CN108535239B - Micro-fluidic chip and detection system based on micro-droplets - Google Patents

Abstract

The present invention provides a micro-droplet based microfluidic control system for biological sample detection. The microfluidic chip comprises a microfluidic flow channel, a reaction micro-droplet generation module, a cleaning micro-droplet generation module, a shearing micro-droplet generation module, a capturing-cleaning-shearing module, a signal reading and detecting module and a droplet queue control module, and optionally comprises an incubation control module and a waste liquid collecting module.

Description

Micro-fluidic chip and detection system based on micro-droplets

Technical Field

The invention belongs to the technical field of biotechnology and clinical in-vitro detection, and particularly relates to a micro-fluidic chip and a detection system based on micro-droplets.

Background

In Vitro Diagnosis (IVD) refers to products and services for determining diseases or body functions by detecting human body samples (blood, body fluids, tissues, etc.) to obtain clinical Diagnosis information, outside the human body. In the prior art, there are many conventional methods for biological detection and diagnosis, such as biochemical detection, molecular detection and analysis, immunoblotting, chemiluminescence immunoassay, enzyme-linked immunosorbent assay (ELISA), and the like. However, these detection methods require a relatively long analysis time, and the liquid treatment process is relatively troublesome, and has relatively low flux and insufficient accuracy.

Recently, a microfluidic system has been widely used in biological and chemical fields as a platform capable of integrating a plurality of functional modules, and is known as "one of the 15 most important inventions affecting the future of human beings". Compared with the traditional biological detection and diagnosis method, the method based on the microfluidic system has several obvious advantages, including the capability of integrating various functional parts, low consumption of expensive biological reagents, capability of avoiding sample cross contamination, short analysis time and the like.

Some immunoassay platforms using microfluidic technology are available. However, there are still some significant drawbacks to the current microfluidic platform based immunodetection diagnostic methods or systems. For example, the antibody is pre-embedded in a channel or chamber of the solid phase carrier, the reaction is heterogeneous, and thus it is not possible to efficiently and accurately determine whether the antigen-antibody is sufficiently reacted; the required sample amount is slightly reduced compared with the traditional method, but the required amount is still more; most microfluidic chips are disposable products, which cannot be reused or have high cost, and the manufacturing cost of disposable microfluidic chips is also high.

In addition, some droplet-based microfluidic high-throughput bioanalytical platforms have been published. The immunoassay platform using the droplet microfluidic technology is typically as follows: a method for detecting biomacromolecules based on nano homogeneous time-resolved fluorescence immunization and a droplet microfluidic technology, a high-flux droplet microfluidic system or method based on a novel sensor (such as a capsule-packaged sensor), a droplet microfluidic system and method for detecting the interaction between quantum dots and biomolecules, and the like. However, these systems are not satisfactory and suffer from various drawbacks, such as: some microfluidic chips can perform high-flux detection, but have complex structures, high cost and low sensitivity; some adopt heterogeneous reaction, so the sample needs a lot of quantity and repeatability is lower; and most of the microfluidic chips are disposable, which is not beneficial to popularization.

In view of the foregoing, there is a strong need in the art to develop a micro-droplet-based microfluidic chip and a detection system that have the advantages of high throughput, high accuracy, high sensitivity, low cost, and the like, and are reusable.

Disclosure of Invention

The invention aims to provide a micro-fluidic chip and a detection system based on micro-droplets, which have the advantages of high throughput, high accuracy, high sensitivity and the like and can be repeatedly used.

In a first aspect of the present invention, there is provided a microfluidic chip comprising:

(a) a microfluidic flow-through channel for the flow of the continuous phase and microdroplets carried therein;

(b) a reaction microdroplet generation module for generating microdroplets for reaction, the reaction microdroplets comprising: the kit comprises a sample to be detected and a reaction reagent which reacts with the sample to be detected, wherein a substance to be detected in the sample to be detected reacts with the reaction reagent to form a detection product carrying a detectable marker;

(c) the cleaning micro-droplet generation module is used for generating cleaning liquid micro-droplets for cleaning;

(d) the shearing micro-droplet generation module is used for generating shearing liquid micro-droplets for shearing;

(e) a capture-wash-shear module, also referred to as an extraction module, for capturing the detectable label-bearing detection product from the incubated reaction microdroplets and washing the captured detectable label-bearing detection product with the wash solution microdroplets; shearing the washed detection product carrying the detectable marker by using the sheared micro-droplets so as to generate micro-droplets for detection;

(f) the signal reading and detecting module is used for reading the signal of the micro liquid drop for detection; and

(g) the liquid drop queue control module is connected with the cleaning micro liquid drop generation module and the shearing micro liquid drop generation module and is used for controlling the cleaning micro liquid drop generation module and the shearing micro liquid drop generation module to work after the reaction micro liquid drops move to a preset position, so that a micro liquid drop queue of 'reaction micro liquid drops, cleaning micro liquid drops and shearing micro liquid drops' is formed in the microfluid flow channel in the liquid flow direction, wherein the reaction micro liquid drops are positioned at the forefront of the flow.

In another preferred embodiment, the microfluidic flow channel is a capillary channel.

In another preferred embodiment, the microfluidic flow-through channel is a microchannel having a depth of 10 to 1000 μm and/or a width of 10 μm to 1000 μm; preferably, the microfluidic flow-through channel is a microchannel having a depth of 50 μm and/or a width of 100 μm.

In another preferred embodiment, the microfluidic flow-through channel is selected from the group consisting of: straight channels, annular channels, zigzag channels, cavities or a combination thereof.

In another preferred example, the reaction microdroplet generation module further includes a drop count identification submodule, and the drop count identification submodule is used for counting and numbering the reaction microdroplets.

In another preferred embodiment, the reaction microdroplet generation module further comprises a microdroplet fusion submodule, wherein in the reaction microdroplet generation module, sample microdroplets and reaction reagent microdroplets are generated firstly, and then the reaction reagent microdroplets and the sample microdroplets are fused into the reaction microdroplets through the microdroplet fusion submodule.

In another preferred example, the reaction microdroplet generation module further comprises a valve assembly for controlling the generation of the reaction reagent microdroplets and the sample microdroplets or controlling the generation of the reaction microdroplets.

