine the absolute number of target nucleic acid molecules in the sample. Statistically speaking, droplets that exceed the target molecules by 3-10 times have higher accuracy in detection, but a smaller dynamic range. On the other hand, a larger dynamic range can be obtained using Poisson distribution (Majumdar, 2015).

[0126] Multiple reactions in multiple droplets

[0127] As shown in the figure, the present invention provides a platform for carrying out multiple reactions in all droplets and detecting and quantifying target molecules in each droplet in real time. Unlike endpoint measurements in existing droplet-based technologies, the present invention can provide more efficient and accurate diagnosis.

[0128] In one embodiment, the present invention includes the step of performing multiple reactions in droplets generated by an integrated droplet microfluidics system.

[0129] In one embodiment, the present invention allows for the simultaneous performance of assays of the same or different types in different droplets in a parallel manner on a large scale.

[0130] In one embodiment, the same platform can be used for droplet storage, subsequent reactions or determinations, and detection of target molecules. For example, when the droplets are dispersed into the droplet storage chamber of the present invention, the reaction can be carried out. In another embodiment, the platform used for subsequent reactions or determinations is different from the platform used for detecting target molecules. For example, when DNA needs to be detected, the present invention uses a droplet generator to generate droplets, PCR tubes to store droplets and then place them in a PCR thermal cycler for reaction, and finally a droplet reader detects the target molecules and generates a readable fluorescent signal.

[0131] Of course, the storage droplets and the reaction can be performed in the same storage device. For example, if the storage is in the form of a microporous chip, the same storage chip is used for the amplification reaction during the reaction, and then the result of the chip reaction is detected.

[0132] In one embodiment, after dividing the sample into many individual droplets and dispersing them into the droplet reservoir, the method and apparatus of the present invention simultaneously conducts a reaction in each droplet in the droplet reservoir, and the reaction can be the same or different types. The present invention allows the reaction carried out in one droplet to be independent of any other reactions in other droplets, thus providing high flexibility in performing various biological assays.

[0133] The "reaction" mentioned here refers to the reaction of a single droplet, specifically the substances necessary for the reaction included in the droplet, which can be used to indicate whether one or more target molecules exist. For example, when there are multiple droplets, the reaction of each droplet is different, and the target molecules targeted are also different. For example, one droplet is used for nucleic acid testing, another droplet is used for antibody and antigen reaction, and other droplets have chemical color development reactions. Of course, it can also be considered that in some individual droplets, there is an amplification reaction of DNA of class A specific target DNA fragments, and in other individual droplets, an amplification reaction of class B specific target DNA fragments is carried out. It can also be that different RNA reactions are carried out in different droplets, such as messenger RNA amplification in one droplet and transcription RNA reaction in another droplet. In this way, separate different reactions can be carried out in separate liquids, and the testing of different target molecules can be achieved.

[0134] In one embodiment, the reaction carried out after discretization is a reaction targeting a target molecule, so that the target biomolecule can be detected or quantified in a subsequent step. In one embodiment, the reaction is any compatible bioassay in the art. In one embodiment, the reaction selected to be carried out depends on the properties of the target biomolecule.

[0135] In one embodiment the biomolecule is a nucleic acid, a protein or a small molecule.

[0136] In one embodiment, the biomolecule is a free molecule including but not limited to free DNA (cfDNA), free protein, exosomes and free molecules circulating in a liquid. In one embodiment, the biomolecule is a molecule attached to a cell surface or inside a cell.

[0137] In one embodiment, the biomolecule is a nucleic acid of various types (e.g., DNA including cDNA, RNA including mRNA and rRNA), forms (e.g., single-stranded, double-stranded, helical, as a plasmid vector, non-coding or coding), and lengths (e.g., oligonucleotides, genes, chromosomes, and genomic DNA) derived from a subject or exogenous organism.

[0138] In one embodiment, the biomolecule is a protein, i.e., a peptide or polypeptide protein, including intact protein molecules, degraded protein molecules, and digested fragments of protein molecules. In one embodiment, the biomolecule includes, but is not limited to, antigens, receptors, and antibodies derived from a subject or an exogenous body.

[0139] In one embodiment, the biomolecule is a small molecule such as a metabolite. In one embodiment, the metabolite is a disease-related metabolite that can indicate the extent of the presence of a disease or a healthy state. In one embodiment, the metabolite is a drug-related metabolite, such as a drug byproduct that changes the drug level after a subject takes the drug.

[0140] In one embodiment, the target biomolecule is a molecule produced by a tumor or cancer, or produced by the subject's body in response to a tumor or cancer.

[0141] In one embodiment, the target biomolecule is not commonly found in healthy subjects. In one embodiment, the target biomolecule is commonly found in healthy subjects but its levels are indicative of a particular disease or health state.

[0142] For example, nucleic acid biomolecules may need to be amplified using polymerase chain reaction (PCR) with complementary primers, while protein biomolecules may need to be hybridized with antibodies that recognize a certain protein epitope.

[0143] In one embodiment, nucleic acids can be detected and quantified by reactions including, but not limited to, polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), real-time PCR and real-time RT-PCR, reverse transcription, labeling, digestion, blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoassay, and enzymatic assays. For example, ddPCR TM The EGFR Exon 19 Deletion Screening Kit (Bio-Rad Laboratories, Inc.) is used to screen for deletion mutations in exon 19 of the EGFR gene. 15 This kit can also detect other deletions in this region of EGFR exon 19. EGFR exon 19 deletions are commonly associated with melanoma, colorectal cancer, and lung cancer.

[0144] In one embodiment, biomolecules (e.g., proteins, peptides, antibodies) whose protein properties are to be detected and quantified, reactions include, but are not limited to, ELISA-based reactions, labeling of target proteins by target-specific signal groups, and reactions catalyzed or inhibited by target proteins. For example, the α-synuclein discovery kit (QuanterixTM) can be used to detect the presence of α-synuclein (α-synuclein), which has a tendency to form toxic soluble oligomers (i.e., fibrils), which eventually aggregate into insoluble fibrils. α-Synuclein has been reported to be associated with the pathogenesis of Parkinson's disease, Parkinson's disease dementia, Lewy body dementia, and possibly Alzheimer's disease.

[0145] In one embodiment, in the case of detecting and quantifying exosomes, the reactions include but are not limited to those for labeling, detecting or quantifying biomolecules with specific exosomes. For example, exosomes are quantified using target proteoglycan (GPC-1), an exosome membrane protein that is more expressed on cancer exosomes than on non-cancer exosomes, and serum samples from breast cancer patients and healthy people can be distinguished from various exosome grouping protein biomarkers (Liu, 2018). In one embodiment, the absolute number of exosomes carrying one or more specific biomolecules can be determined digitally by the ExoILISA method. In one embodiment, the method used is as described in Liu (2018), which is fully referenced as the present invention.

[0146] In one embodiment, the target molecule (optionally together with non-target molecules) is extracted from the exosomes and then detected and quantified using the present invention (Chen, 2013; Li 2018), instead of detecting and quantifying the exosomes in the sample. However, in another embodiment, the method of the present invention is used to simultaneously detect and quantify the exosomes and the target molecule, including but not limited to the method described in Example 2. For a particular disease or in view of the disclosure of the present invention and the content known in the art, those skilled in the art will be able to select an appropriate analysis method.

[0147] In one embodiment, when pathogens are to be detected and quantified, the reactions include, but are not limited to, those for labeling, detecting or quantifying pathogen-specific biomolecules such as DNA, RNA, or viral / bacterial antigens. For example, human immunodeficiency virus (HIV) DNA can be quantified using appropriate kits and droplet digital PCR to detect and quantify HIV. It is believed that HIV DNA provides the most sensitive measurement of residual infection in patients in combination with antiretroviral therapy (cART) (Strain 2013).

[0148] In one embodiment, the reaction is performed in every droplet produced.

[0149] In one embodiment, the reactions performed in each droplet are of the same type. In another embodiment, the reactions performed in each droplet are of different types. Yet in another embodiment, the reactions performed in some droplets are of the same type, while the reactions performed in other droplets are of different types.

[0150] In one embodiment, the operating conditions (such as temperature, pressure and duration) used to carry out the reaction in each droplet are the same. In one embodiment, one or more operating conditions for carrying out the reaction in each droplet are different.

[0151] In one embodiment, in order to identify and label the target biomolecule in the droplet, a target-specific component is included in the reaction. In one embodiment, the target-specific component is a molecule that can specifically recognize the target biomolecule, which can be recognized by structure, function, or both.

[0152] In one embodiment, the target specific component is used to identify and label specific types and species of biomolecules in the droplet. In one embodiment, the target specific component is a primer or probe comprising nucleic acids that contain sequences complementary to the target nucleic acid. In one embodiment, the target specific component is a probe, antibody, or equivalent that can recognize a specific epitope or spatial configuration of a target biomolecule such as a protein, peptide, or viral particle.

[0153] In one embodiment, the target specific component is a molecule that has been processed (e.g., digested, degraded, oxidized or otherwise modified) by the target biomolecule. For example, when an enzyme is the target biomolecule, the target specific component can be a small molecule substrate that is catalyzed by the enzyme's enzymatic reaction.

[0154] In one embodiment, the target-specific component includes one or more signal-generating groups that are capable of generating a detectable signal so that the target biomolecule can be detected and quantified in subsequent assays and analyses.

[0155] In one embodiment, signal generating groups include, but are not limited to, chemiluminescent, fluorescent, and chromogenic substrates, as well as other substrates that convert to detectable products.

[0156] Digital detection and quantification of target biomolecules

[0157] In one embodiment, the invention includes detecting a target molecule using a method or device that produces a detectable signal indicative of the target molecule.

[0158] In one embodiment, the method of the present invention comprises the step of measuring the absolute number of indicative signals indicative of the presence of target biomolecules and then quantifying the absolute number of target biomolecules. In one embodiment, the present invention measures the signal generated by a signal generating group as described herein.

[0159] In one embodiment, the method of the present invention includes a single digital measurement step of a specific signal from a large number of droplets. Digital means that the signal is either one or zero. For example, a droplet with fluorescence is called "positive" (i.e., the droplet contains the target molecule), and a droplet without fluorescence is "negative" (i.e., the target molecule is not present in the droplet).

[0160] In one embodiment, the signal to be measured is a fluorescent signal, and a system or device that can capture the fluorescent signal and measure the intensity of the fluorescent signal is used. In one embodiment, a charge coupled device (CCD) is used to capture the fluorescent signal and generate an image of the fluorescent droplets in the chamber or on the chip. The fluorescent signal can be processed and analyzed by counting the number of fluorescent droplets and the intensity of the fluorescent signal of each droplet. In one embodiment, a proprietary image processing code is used to process and analyze the measured fluorescent signal. Figure 8 A fluorescence image of the droplet obtained by a CCD camera is shown.