In another preferred embodiment, the dispersed phase sample injection submodule comprises a dispersed phase sample injection port, a waste liquid outlet and two valves; the dispersed phase sample inlet is used for adding a sample, the valve and the waste liquid outlet are used for rapidly cleaning the dispersed phase sample inlet module, and the waste liquid outlet is connected with the waste liquid collecting module.

In another preferred embodiment, the dispersed phase sample inlet can be attached and sealed with the sample needle, so that sufficient positive pressure is ensured while the sample is added.

In another preferred embodiment, the reaction micro-droplet generation module, the cleaning micro-droplet generation module and the shearing micro-droplet generation module have a T-shaped interface structure, a flow confocal structure, a cross-shaped droplet generation structure, a coaxial flow structure or a combination thereof, and the structures are used for generating micro-droplets; preferably, a T-shaped droplet generating structure, a cross-shaped droplet generating structure, or a combination thereof is employed.

In another preferred embodiment, the signal is selected from: a chemiluminescence signal, a light signal emitted by excitation of a fluorescent group, a visible light signal emitted by light excitation of a quantum dot microsphere, an absorbance change signal, a magnetic field strength signal or a combination thereof.

In another preferred example, the number of the cleaning micro-droplets is one or more.

In another preferred embodiment, the reaction liquid drop is one or more.

In another preferred embodiment, there is one sheared droplet.

In another preferred embodiment, the microfluidic flow channel comprises the following sections: a continuous phase sample injection section, a detection sample injection section, a reaction reagent addition section, an incubation reaction section, a cleaning micro-droplet addition section, a shearing micro-droplet addition section, a capturing-cleaning-shearing treatment section, a signal detection section and an optional waste liquid channel section.

In another preferred example, the capture-wash-shear module further comprises a magnetic field region, and the magnetic field region is a magnetic field region which can be controlled to be opened or closed.

In another preferred embodiment, the magnetic field area is provided with a movable permanent magnet or electromagnet, and the magnetic field of the magnetic field area is generated by the movable permanent magnet or electromagnet.

In another preferred embodiment, the cleaning micro-droplet generation module and the shearing micro-droplet generation module further include: a control valve assembly controlled by a drop train control module.

In another preferred embodiment, the control valve assembly is selected from: electromagnetic valve, paraffin hot melt valve, magnet moving valve, pneumatic valve, diaphragm valve, drain valve, mechanical valve or their combination; preferably, the control valve assembly is a solenoid valve.

In another preferred example, the driving force assembly is a pressure driving assembly.

In another preferred embodiment, the microfluidic chip further includes: (i) an incubation control module for controlling incubation parameters of the microfluidic flow-through channel of the incubation section.

In another preferred embodiment, the microfluidic chip further includes: (j) the waste liquid collecting module is used for collecting waste liquid generated in the detection process.

In another preferred example, the signal reading detection module further comprises a post-processing sub-module, in which a substance for generating a detection signal is added to the microdroplets for detection.

In another preferred example, the post-processing sub-module is further provided with a sensor for identifying whether the microdroplet for detection is a valid microdroplet, and a substance for generating a detection signal is added to the valid microdroplet for detection in the post-processing sub-module.

In another preferred embodiment, the material of the microfluidic chip is selected from a glass substrate material, glass, a silicon-based material, polydimethylsiloxane, acrylic plastic, a cyclic olefin copolymer material, polypropylene plastic, polystyrene plastic, or a combination thereof; preferably, a glass substrate material.

The second aspect of the present invention provides a detection method based on a microfluidic chip, the method comprising the following steps:

a. adding a mobile phase and filling the microfluidic flow channel with the mobile phase;

b. pretreating the reagent;

c. preparing a biological sample to be tested, said sample containing a target substance to be tested;

d. adding a sample and a pretreated reagent, generating a sample micro-droplet and a reagent micro-droplet through the reaction micro-droplet generation module, and fusing the sample micro-droplet and the reagent micro-droplet in the reaction micro-droplet generation module to form a reaction micro-droplet; or the sample and the pretreated reagent are mixed in advance to form reaction liquid, and the reaction liquid is added to generate reaction micro-droplets through the reaction micro-droplet generation module;

e. under the control of the liquid drop queue control module, a cleaning micro liquid drop generation module generates cleaning micro liquid drops;

f. under the control of the liquid drop queue control module, a shearing micro-liquid drop generation module generates shearing micro-liquid drops;

g. e, forming a micro-droplet queue by the micro-droplets generated in the step f and the reaction micro-droplets;

h. the micro liquid drop queue enters a capture-cleaning-shearing module and forms micro liquid drops for detection;

i. the micro liquid drops for detection pass through a signal reading and detecting module, and the reading and detecting module detects and reads signals.

In another preferred embodiment, the reagent is a reagent that can react with a target substance in a sample.

In another preferred embodiment, the reagent is a reagent that can bind to a magnetic bead or generate a detectable signal.

In another preferred embodiment, the target substance is selected from: proteins, nucleic acids, liposomes, peptide fragments, nucleotides, amino acids, viruses, bacteria, parasites, cells; preferably a peptide fragment or a proteinaceous substance.

In another preferred embodiment, the sample is selected from: blood, plasma, serum, interstitial fluid, lymph, urine, or culture fluid for culturing microorganisms/cells of a human or other animal.

In another preferred embodiment, the biological sample is pretreated.

In another preferred embodiment, after the step d, the sample inlet is cleaned by using a micro-fluidic chip cleaning agent, the waste liquid of the cleaning agent enters the waste liquid collection module, and the step d is repeated to generate a plurality of reaction micro-droplets with different samples.

In another preferred embodiment, after step d, the microfluidic chip cleaning agent is selected from a buffer system, a surfactant, a protein or a combination thereof; preferably, the buffer system is selected from: a phosphate system, an acetate system, a borate system or a Tris-HCl system, and/or a surfactant selected from: PEG, PVP, Tween 20, Tween 80, Triton X100 or a combination thereof, and/or the protein is selected from serum albumin, casein or a combination thereof.

In another preferred embodiment, the reaction micro-droplets generated in step d are fully reacted/uniformly mixed in the incubation reaction zone.

In another preferred embodiment, the conditions of the incubation reaction section are controlled by the incubation control module.

In another preferred embodiment, the incubation reaction section is under temperature conditions.

In another preferred embodiment, 1-3 wash microdroplets are generated in a single assay.

In another preferred example, 1 sheared microdroplet is generated in a single assay.

In another preferred embodiment, when the droplet queue enters the capture-wash-shear module, the magnetic field is turned on, the sample containing the magnetic beads in the reaction micro-droplets is captured by the magnetic field, then the micro-droplets are washed to wash the captured sample, and then the shear droplets shear off the detectable labels and form the micro-droplets for detection.

In another preferred example, the used washing micro-droplets and the reaction micro-droplets without the sample enter the waste liquid module through the waste liquid channel.

In another preferred example, the magnetic field is closed, and the waste magnetic beads enter the waste liquid module along with the continuous phase.

In another preferred example, step i further comprises a microdroplet post-processing step for detection, and a substance for generating a detection signal is added to the microdroplet for detection.

In another preferred embodiment, the substance for exciting the detection signal is a chemiluminescent excitation substrate.

In another preferred embodiment, the micro-droplets of the read signal enter the waste liquid treatment module through the waste liquid channel.

In another preferred example, in step b, the pretreatment of the reagent is coating or coupling magnetic beads.

In another preferred example, in the step b, the magnetic beads are compounds of iron, cobalt and nickel; preferably ferric oxide or ferroferric oxide compound;

in another preferred example, in the step b, the magnetic beads are superparamagnetic particles.

In another preferred embodiment, the size of the magnetic beads in step b is 0.01-10 μm, more preferably 1-2.8 μm.

In another preferred embodiment, the volume ratio of the sample micro-droplets to the reagent micro-droplets is 1:0.5 to 1: 2.

In another preferred embodiment, the method is a detection method based on a double antibody sandwich method, or a competitive binding method.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Fig. 1 shows a schematic structure of a microfluidic chip according to an embodiment of the present invention. Wherein, each mark is as follows:

1-a continuous phase sample inlet, 2-a control valve a, 3-a flow pipeline, 4-a waste liquid inlet, 5-a control valve B, 6-a dispersed phase sample inlet, 7-a control valve C, 8-a droplet generation inlet, 9-a sensor A, 10-a sensor B, 11-a reagent fusion inlet, 12-a reagent fusion inlet 1, 13-a reagent fusion inlet 2, 14-a control valve D, 15-a first incubation pipeline, 16-a first droplet treatment liquid sample inlet (or a pretreatment liquid sample inlet), 17-a control valve e, 18-a first cylindrical structure region, 19-a magnetic field region, 20-a sensor C, 21-a sensor D, 22-a droplet treatment liquid fusion inlet, 23-a control valve f, 24-a second droplet treatment liquid sample inlet (or a post-treatment liquid sample inlet), 25-second incubation channel, 26-second cylindrical structured zone, 27-sensor E, 28-outlet.

FIG. 2 shows a schematic cross-sectional view of a dispersed phase injection port in one embodiment.

Fig. 3 shows a flow chart of the operation of the microfluidic chip of the present invention.

Fig. 4 shows a schematic diagram of the movement of micro-droplets at various stages in a microfluidic chip according to an embodiment of the present invention. The method comprises an A-droplet generation stage, a B-reagent fusion stage, a C-mixing incubation stage, a D-droplet extraction stage and an E-droplet shearing stage. In C, three different representative reactions are shown: c1-mixing incubation stage based on antigen-antibody reaction in the droplet, C2-mixing incubation stage based on loop-mediated isothermal amplification reaction in the droplet and C3-BCA method in the droplet for determining total protein concentration.

FIG. 5 shows a schematic of the state of micro-droplets in an extraction process in one embodiment of the invention.

FIG. 6 shows the results of an extraction experiment in one embodiment of the present invention.

FIG. 7 shows a histogram of the gray scale values of the optical signals detected at different extraction times in the present invention.

FIG. 8 shows a standard curve for different extraction times.

Detailed Description

The inventors of the present invention have conducted long and intensive studies to design a microfluidic chip based on micro-droplets with high module integration, and a control system, device and method thereof. By the design, the precise control of the liquid drops can be realized, and various required processes can be completed on a single chip. On the basis of this, the present invention has been completed.

Term(s) for

As used herein, the terms "microfluidic chip of the invention", "microfluidic chip based on micro-droplets of the invention", or "chip of the invention" are used interchangeably to refer to a microfluidic chip of the invention.

As used herein, the term "droplet microfluidic chip" refers to a chip (or system) based on droplet microfluidic technology, wherein a series of monodisperse droplets, each containing a specific biochemical reaction, are formed by the dispersion of one phase in another phase under the action of fluid shear forces and interfacial tension using immiscible two-phase flows. Compared to continuous fluid microfluidic systems, droplet-based microfluidic systems can produce droplets with nanoliter to picoliter volumes at frequencies up to kilohertz, each droplet can act as a microreactor, provide a well-defined solution for high throughput, and enable independent manipulation of individual droplets in a short time, including droplet generation, fusion, incubation, aggregation, splitting, sorting, extraction, and analysis. The generation of droplets relies on the combined shear and surface tension of the two phases.

As used herein, the term "fusion of droplets" refers to the fusion of two or more droplets together to allow a mixing reaction between the droplets of different components.

As used herein, the term "splitting of a droplet" refers to splitting a droplet into two or more droplets, enabling further manipulation of droplet volume and concentration of the contents within the droplet after droplet generation.

As used herein, the term "incubation of a droplet" refers to mixing the contents of the droplet uniformly over a period of time.

As used herein, the term "continuous phase" refers to a carrier liquid, for example, in the examples herein, an oil phase, but is not limited to an oil phase.

As used herein, the term "dispersed phase" refers to a liquid comprising a test sample, a reagent, a wash solution, or a shear fluid, etc., which can produce microdroplets in the continuous phase, e.g., in the examples herein, a water-soluble liquid, but is not limited to water-soluble liquids. Wherein the continuous phase is selected based on the nature of the dispersed phase, and the continuous phase is immiscible with the dispersion.