[0161] In one embodiment, only one type of biomolecule is detected and quantified in each individual droplet.

[0162] In one embodiment, two or more types of biomolecules are detected and quantified in each individual droplet. For example, proteins, nucleic acids, exosomes and / or other types of biomolecules can be measured and quantified one after another in a single droplet.

[0163] In one embodiment, each individual droplet has two or more biological molecules of the same type that need to be measured and quantified, for example, each individual droplet has two or more nucleic acids (ie, DNA molecules and RNA molecules) that need to be measured and quantified.

[0164] In one embodiment, when there are two or more biomolecules to be measured and quantified in each individual droplet, one type of biomolecule is measured and quantified in each individual droplet first, then another type of biomolecule is measured and quantified in each individual droplet, etc. For example, one or more nucleic acids are measured and quantified in each individual droplet first, then one or more peptides are measured and quantified in each individual droplet.

[0165] In one embodiment, the type of biomolecule measured and quantified in one droplet is different from the type of biomolecule measured and quantified in other droplets.

[0166] In one embodiment, the present invention detects 1-5 biomolecule types per run. In another embodiment, the present invention detects 6-10 biomolecule types per run. In yet another embodiment, the present invention detects 11-20 biomolecule types per run.

[0167] In one embodiment, the present invention detects 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 types of biomolecules per run.

[0168] Downstream Uses

[0169] In one embodiment, the droplets generated can be collected at will for subsequent storage or downstream analysis. In one embodiment, the droplets are collected from the outlet of the integrated droplet microfluidic system of the present invention. The collected droplets can be lysed to extract and recover the biological material in the droplets. For example, DNA products amplified in previous reactions can be recovered from the droplets and used for further processing such as next-generation sequencing.

[0170] In one embodiment, all generated droplets are collected from the outlet, lysed to obtain a suspension of their contents, and then the suspension is divided into droplets and analyzed using the methods and apparatus described herein.

[0171] In one embodiment, all the generated droplets are collected from the outlet and then the droplets containing the target molecule are sorted. For example, the collected droplets can be loaded into a microfluidic sorting device for screening. In short, the collected droplets are injected into the sorting device as a monodisperse emulsion, and the droplet emulsion is kept separate from the fluorinated oil without surfactant. The droplets are diverted when they pass through a sorting node with an alternating current electric field (AC) (Baret, Jean-Christophe, 2009).

[0172] In one embodiment, the droplets collected or sorted from the outlet are further processed, such as by adding reagents and incubating under appropriate conditions.

[0173] Examples 2-5 describe the use of the present invention to detect different types of biomolecules. The following examples show how the present invention can be used to detect or quantify a variety of biomolecules that are indicative of disease states. However, those skilled in the art will readily appreciate that the embodiments and descriptions provided herein are for illustrative purposes only and are not intended to limit the scope of the present invention, which is defined by the claims that follow.

[0174] Detection of microorganisms by co-detection and quantification of DNA and RNA

[0175] In one embodiment, the present invention provides a method for detecting pathogenic microorganisms present in samples such as food, feed and plants or any solution or suspension derived therefrom. This method plays an important role in ensuring food safety and preventing the spread of microorganisms.

[0176] In one embodiment, a method for detecting pathogenic microorganisms includes the steps of detecting and quantifying biomolecules indicative of the presence of suspected pathogenic microorganisms, wherein the method divides a sample into droplets, and digitally detects and analyzes the target pathogenic microorganism using the methods and devices described herein. In one embodiment, the biomolecule to be detected is a nucleic acid and / or protein possessed by the target pathogenic microorganism.

[0177] In one embodiment, multiple nucleic acid types of target pathogenic microorganisms are detected and quantified by the present invention. Because the genome composition of pathogens is DNA, RNA or both, multifunctional detection or diagnostic tools that can simultaneously efficiently and sensitively amplify and detect target RNA and DNA are very useful in clinical practice. Example 2 provides an example of jointly detecting DNA and RNA at the single molecule level. By detecting or quantifying biomarkers related to DNA or RNA, the present invention can be used to detect any living or dead organisms, tissues, cells and groups containing these DNA or RNA biomarkers.

[0178] In one embodiment, nucleic acids and proteins of pathogenic microorganisms are simultaneously detected and quantified by the present invention.

[0179] Co-detection and quantification of nucleic acids and proteins

[0180] In one embodiment, the present invention provides a method for co-detecting nucleic acids and proteins in a sample.

[0181] In one embodiment, the present invention provides a method for co-detecting and / or quantifying one or more protein markers and one or more nucleic acid markers indicative of a disease or health state, thereby determining the presence of a disease or health state in a subject.

[0182] Example 3 provides an example of co-detection and quantification of a certain protein marker and a nucleic acid marker for the detection of solid tumor metastasis.

[0183] For example, it has been reported that combining four protein biomarkers with a single gene signature (KRAS mutants) increases the sensitivity of pancreatic cancer detection (Cohen, 2017), and a blood test can detect eight common cancer types (ovarian, liver, gastric, pancreatic, esophageal rectal, lung, and breast) by assessing circulating protein levels (eight proteins) and mutations in cell-free DNA (16 genes) (Cohen, 2018).

[0184] RNA(mRNA)Co-detection and quantification of protein and messenger RNA(mRNA)

[0185] In one embodiment, the present invention provides a method for co-detection of protein and mRNA in a sample. Example 4 provides a method for co-detection and quantification of protein and messenger RNA in a sample.