As used herein, the term "microdroplet queue" merely represents the order in which microdroplets pass through the predetermined locations, and the microdroplets in the "microdroplet queue" may be present in the microchannel at the same time and flow sequentially through the predetermined locations; or generating micro-droplets sequentially and sequentially flowing through the preset positions.

Micro-fluidic chip

The invention provides a micro-fluidic chip which has high function integration level and can automatically realize a series of functions including generation, fusion, extraction, incubation, splitting, signal detection and the like.

The number and sequence of each module of the microfluidic chip are not particularly limited, and can be adjusted according to the specific detection condition.

In another preferred embodiment, the microfluidic chip of the present invention employs an optimized valve structure, and the droplets can be precisely controlled under the control of the valve structure.

In another preferred embodiment, the microfluidic chip of the present invention is equipped with a power source, wherein the power source is used for moving each micro droplet (dispersed phase) and continuous phase to a desired direction, and the power source can provide a forward driving force or a reverse driving force; the power source may be various power sources common in the art, such as, but not limited to, power obtained by centrifugal force driving, electrowetting driving, pressure driving; preferably pressure driven (the pressure is selected from the group consisting of electrolysis pressure, compressed gas pressure, direct gas pressure difference).

In another preferred example, the reading content of the signal reading detection module of the microfluidic chip of the present invention includes but is not limited to: chemiluminescence signals, optical signals emitted by excited fluorescent groups, visible light signals emitted by light excitation of quantum dot microspheres and the like.

In another preferred embodiment, the signal reading and detecting module of the microfluidic chip of the present invention can be determined according to a specific detecting method, and representative examples include (but are not limited to): a laser-induced multi-channel spectral camera, a laser-induced multi-channel spectral sensor, a photomultiplier tube, or a combination thereof.

In another preferred embodiment, sensors are provided at multiple locations in the chip of the invention (e.g., at the inlet and outlet of each of the micro-droplet generation module, capture-wash-shear module); the sensor can count and mark the drops, confirm that the drop size is consistent with the requirements of the test, calculate the passing frequency (or velocity) of the drops and feed back to the control system.

In another preferred embodiment, the microfluidic chip of the present invention can realize the matching of each module through a general control system (e.g., self-programming software or commercial software); the general control system receives signals obtained from all sensors in the chip and processes the signals to generate control signals, and the general control system controls all parts of the whole chip, such as a power source, all valves and the like, and is matched with the liquid drop queue control module of the chip to realize the detection of various organisms.

In another preferred embodiment, the microfluidic chip of the present invention can be made of materials commonly used in the art, including (but not limited to): glass, silica-based materials, polydimethylsiloxane, acrylic plastics, cyclic olefin copolymer materials, polypropylene plastics, polystyrene plastics, rubber, or combinations thereof; these materials themselves may be selected, or doped, modified, or modified materials may be selected.

In another preferred embodiment, the shape of the microfluidic chip microfluidic flow channel of the present invention can be various channel shapes commonly used in the art including, but not limited to, a channel, a circular channel, a zigzag channel or a cavity; wherein the cavity structure includes, but is not limited to, a single layer height, a double layer height, or a multiple layer height.

In another preferred embodiment, the microfluidic chip microfluidic flow channel of the present invention can be formed by etching the inside of the material or by capillary connection.

In another preferred embodiment, each micro-droplet generation module has a droplet structure that can be formed by fluid shear force, and the structures include, but are not limited to, a T-junction structure, a cross-junction structure, a flow confocal structure, or a coaxial flow structure.

In another preferred embodiment, the micro-droplet generating module of the microfluidic chip of the present invention forms a micro-droplet structure, and the diameter of the generated micro-droplet is about 1 μm to 1000 μm, preferably about 50 μm, and if necessary, a liquid column can also be produced.

In another preferred embodiment, the microfluidic chip of the present invention can perform different detection on the chip according to different targets, including but not limited to nucleic acid-based detection methods (e.g., PCR, PT-PCR, loop-mediated isothermal amplification technique or strand displacement) or protein-specific reaction-based detection methods (e.g., double antibody sandwich method, competitive immunological binding method, etc.).

In another preferred embodiment, the microfluidic chip of the present invention is suitable for disposable use and also suitable for repeated use.

Detection method

The invention also provides a detection method based on the microfluidic chip.

A prominent feature of the process of the invention is the provision of a highly automated extraction operation. For ease of understanding, the following description is made in conjunction with the accompanying drawings. It should be understood that these drawings do not in any way limit the scope of the present invention.

Referring to fig. 3, the working process of the microfluidic chip of the present invention includes sample introduction, droplet generation, reagent fusion, incubation, droplet pretreatment, droplet post-treatment, signal observation, and entering into a waste liquid pool.

Referring to fig. 4, on the chip of the present invention, microdroplets are generated as shown in fig. 4A, the sample and the continuous phase flow along the microfluidic flow channel under the driving of an external force, and at the drop generation interface, the sample forms microdroplets under the shearing action formed by the dispersed phase at the generation interface. The process of fusion between the micro-droplets is shown in fig. 4B, where the micro-droplets containing the sample and the reagent micro-droplets are fused one-to-one. The various incubation reactions inside the microdroplets are shown in fig. 4C. As shown in fig. 4D, a microdroplet containing a sample with magnetic beads, the material with magnetic beads remains in the region when passing through the magnetic field region. As shown in fig. 4E, shearing the microdroplets shears the target analyte droplets (i.e., the shearing fluid after the shearing reaction).

Referring to fig. 5, the extraction process is described, which mainly includes capturing, washing and shearing, taking 3 micro-droplets, 1 washing liquid droplet and 1 shearing liquid droplet, which have undergone the corresponding reactions, as an example.

Taking the magnetic bead method as an example, in a chip channel, a generated sample droplet and a reagent droplet generated by an added reagent are fused to form a mixed droplet, i.e., a reaction micro droplet (i.e., a micro droplet for reaction), and the mixed droplet enters a chip extraction module after incubation is completed.