[0186] Integrated co-detection of proteins and mRNA from the same cell may not only reveal the connection between two important classes of biomolecules, but also help understand the mechanisms of gene regulation at the transcriptional and translational levels. Co-detection and quantification of DNA, RNA and protein in exosomes

[0187] In one embodiment, the present invention provides a method for the joint detection and quantification of DNA, RNA and protein in exosomes. In another embodiment, the present invention provides a method for quantifying exosomes in a sample in an absolute manner.

[0188] Example 5 provides an example of the co-detection and quantification of DNA, RNA, and protein in individual exosomes, as well as the absolute number of exosomes in a sample.

[0189] Exosomes containing biomolecules have DNA, RNA and proteins in their lumen and lipid membrane. These molecules can be extracted from exosomes isolated from samples such as serum. Using the present invention, the absolute number of exosomes can be obtained digitally by the ExoELISA method, or the absolute number of single biomolecules such as DNA, RNA or protein can be obtained by digital PCR or digital ELISA. Information about the number of biomolecules can be coupled or combined for diagnostic purposes or other uses.

[0190] Validity Assessment of Bioassay Results

[0191] In one embodiment, based on the number of target biomolecules determined in the above steps, the present invention provides a method for evaluating the validity of the bioassay results. For example, if the number of target biomolecules in a sample is found to be below the detection limit of a molecular assay, or if the relative number of DNA and RNA molecules in a sample is found to be below the normal range, the results obtained by the bioassay may be inaccurate. The concentration of the most recently collected sample or the original sample may be required to confirm the result.

[0192] Determine and assess the subject's health status

[0193] In one embodiment, the present invention provides a method for determining or assessing the health status of a subject based on the amount of a target biomolecule determined by the methods or devices described herein.

[0194] Because the present invention provides accurate quantification of target molecules in a highly specific and sensitive manner, the present invention can significantly improve the sensitivity, specificity and accuracy of biomarker diagnostic assays, especially those that rely on the absolute number of biomarkers. For example, by providing accurate absolute counts of cancer cell types or pathogens in a subject, the present invention can distinguish patients with various risk levels, infection levels or different cancer stages.

[0195] In one embodiment, the methods of the present invention include the step of determining the presence or absence of a state indicative of a disease or health condition in a subject based on the amount of a target biomolecule determined by the methods or devices described herein.

[0196] In one embodiment, the amount of a target biomolecule is determined according to the methods or devices described herein, and the methods of the invention include the step of determining the stage of disease progression.

[0197] In one embodiment, the amount of a target biomolecule is determined according to a method or device as described herein, and the method includes the step of assessing the subject's response to the drug.

[0198] In one embodiment, diseases that can be detected or diagnosed using the methods or devices of the present invention include, but are not limited to, cancer, infectious diseases, endocrine diseases, mitochondrial diseases, genetic diseases, nervous system and sensory organ diseases, circulatory system diseases, respiratory system diseases, digestive system diseases, genitourinary system diseases, skin and subcutaneous tissue diseases, musculoskeletal system and connective tissue diseases, and congenital anomalies.

[0199] In one embodiment, the present invention provides a real-time and digital counting method for target molecules in a sample containing the target biomolecule. The method comprises the following steps:

[0200] a) providing a droplet generator with the sample and reagents suitable for the method;

[0201] b) generating droplets using the droplet generator, wherein some of the droplets contain the reagent and the target biomolecule;

[0202] c) allowing the reagent to label the target biomolecule to generate a fluorescent signal;

[0203] d) detecting fluorescent signals from all droplets; and

[0204] e) converting the signal into a numerical value to obtain the total number of target biomolecules in the sample.

[0205] In one embodiment, the present invention provides a method for real-time and digital counting of cells or exosomes containing target biomolecules in a sample, the method comprising the following steps:

[0206] a) providing a droplet generator with the sample and reagents suitable for the method;

[0207] b) generating droplets using the droplet generator, wherein some of the droplets contain the reagent and the cells or exosomes;

[0208] c) allowing some reagent to lyse the cells or exosomes to release the target biomolecule, and then some reagent is used to label the target biomolecule to generate a fluorescent signal;

[0209] f) detecting fluorescent signals from all droplets; and

[0210] g) converting the signal into a numerical value to obtain the total number of cells or exosomes in the sample.

[0211] In one embodiment of the method of the present invention, each of the plurality of droplets in step b contains no more than one copy of the same type of target biomolecule. In one embodiment, each of the plurality of droplets contains no more than one copy of a cell or exosome.

[0212] In one embodiment of the method of the present invention, the target biomolecules to be labeled in step c are of the same type or of different types.

[0213] In one embodiment of the method of the present invention, the method can detect 1-10 types of target biomolecules.

[0214] In one embodiment of the method of the present invention, the target biomolecule can be selected from the group consisting of nucleic acids, peptides, proteins, enzymes, viruses and microorganisms.

[0215] In one embodiment of the method of the present invention, the nucleic acid can be selected from the group consisting of coding DNA, non-coding DNA, messenger RNA, ribosomal RNA, small RNA and transcribed RNA.

[0216] In one embodiment of the method of the present invention, steps c and d can be performed simultaneously in real time to detect the fluorescent signal.

[0217] In one embodiment of the method of the present invention, when the target biomolecule is labeled in step c, step d can be performed continuously or intermittently.

[0218] In one embodiment of the method of the present invention, after any step of steps c to e, the method further comprises a step of collecting droplets.

[0219] In one embodiment of the method of the present invention, the collected droplets are split to obtain a suspension containing labeled target biomolecules.

[0220] In one embodiment of the method of the present invention, the collected droplets are introduced into a droplet sorting unit to select droplets containing a specific type or having a specific property of target biomolecules.

[0221] In one embodiment of the method of the present invention, the structure is selected from the group consisting of a flow focusing structure, a cross-flow structure, a co-flow structure, a step emulsification structure and a microchannel emulsification structure.

[0222] In one embodiment of the method of the present invention, the sample and the reagent are introduced into the droplet generator through the same inlet or different inlets.

[0223] In one embodiment of the method of the present invention, the fluorescent signal is detected by a charge coupled device.

[0224] In one embodiment of the method of the present invention, the diameter of the droplets ranges from 5 μm to 200 μm.

[0225] In one embodiment of the method of the present invention, the number of droplets generated ranges from hundreds to millions.

[0226] In one embodiment of the method of the present invention, wherein the method is performed in an integrated droplet microfluidic system, the integrated droplet microfluidic system comprises the droplet generator, a droplet storage chamber, a detection unit and a plurality of microfluidic channels.

[0227] In one embodiment of the method of the present invention, the total number of the target biomolecule is indicative of the absence and severity of a disease.

[0228] In one embodiment, the present invention provides a method for diagnosing a disease in a subject, the method comprising determining the absolute amount of a biomarker for the disease in a sample from the subject, wherein the absolute amount is determined by using any of the methods described herein, wherein the biomarker is a target biomolecule present in the sample or in cells or exosomes of the sample, wherein the absolute amount of the marker as compared to a control indicates the presence, severity or absence of the disease in the subject.

[0229] Throughout this application, various publications are cited. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0230] Throughout this application, it should be noted that the transitional term "comprising", which is synonymous with "including", "containing" or "characterized by", is inclusive or open-ended and does not exclude other, unrecorded principles or method steps.

[0231] The present invention will be better understood by reference to the following examples. However, it will be readily appreciated by those skilled in the art that the examples provided are only for the purpose of illustrating the present invention and are not meant to limit the scope of the present invention, which is defined by the claims thereafter.

[0232] Implementation Example

[0233] Example 1 - Integrated droplet microfluidics system

[0234] This example shows an embodiment of a digital quantification system integrated in a microfluidic chip. The microfluidic chip consists of a droplet generator and a droplet reservoir. The droplet generator has a fluid focusing structure, a T-type or a step emulsion emulsion structure. The droplet reservoir design depends on the total number and volume of droplets required for a specific assay. The height of the chamber is usually 1-1.5 times the droplet diameter, and the size of the chamber is larger than the total droplet volume. The generated droplets are spread in a monolayer configuration on the microfluidic chip. After the sample partitioning is completed, parallel in situ reactions are carried out in all droplets. Once the reaction is completed, a microscope camera and an XY motion station are coupled to obtain images of the droplets on the entire microfluidic chip. The image is processed by a computer for digital counting and data analysis.

[0235] Single-layer configuration means that the chip contains a layer of carrier, and multiple micropores are distributed on the carrier, and each of these micropores includes a droplet. Of course, not all micropores include droplets, and some or none of them may have droplets. It can be understood here that the chip contains multiple layers of carriers, and each layer of carriers includes multiple micropores, and each of some or all of the micropores includes a droplet. The multi-layer carriers and the micropores are connected by capillary channels to form liquid. The so-called liquid connection means that the liquid can flow from one place to another.

[0236] Figure 1 One embodiment of an integrated droplet microfluidics system of the present invention is shown.

[0237] Figure 2 An embodiment of the present invention is shown, including an integrated droplet microfluidic system for digital quantification of biomolecules within each droplet. The microfluidic system includes four parts (from left to right): an inlet, a droplet generator, a droplet reservoir, and an outlet. The inlet is used to introduce various droplets (such as oil, samples for carrying out reactions, and reagents) into the droplet generator, where these liquids can be loaded separately through different inlets (such as Figure 2 As shown), or pre-mixed as a mixture and loaded through the same inlet. A sample containing biomolecules is prepared and divided into individual droplets by a droplet generator; the generated droplets are then spread out in a single-layer configuration in a droplet reservoir (or on a chip). After the sample is divided, parallel separate in situ reactions are carried out in individual droplets, wherein these reactions are used to analyze a portion of the target biomolecule assay, and the reactions in different droplets can be the same or different. Once the reaction is completed, a signal indicating the presence of the target biomolecule is detected, such as by capturing an image of the droplets in the droplet reservoir with a microscopic camera. The signal is then processed by a computer for digital counting and data analysis. The outlet is used to remove liquid or gas from the system. In one embodiment, the outlet is used to exhaust air or oil. In another embodiment, when the droplets need to be collected after the reaction, they can be collected from the droplet reservoir through the outlet.

[0238] Example 2 - Co-detection and quantification of DNA and RNA from single cells / exosomes at the molecular level

[0239] This example shows the use of the present invention to detect pathogenic microorganisms present in food, feed, plants or other samples. This is of great significance for providing safe food and preventing the spread of microorganisms. Since the genome of pathogens is composed of DNA and / or RNA, multifunctional diagnostic tools that can efficiently and sensitively amplify and detect DNA and RNA targets are particularly useful in clinical practice.