After the mixed droplets flow through the predetermined locations, the desired valves are sequentially opened under precise signal transmission and operational control to generate cleaning fluid droplets and shear fluid droplets, thereby forming a sequence of micro-droplets, cleaning fluid droplets and shear fluid droplets (fig. 5a) in the microfluidic channel.

When a droplet containing magnetic beads with bound antigen-antibody and fluorescent label flows through the magnetic field region under the action of the magnetic field, the magnetic beads in the first mixed droplet are attracted to the side wall of the chip channel (FIG. 5b) and separated from the flowing droplet (FIG. 5c, 5 d). Similarly, the magnetic beads in the second and third mixed droplets are also attracted to the side walls of the chip channel (not shown) and separated from the flowing droplets.

Following the flow of the continuous phase (oil phase) in the chip channel, the bead clusters in the mixed droplet were separated from the other droplet contents by droplet splitting, and then the bead clusters were washed sequentially with droplets containing wash liquid, sufficiently removing interfering residues (fig. 5e, 5 f).

Finally, the shear drop is fused in contact with the cluster of beads in the same way, then it is split, during which the material for signal detection is sheared and carried away (fig. 5g, 5 h). Thereafter, the sheared liquid drops continue to flow forward in the state of liquid drops for signal detection, or enter a chip post-processing area for further operation (not shown). After the magnetic field is switched off, the magnetic bead clusters flow forward into the waste liquid pool in the state of waste liquid drops. (not shown)

In another preferred embodiment, the original biological sample or the processed biological sample enters the droplet microfluidic chip through the sample injection port, and the control of the sample flow is realized through the valve. The continuous phase (such as oil phase) enters the droplet microfluidic chip at the continuous phase injection port, and the continuous phase flow is controlled through the valve.

In another preferred example, the biological sample is a biological fluid sample, and may be blood, plasma, serum, tissue fluid, lymph, urine, etc. of a human or other animal, or a culture fluid for culturing microorganisms or cells, or other more precious or rare environmental samples.

In another preferred embodiment, the biological fluid sample contains a target analyte, and the method of the present invention can perform qualitative, semi-quantitative or quantitative detection on the target analyte.

The target sample may be, but not limited to, a protein, a nucleic acid, a liposome, a peptide fragment, a nucleotide, an amino acid, a virus, a bacterium, a parasite, a cell, other single molecules or complexes, and the like.

In another preferred embodiment, the biological sample is pretreated by a pretreatment method that is conventional in biological detection (e.g., adding an anticoagulant, adding a lysis solution, adjusting the concentration, etc.).

In another preferred embodiment, after one sample is added to the sample injection port, a chip cleaning agent can be added to clean the sample injection port, and the sample injection port is controlled by a valve or a driving force and discharged into the waste liquid module from the connected waste liquid channel section.

In another preferred example, the microdroplet for detection stays in the signal detection section and is read by signal, or the microdroplet for detection flows in the signal detection section and is read by signal.

In another preferred example, when the micro-droplets are in the incubation period, the micro-droplets can be controlled to stay in the incubation reaction section for sufficient mixing or reaction; alternatively, the microdroplets may be kept in a fluid state for thorough mixing or reaction during the incubation period.

In another preferred embodiment, the method of the present invention can prepare for the next extraction process by turning off the magnetic field to allow the mobile phase to carry away the magnetic beads remaining in the capture-wash-shear processing section.

In another preferred example, the incubation module can be controlled to maintain a constant temperature (temperature is selected according to the specific reaction).

In another preferred embodiment, the method of the present invention can be monitored and controlled by computer software during the whole testing process.

In another preferred embodiment, the volume of the sample volume for a single assay of the method of the invention is 0.5-10. mu.L, preferably 0.5-5. mu.L.

In another preferred embodiment, the single detection time of a single sample in the method is 3-15 min.

In another preferred embodiment, the method of the present invention can simultaneously detect a plurality of samples.

Microfluidic control chip or method based on micro-droplets

A microfluid control chip or a method based on micro-droplets is disclosed, wherein the microfluid control chip comprises a fluid sample introduction module, a micro-droplet generation module, a reagent fusion module, an incubation module, a micro-droplet processing module, a signal reading and detecting module and a waste liquid pool module. But is not limited to such modules being combined in sequence and also includes mixed use and multiplexed systems of such modules. The method is characterized by comprising the following detailed module design and steps:

a) a fluid sample introduction module: the biological original liquid sample or the processed biological liquid sample enters the liquid drop microfluidic chip through the disperse phase inlet, and the flow of the sample is controlled through the valve. And the oil phase used as the continuous phase enters the droplet microfluidic chip at the sample inlet of the continuous phase, and the flow of the continuous phase is controlled by a valve.

Optionally, a waste liquid channel may be added at the inlet of the dispersed phase, and the waste liquid channel may discharge the residual sample.

And optionally, adding a cleaning solution from the dispersed phase inlet to clean the dispersed phase inlet, and discharging the cleaning solution through the waste liquid channel. And optionally, a valve may be used to control the opening or closing of the waste channel.

The power source for the flow of the dispersed phase and the continuous phase and the power source for discharging the waste liquid provide forward thrust for the fluid in the droplet microfluidic chip, so that the fluid flows forward uniformly, and the difference of test values caused by different flow rates is avoided. Wherein optionally, the power source may be, but not limited to, centrifugal force drive, electrowetting drive, pressure drive (electrolytic pressure, compressed gas pressure, chemical decomposition pressure, direct gas pressure difference drive). Optionally, the power source may be a forward driving force or a reverse driving force.

The dispersed phase and the continuous phase and the valve in the waste liquid discharge channel can be but not limited to a paraffin valve, a paraffin hot melting valve, a magnet moving valve, a pneumatic valve, a diaphragm valve, a drain valve and a mechanical valve, can be used in combination of one or more of the valves, or can be directly controlled by power without using the valves.

b) A micro-droplet generation module: the disperse phase and continuous phase liquid are connected together through a channel, and form liquid drops through the shearing force of flowing fluid, and the liquid drops can be formed in a mode of all structures capable of forming the liquid drops, preferably, the T-shaped interface, the flowing confocal structure and the coaxial flowing structure are included.

c) A reagent fusion module: adding a labeled substance capable of being combined with a target substance to be detected into a liquid drop formed by the biological sample, and realizing the fusion of the reagent and the sample liquid drop through a liquid drop fusion and contact fusion method.