[0240] For DNA targets, digital PCR can be used for absolute counting of DNA molecules using specific primers and reagents co-encapsulated in droplets.

[0241] For RNA targets, digital RNA counting can be achieved by reverse transcribing RNA extracted from cells and then performing digital PCR. However, this absolute RNA counting method only provides the average value of the total number of cells, and therefore only reveals the average genotype / phenotype of the cell population. For absolute counting of RNA from individual cells or exosomes, one can use Fig. 9 The system shown includes two encapsulation circles: one for single cells or exosomes and the other for single mRNA molecules.

[0242] like Fig. 9 As shown in the figure above, a single exosome (or a single cell), a primer specific to the target RNA, and a lysis buffer for lysing the exosome or cell are attached to the magnetic bead and encapsulated in a droplet by the droplet generator. The droplets generated are as follows Figure 4 As shown. After the droplets are generated, they are stored in the droplet storage chamber on the right. In each individual droplet, a single exosome is lysed, and the RNA contained in it is released and paired with the target-specific primers on the magnetic beads. The droplets are then collected from the outlet for subsequent analysis. All collected droplets are then lysed with a solvent (such as perfluoro-1-octanol) to dissolve the oil phase and obtain an aqueous magnetic bead suspension with single cell / exosome RNA. An elution solution (such as PBS) is added to the suspension and vortexed to mix. The mixture is then placed on a magnetic rack, and components that are useless for subsequent RT-PCR are pipetted away together with the elution solution, while those magnetic beads bound to the target RNA are retained.

[0243] like Fig.10 As shown, after exosome lysis, mRNA molecules from exosomes are released, and the mRNA molecules will bind to the target primers on the magnetic beads. Unnecessary components are removed by washing steps. The resulting sample contains magnetic beads with primers bound to mRNA, which are then encapsulated into droplets for digital quantification of mRNA.

[0244] like Fig. 9 As shown in the lower part of the figure, the magnetic beads with primers bound to mRNA are mixed with the reverse transcription mixture and the PCR mixture (collectively referred to as the rt-PCR mixture), and then loaded into the integrated droplet microfluidics system for in situ reverse transcription and PCR temperature cycling. Figure 3 The droplets are generated in the form shown. Since reverse transcription and PCR are hot-start reactions, the mRNA sample and rt-PCR reaction mixture can be pre-mixed before packaging. The fluorescence signal is then digitally detected by a microscopic camera (such as Fig. 9 By using the RNAi technology (shown as black dots in the droplet reservoir in the lower middle panel), absolute counts of RNA targets from single cells or exosomes can be calculated.

[0245] Fig.11 Data for the digital detection of DNA using the endpoint of the present invention is shown. PCR variable temperature cycles are implemented to amplify the target DNA of the sample. The resulting mixture is then divided into 100,000 droplets, which are then deployed in a droplet storage chamber configured in a single layer. The fluorescent signal indicating the presence of the target DNA is recorded by a camera, and the intensity of the fluorescent signal in each droplet is measured using an image processing program. The image on the left records the fluorescent signal detected on the droplets in one area of ​​the droplet storage chamber, and then the image on the right shows the intensity of the fluorescent signal generated by each droplet in another area of ​​the droplet storage chamber (each circle represents a droplet, and a higher value means a higher level of fluorescent signal). The absolute count of the target DNA is then counted based on the detected signal.

[0246] In the case of detecting multiple RNA molecules from a cell or exosome population, some cells or exosomes may not contain every type of target RNA molecule, or some cells or exosomes may contain fewer or more types of target RNA molecules than other cells or exosomes. Through the two rounds of encapsulation process described herein, the method of the present invention can extract single molecules from a cell or exosome population and analyze the target RNA molecules from each cell or exosome. The number of cells or exosomes in the population, the number of cells or exosomes containing the target RNA, and the number of target RNA molecules from each cell or exosome can all be determined using the assays described herein (such as ExoELISA and rt-PCR reactions).

[0247] Example 3 - Co-detection and quantification of nucleic acids and proteins

[0248] This example shows one use of the present invention for detecting nucleic acids and proteins using the integrated droplet microfluidics system of the present invention.

[0249] In this example, a panel of protein and gene markers was evaluated for the detection of pre-metastatic disease in a variety of solid tumors. Diagnostics based on protein and gene markers generally provide a more accurate diagnosis.

[0250] Digital PCR assays are designed to simultaneously quantify multiple relevant DNA mutants, or multiple regions of driver genes that are commonly mutated in various cancer types. To confirm downstream applications, assays are designed to quantify relevant free protein markers. Droplet digital ELISA can be used in conjunction with the integrated droplet microfluidics system of the present invention for absolute counting of free proteins.

[0251] Compared with the use of either marker alone, the quantitative results of the combined use of protein markers and gene markers will give a more accurate and sensitive diagnosis.

[0252] Example 4 - Co-detection and quantification of protein and messenger RNA (mRNA)

[0253] This example shows the use of the present invention to detect protein and mRNA.

[0254] Integrated co-detection of proteins and mRNAs in the same cells not only reveals the association between two important types of biological molecules, but also helps to understand the mechanism of gene regulation at the transcriptional and translational levels.

[0255] The process described in Example 2 can be applied to this example to detect and quantify mRNA. The process described in Example 3 can be applied to this example to detect and quantify protein using droplet digital ELISA.

[0256] Example 5 - Co-detection and absolute counting of DNA, RNA and protein in exosomes

[0257] This example shows the use of the present invention to detect DNA, RNA and protein in single exosomes.

[0258] Exosomes contain biomolecules such as DNA, RNA and proteins in their lumen or on their lipid membranes. These biomolecules can be extracted from exosomes isolated from serum samples. According to the present invention, the absolute count of exosomes with specific targets can be digitally determined using the ExoELISA method, or the absolute count of single biomolecules such as DNA, RNA or protein can be determined using digital PCR or digital ELISA. Technical information about these biomolecules can be combined or summarized for diagnostic analysis of cancer types.

[0259] The process described in Example 2 can be applied to this example to detect and quantify DNA and RNA of individual exosomes, and the process described in Example 3 can be applied to this example to detect and quantify protein using droplet digital ELISA.

[0260] References:

[0261] 1.P.Zhu and L.Wang,“Passive and active droplet generation withmicrofluidics:a review”Lab Chip,2017,17,34-75.

[0262] 2.Baret,Jean-Christophe."Surfactants in droplet-based microfluidics."Lab on a Chip 12.3(2012):422-433.

[0263] 3.Liu,Chunchen,Xiaonan Xu,Bo Li,Bo Situ,Weilun Pan,Yu Hu,Taixue An,Shuhuai Yao,and Lei Zheng."Single-exosome-counting immunoassays for cancerdiagnostics."Nano letters(2018).

[0264] 4.Matthew C.Strain,Steven M.Lada,Tiffany Luong,Steffney E.Rought,SaraGianella,Valeri H.Terry,Celsa A.Spina,Christopher H.Woelk,and DouglasD.Richman"Highly precise measurement of HIV DNA by droplet digital PCR."PloSone 8.4(2013):e55943.

[0265] 5.Baret,Jean-Christophe,et al."Fluorescence-activated droplet sorting(FADS):efficient microfluidic cell sorting based on enzymatic activity."Labon a Chip 9.13(2009):1850-1858.

[0266] 6.Cohen,Joshua D.,et al."Combined circulating tumor DNA andproteinbiomarker-based liquid biopsy for the earlier detection of pancreaticcancers."Proceedings of the National Academy of Sciences 114.38(2017):10202-10207.

[0267] 7.Cohen,Joshua D.,et al."Detection and localization of surgicallyresectablecancers with a multi-analyte blood test."Science(2018):eaar3247.

[0268] 8.Xu,X.,Yuan,H.,Song,R.,Yu,M.,Chung,H.Y.,Hou,Y.,Shang,Y.,Zhou,H.andYao,S.,2018.“High aspect ratio induced spontaneous generation ofmonodispersepicolitre droplets for digital PCR”.Biomicrofluidics,12(1),p.014103.

[0269] 9.Liu,C.,Xu,X.,Li,B.,Situ,B.,Pan,W.,Hu,Y.,An,T.,Yao,S.and Zheng,L.,2018.“Single-exosome-counting immunoassays for cancer diagnostics”.Nanoletters.

[0270] 10.Cohen,Limor,and David R.Walt."Single-molecule arrays for proteinandnucleic acid analysis."Annual Review of Analytical Chemistry 10(2017):345-363.

[0271] 11.Abbyad,Paul,et al."Rails and anchors:guiding and trappingdropletmicroreactors in two dimensions."Lab on a Chip 11.5(2011):813-821.

[0272] 12.Huebner,Ansgar,et al."Static microdroplet arrays:a microfluidicdevice fordroplet trapping,incubation and release for enzymatic and cell-based assays."Lab ona Chip 9.5(2009):692-698.

[0273] 13.Nivedita Majumdar,Thomas Wessel,Jeffrey Marks,‘Digital PCRModelingfor Maximal Sensitivity,Dynamic Range and Measurement Precision’(2015),PLoSONE 10(3):e0118833.

[0274] 14.Chen,Walter W.,et al.BEAMing and droplet digital PCR analysis ofmutantIDH1 mRNA in glioma patient serum and cerebrospinal fluidextracellularvesicles.Molecular Therapy-Nucleic Acids 2(2013).

[0275] 15.Li,Tianwen,et al.Plasma circular RNA profiling of patients withgastriccancer and their droplet digital RT-PCR detection.Journal ofMolecularMedicine 96.1(2018):85-96。

Claims

1. A droplet microfluidic system, comprising: A droplet generator and a storage device capable of storing a plurality of droplets, wherein the droplet generator and the droplet storage device are in fluid communication, wherein: After the droplet generator generates droplets, the droplets are dispersed in the storage in the form of a single layer or multiple layers.

2. A droplet microfluidic system, comprising: A generator for generating droplets and a storage for storing droplets, wherein the storage comprises a plurality of micropores, and some of the plurality of micropores are used to store the droplets, wherein the droplet generator and the droplet storage are fluidically connected, and each micropore is fluidically connected, wherein: When droplets need to be stored, the droplets are dispersed in the storage in the form of a single layer or multiple layers.

3. A droplet microfluidic system, comprising: A generator for generating droplets and a storage for storing droplets, wherein the storage comprises a multi-layer carrier, each layer of the carrier comprises a plurality of micropores, and some of the plurality of micropores are used to store the droplets, wherein the droplet generator and the droplet storage are fluidically connected, each micropore is fluidically connected, and the multi-layer carriers are fluidically connected, wherein When it is necessary to store the droplets, the droplets are dispersed in each multilayer carrier in the form of a monolayer distribution.