Optionally, the reagent is a labeled substance that is added to the droplet and can bind to the target substance to be detected, a medium that assists the substance to be detected to bind to the labeled substance, a reactant that can react with the substance to be detected to generate a detectable signal, or other reagents with similar functions.

Optionally, the fusion mode of the reagent and the droplet can be realized by the structure of a chip channel or the speed generated by the droplet, or the fusion of the reagent and the droplet is performed by a method of contacting the droplet and the reagent with each other, where the active control fusion or the passive contact fusion can be selected.

d) An incubation module: the module provides an area, so that the solution in the liquid drop can be uniformly mixed or fully reacted, the reaction can be completed in the flow of the liquid drop, and the flow of the liquid drop can also be stopped to be fully completed.

Wherein optionally, the module provides or does not provide a constant temperature module to assist in the sufficient reaction of the reagents within the droplets or to achieve reaction conditions for the reaction.

And optionally, the module may provide one or more constant temperature modules to assist in the reaction.

e) The liquid drop processing module realizes the extraction of the incubated reaction micro-liquid drops through various modules (such as a capture-cleaning-shearing module, a liquid drop queue control module and the like) in the microfluidic chip of the invention: the droplet processing module includes a droplet processing chamber (e.g., a capture-clean-shear process section) and an input of droplet cleaner/treatment agent. The module provides an area for processing a target detection object which has fully reacted in the liquid drop, so that the target detection object can be more accurate, faster and more efficient in signal reading.

In another preferred embodiment, the method can be divided into pre-treatment and post-treatment, wherein the pre-treatment is extraction or capture-cleaning-shearing.

Optionally, the droplet processing chamber provided by the module can realize the control of the droplet in the region through the design of the channel or the applied driving force, so as to facilitate the further processing of the droplet.

And optionally, the channel can be designed to be a double-layer structure to control the liquid drop to stay in the liquid drop processing chamber, a columnar structure can be designed to slow down the movement speed of the liquid drop, and other designs can be used for realizing the same or similar functions.

And optionally, the motion of the micro-droplets can be controlled by an external driving force, such as a magnetic force or other force capable of performing the same or similar function.

Wherein optionally, the input of the droplet cleaning/treating agent in the module can treat the droplets. The input of the cleaning agent can clean the unreacted substances which can influence the reading of the signals in the liquid drops and remove the influence of impurities. The droplet processing agent can perform other functions such as shearing between magnetic beads and antibodies, and the like, as required.

Optionally, a step of reading the auxiliary signal may be provided in the droplet processing chamber, such as a photo-excitation process in a homogeneous immunoassay, or other similar or analogous step processes.

f) The signal reading detection module: the module is a region provided in a chip, liquid drops after reaction are pretreated in the region, and then signal detection equipment or instruments such as a sensor or a sensor system outside the chip read the index of the content of a reaction target detection object in the region in the chip, wherein the index can be but is not limited to an optical signal, a fluorescent group signal, a quantum dot signal, a fluorescent microsphere signal and the like.

Wherein optionally, the sensor may be, but is not limited to, a microscope, an optical fiber, and the like.

The pretreatment of the liquid drops after the internal reaction is to remove other substances in the liquid drops interfering with the detection object of the reaction target by using a cleaning solution so as to avoid causing false positive or false negative signals.

g) A waste liquid tank module: the module is used to collect all waste generated during the sample assay.

In another preferred example, the operation states of all modules in the micro-droplet based microfluidic control system or device or method are monitored and controlled by computer software. For example, controlling the opening and closing of valves in a system-on-chip; and the control signal reading and detecting module is used for processing the measured data to give visual information.

Optionally, the computer control software may be, but is not limited to, a commercial control software and an autonomous programming software, and one or a combination of the two may be used.

In another preferred embodiment, the fluid for generating droplets described in the foregoing is a biological raw liquid sample, or other processed biological raw liquid sample. Including but not limited to, human or other animal blood, plasma, serum, interstitial fluid, lymph fluid, urine, and the like.

Wherein, optionally, the sample may be a culture solution for culturing microorganisms or cells.

Optionally, the sample may be other precious or rare environmental samples.

In another preferred embodiment, the fluid for generating droplets described in the foregoing may contain a target analyte, and the system is used to perform qualitative, semi-quantitative or quantitative detection on the target analyte.

The target sample may be, but not limited to, a protein, a nucleic acid, a liposome, a peptide fragment, a nucleotide, an amino acid, a virus, a bacterium, a parasite, a cell, other single molecules or complexes, and the like.

In another preferred embodiment, the reagent of the reagent fusion part in the above description contains a label capable of binding with the target detection object, such as an antibody bound with a magnetic bead or a fluorescent signal substance.

And optionally, the reagent is another substance or chelate that can react with the sample to produce a detectable signal.

Optionally, the magnetic beads are superparamagnetic particles, and include compounds of iron, cobalt, and nickel, mainly including, but not limited to, iron oxide and ferroferric oxide compounds. Wherein the size of the magnetic beads is 0.1-10 μm, preferably 0.5-3 μm.

And optionally, a permanent magnet or an electromagnet may be used to manipulate the movement or aggregation of the magnetic beads.

Optionally, the fluorescent signal substance mainly includes, but is not limited to, fluorescent microspheres and fluorescent signal groups.

In another preferred embodiment, the modules of the microfluidic chip system described in the foregoing are connected by microchannels, and the droplets generated by the sample can flow along the channels to each module. The microchannels may be, but are not limited to, those described as: micro-channels etched or otherwise formed within the material, micro-channels formed between different components, micro-channels fabricated using capillaries, and the like.

Wherein, optionally, the shape of the micro-channel can be but not limited to a straight channel, a circular channel, a zigzag channel, and the like.

Where optional, the depth of the microchannels may be, but is not limited to, 1 μm to 10 μm, 5 μm to 50 μm, and 30 μm to 300 μm and 100 μm to 1000 μm, etc.

And optionally, the width of the microchannel can be, but is not limited to, 1 μm to 10 μm, 5 μm to 50 μm, and 30 μm to 300 μm and 100 μm to 1000 μm, etc.

7: the fluid in the invention 1 forms micro-droplets under the action of the continuous phase to flow in the channel in the invention 5, and the diameter of the generated droplets can be but is not limited to 1 μm-50 μm, 50 μm-1000 μm and the like.

In another preferred embodiment, the modules described in the above summary of the invention may be combined into a system in sequence, or some of them may be combined into a system, or they may be mixed to form a system, or a certain module and other modules may be used several times to form a system, or these modules may be used to form a multiplexing system, etc.

Optionally, the system also includes a part of module combinations for multiple uses in the system, for example, a combination of a reagent fusion module and an incubation module, which can be incubated again after adding different reagents respectively, and the like.

In another preferred embodiment, the continuous phase mentioned in the foregoing description may be the oil phase mentioned in the disclosure 1, or another liquid immiscible with the dispersion, and the continuous phase may be selected according to the property of the sample to be tested.

In another preferred embodiment, the sample inlet cleaning solution provided in the foregoing disclosure can be used to clean the residue of the sample at the sample inlet, remove the non-specifically adsorbed analyte and other substances affecting the detection result, and avoid cross contamination.

Wherein optionally, the cleaning solution comprises a buffer system, a surfactant and a protein, wherein optionally, the buffer system can be but is not limited to phosphate, acetate, borate, Tris-HCl and the like; the protein may be, but is not limited to, bovine serum albumin, casein, and the like; the surfactant can be, but is not limited to, PEG, PVP, Tween 20, Tween 80, Triton X100, etc.; the pH range of the cleaning solution is 6-10.

In another preferred embodiment, the waste liquid channel described in the foregoing can be, but is not limited to, connected to the sample inlet of the dispersed phase, and the waste liquid channel can be used to discharge the excess sample or the cleaning solution for cleaning the sample inlet.

Optionally, the waste liquid channel may be, but is not limited to: micro-channels etched or otherwise formed within the material, micro-channels formed between different components, micro-channels fabricated using capillaries, and the like.

And optionally, the shape of the waste liquid channel can be, but is not limited to, a straight channel, a circular channel, a zigzag channel, and the like.

In another preferred embodiment, the incubation module described in the above can be used for mixing the droplets and performing sufficient reaction.

Optionally, the module may be, but not limited to, split into two modules, a blending module and an incubation module, but performs the same or similar functions as the incubation module described in summary 1.

Optionally, the module structure may be, but is not limited to, a straight channel, a circular channel, a zigzag channel, a storage cavity, or the like.

In another preferred embodiment, the signal reading and detecting module described in the foregoing is to detect the incubated liquid drop by using a sensor or a sensor system outside the chip. After the sample is subjected to micro-droplet generation, a reagent capable of detecting the target detection object is added into the droplet, and the reaction of the incubation module is completed, the signal reading and detecting module detects the signal of the target detection object by using the sensor or the sensor system.

Alternatively, the method of detecting the signal may be based on different target detection agents, including but not limited to nucleic acid-based detection methods, antibody and enzyme based detection methods, molecular beacons, sensors for detecting fluorescence, immunostaining assays, sandwich assays, small molecule based chemical detection methods, and the like, as well as combinations thereof.

And optionally, the nucleic acid-based detection methods include, but are not limited to, PCR, PT-PCR, loop-mediated isothermal amplification techniques, strand displacement, and the like.

And optionally, the antibody-based detection methods include, but are not limited to, ELISA, sandwich-based assays, immunostaining assays, antibody capture assays, and the like.

Optionally, the signal of the target detection object includes, but is not limited to, an electrical signal, mass, turbidity (absorbance), a magnetic signal, fluorescence, quantum dots, chemiluminescence, fluorescent microspheres, and the like

In another preferred example, the signal acquisition module described in the foregoing can acquire and read signals in the flow of the droplets, or acquire and read signals after the droplets are stored or stay.

Optionally, the signal acquisition in the droplet flow means that a valid signal in the droplet is acquired when the droplet flows through the sensor or the sensor system.

And optionally, the channel flowing through the sensor may be, but is not limited to being, a channel of: micro-channels etched or otherwise formed within the material, micro-channels formed between different components, micro-channels fabricated using capillaries, and the like.

And optionally, the shape of the flow-through sensor channel may be, but is not limited to, a straight channel, an annular channel, a folded channel, a serpentine channel, etc.

Wherein optionally, the portion for holding or stopping the liquid drop can be a storage chamber or a storage channel or other components with similar functions. The storage chamber can be single-layer height, double-layer height and multi-layer height, and the storage channel can adopt a trapping design, can be directly controlled by a power source, or can realize the same function in other modes.

In another preferred example, the materials of the droplet microfluidic chip module described in the foregoing may be, but are not limited to, glass, silicon wafer, ceramic, plastic, paper, and rubber, and may be one or more combinations thereof.

The material itself can be selected, or the material can be doped, modified or modified.

And optionally, the droplet microfluidic chip made of the material is suitable for disposable use and also suitable for repeated use.

The micro-droplet microfluidic control system or device or method based test flow comprises:

step 1) adding a test sample and an oil phase which are used as dispersed phases into a fluid sample injection module, opening a valve at a dispersed phase sample injection port, and enabling the test sample and the oil phase to enter a micro-droplet generation module in sequence;

step 2) generating micro liquid drops of the test sample in a micro liquid drop generating module;

step 3), the micro liquid drops enter a reagent fusion module, and a test sample and a detection object capable of being specifically combined with the object to be detected are fused to start reaction;

step 4), allowing the micro-droplets to enter an incubation module, and allowing the object to be detected inside the micro-droplets to fully react with the detected object;

step 5) the micro liquid drops with complete internal reaction enter a signal reading and detecting module to preprocess the micro liquid drops, a sensor or a sensor system is used for reading a signal to be detected in the micro liquid drops, and the signal to be detected is synchronously transmitted to a computer for numerical processing to give visual information;

step 6) adding cleaning liquid at the inlet of the dispersed phase under the state that the valve of the dispersed phase is closed in step 3), wherein the cleaning liquid flows out from the waste liquid channel and can start a second test, and the step can be repeated;

step 7), collecting all waste liquid by the waste liquid pool module in the test process;

and 8) monitoring and controlling the computer software in the whole testing process.