4. The droplet microfluidic system according to any one of claims 1 to 3, wherein: The droplet generator comprises an inlet, and the droplet storage comprises an outlet.

5. The droplet microfluidic system according to any one of claims 1 to 4, wherein: The system also includes a sorting device, which is used to sort the droplets containing target molecules.

6. The droplet microfluidic system according to claim 5, wherein: The sorting device also includes a sorting node with an alternating current (AC) electric field. When the droplets are at the sorting node, the droplets are diverted.

7. The droplet microfluidic system according to claims 1-6, wherein: The droplet generator comprises a plurality of inlets, each of which is used to receive a different reagent component, and one of the inlets is used to receive a sample.

8. The droplet microfluidic system according to any one of claims 1 to 7, wherein: The droplet generator and the droplet reservoir are in fluid communication via a microfluidic channel.

9. The droplet microfluidic system according to any one of claims 1 to 8, wherein: The material for making the microfluidic channel is selected from one or more of silicon, glass, plastic and polydimethylsiloxane (PDMS).

10. The droplet microfluidic system according to any one of claims 1 to 9, wherein: The diameter of the microfluidic channel is 1-2 times the diameter of the droplet.

11. The droplet microfluidic system according to any one of claims 1 to 10, wherein: The droplet generator comprises one of the following structures to generate droplets: a droplet generating device based on surface tension; a cross-flow structure, a device that allows the continuous phase and the dispersed phase to intersect at a specific angle θ, or a step emulsification structure.

12. The droplet microfluidic system according to claim 1, wherein: The droplet generator includes a droplet sorting and separation unit.

13. The droplet microfluidic system according to any one of claims 1 to 12, wherein: The height of the micropores for storing the droplets is 1-1.5 times the diameter of the droplets.

14. The droplet microfluidic system according to any one of claims 1 to 13, wherein: The droplet reservoir includes a plurality of rows of anchoring structures for anchoring droplets to predetermined positions in the droplet reservoir.

15. The droplet microfluidic system according to claim 14, wherein: The anchoring structure includes a strut form to capture individual droplets.

16. The droplet microfluidic system according to claim 14, wherein: The anchoring structure consists of a groove pattern that captures individual droplets via surface tension.

17. The droplet microfluidic system of claims 1-16, wherein the droplet contains no more than one copy or one target molecule to be analyzed in a subsequent step.

18. The droplet microfluidic system according to any one of claims 1 to 16, wherein: The droplets include cells.

19. A method for storing droplets, the method comprising: A droplet generator and a droplet storage are provided, the droplet generator generates droplets, and then the droplets flow to the droplet storage, so that the droplets are stored in the storage in the form of a single layer or a multi-layer distribution.

20. A method for storing droplets, the method comprising: A droplet generator and a droplet reservoir are provided, and the droplet generator is allowed to generate droplets, and the generated droplets are then spread into the monolayer device in the droplet reservoir.

21. A method for storing droplets, the method comprising: A droplet generator and a droplet storage are provided, the droplet generator is allowed to generate droplets, and the generated droplets are allowed to spread in a droplet storage chamber configured in a single layer.

22. The method according to any one of claims 19 to 21, wherein the droplet generator and the droplet reservoir are in liquid communication via a microfluidic channel.

23. The method according to any one of claims 19 to 22, wherein: The droplet reservoir includes a plurality of rows of anchoring structures for anchoring droplets to predetermined positions in the droplet reservoir.

24. The method according to claim 23, wherein: The anchoring structure includes a strut form to capture individual droplets.

25. The method according to claim 23, wherein: The anchoring structure consists of a groove pattern that captures individual droplets via surface tension.

26. The method according to any one of claims 19 to 25, wherein: The droplet generator comprises an inlet, and the droplet storage comprises an outlet.

27. The method according to any one of claims 19 to 26, wherein: The method further includes sorting the droplets flowing out of the storage outlet.

28. The method according to any one of claims 19 to 26, wherein: The method further comprises using a sorting device for sorting droplets containing target molecules.

29. The method according to claim 28, wherein: The sorting device also includes a sorting node with an alternating current (AC) electric field. When the droplets are at the sorting node, the droplets are diverted.

30. The method according to any one of claims 19 to 29, wherein: The droplet generator comprises a plurality of inlets, each of which is used to receive a different reagent component, and one of the inlets is used to receive a sample.

31. The method according to any one of claims 19 to 30, wherein: The droplet generator and the droplet reservoir are fluidically connected via a microfluidic channel.

32. The method according to claim 31, wherein: The material for making the microfluidic channel is selected from one or more of silicon, glass, plastic and polydimethylsiloxane (PDMS).

33. The method according to any one of claims 31-32, wherein: The diameter of the microfluidic channel is 1-2 times the diameter of the droplet.

34. The method according to any one of claims 19 to 33, wherein: The droplet generator comprises one of the following structures to generate droplets: a droplet generating device based on surface tension; a cross-flow structure, a device that allows the continuous phase and the dispersed phase to intersect at a specific angle θ, or a step emulsification structure.

35. The method according to any one of claims 19 to 34, wherein: The droplet generator includes a droplet sorting and separation unit.

36. The method according to any one of claims 19 to 35, wherein: The droplet storage device comprises micropores, and the height of the micropores is 1-1.5 times of the diameter of the droplet.

37. The method of any one of claims 19-36, wherein the droplet contains no more than one copy or one target molecule to be analyzed in a subsequent step.

38. The method according to any one of claims 19 to 36, wherein: The droplets include cells.

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