In another preferred embodiment, the volume of the test sample volume in step 1) of the aforementioned test procedure is 0.5-10. mu.L, preferably 0.5-5. mu.L.

In another preferred example, in the aforementioned testing process, the testing time of a single sample is 3-10min, but the testing process can be performed simultaneously for multiple samples, so that the overall sample testing time is greatly reduced.

The main advantages of the invention include:

1) the method can realize high-sensitivity detection on the biological sample, and has the advantages of good repeatability, strong anti-interference capability, short detection time, large flux, extremely low sample consumption and the like;

2) the liquid drop micro-fluidic chip technology is adopted, sample generation, mixing, reaction, collection and detection are integrated on the chip, all reagent components required by the reaction can be accurately controlled and added into the chip through a valve, the operation is simple, and a test sample or reagent in the chip is not in direct contact with the chip and an instrument, so that the instrument is not polluted, and cross contamination and serious interference are not generated.

3) The micro-fluidic chip can be recycled and can also be used for one time.

4) The chip of the invention has higher function integration degree, and simultaneously realizes the functions of generation, fusion, extraction, incubation, division, signal detection and the like on the same chip.

5) The chip has the advantages of high flux, high accuracy, high sensitivity and the like, and can be repeatedly used.

6) The chip can also controllably add reagents on line, so that the simultaneous detection of multiple detection objects can be realized on one chip.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: a Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989) or Roitt et al, Immunology (Immunology, 6)^thedition), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1

BNP detection in human blood samples by chemical light-emitting method

In this example, the microfluidic chip shown in fig. 1 was used to perform BNP detection on human blood samples by using a chemical light emission method.

First, experiment method

1. Pre-treating blood sample and reagent, that is, taking human blood samples A and B, and adding EDTA-anticoagulant blood collecting tube to prevent blood coagulation.

Treatment of BNP agents: the BNP antibody is coated with magnetic beads and the BNP antibody is di-labeled with azetidine esters.

3. Preparation of chemiluminescent substrate: equal volume of 0.1M HNO₃，0.1％H₂O₂The luminous excitation liquid A, 0.25M NaOH and 2% Tween-20 luminous excitation liquid B are mixed to form a chemiluminescent excitation substance.

4. Sample introduction: a sample A is added into a disperse phase sample inlet 6 shown in figure 1, continuous phase mineral oil is added into a continuous phase sample inlet 1 shown in figure 1, control valves 2 and 5 are closed at the moment, a control valve 7 is opened, negative pressure is given to a sample outlet 28 and a waste liquid port 4 shown in figure 1 as a power source, and the design of the sample inlet is shown in figure 2.

5. Droplet generation: the droplet generation process is as shown in fig. 4A, the mineral oil and the sample flow along the designed channel under the driving of the external force, at the droplet generation interface 8, the sample forms micro droplets under the shearing action formed by the dispersed phase at the generation interface, and flows forward along the channel under the entrainment of the mobile phase.

6. Cleaning a sample inlet: and after the micro-droplets are formed, closing the control valve 7 and adding a sample inlet cleaning solution. And opening the control valve 5, cleaning the sample inlet and quickly discharging waste liquid. After the sample inlet is cleaned, the control valve 5 is closed, cleaning liquid is added again, the control valve 7 is opened, the sample flow channel is cleaned, the cleaning liquid can form liquid drops at the position 8, the liquid drops are identified by the sensor and the control system, reagents cannot be added or other treatments cannot be carried out, and a waste liquid drop queue is directly formed and is discharged through the sample outlet 28. And adding the sample B to start the sample introduction of a new sample after the sample introduction module is cleaned.

7. Drop counting and identification: the generated droplets are subjected to detection of identification and droplet number by the sensor 9, so as to determine whether the generated droplets are valid sample droplets, and subsequent reagent addition is guided according to the identification and the number. The following steps are performed for valid droplets (samples), and if invalid droplets (waste liquid) are detected, the invalid droplets are discharged from the outlet 28 directly following the droplet queue.

8. Droplet fusion: the sensor 10, the reagent fusion port 11, the reagent fusion sample inlets 12 and 13, the control valve 14 and the pipeline connected with the control valve form a reagent fusion module together. When multiple markers in a sample are detected simultaneously, a reagent fusion module can be added to add corresponding reagents as shown in the figure. In this embodiment, a first antibody coated with magnetic beads is added to the sample inlet 12, a second antibody labeled with azin ester is added to the sample inlet 13, and under the response feedback of the sensor 10, the two antibodies are controlled by the control valve 14 to generate droplets, which are fused with the droplets coated with the sample one to one. The fusion process is shown in fig. 4B, followed by forward flow to the incubation module.

9. Incubation of the samples: the incubation module consists of an incubation pipe 15 and an external temperature controller, and the module enables liquid in the liquid drop to be fully mixed and complete reaction at a proper temperature through the turbulent motion of the liquid drop. The course of the reaction is shown in fig. 4C as C1, and for this experiment, it means that BNP is fully reactive with antibody one and antibody two. And the liquid drops after full reaction enter a liquid drop treatment module.

10. Pretreatment of liquid drops: the droplet processing module is divided into a droplet processing chamber and an input of droplet cleaning/treating agents. The liquid drop treatment chamber is composed of a cavity 18 with a distributed columnar structure and a magnetic field 19 for wrapping the cavity, and the whole cavity is positioned in the magnetic field 19. The input of the droplet cleaner/treatment agent is constituted by the inlet 16 and the valve 17 and the associated conduits, which can be increased in number as required. In this example, a cleaning agent input and a luminescent substrate liquid input are added. The droplet moves to the droplet processing chamber, the columnar structure can slow down the droplet, the magnetic beads in the droplet are subjected to the magnetic field, and the substance with the magnetic beads is retained in the droplet processing chamber, as shown in fig. 4D. At this time, the control valve 17 is opened, the cleaning solution is introduced under the pressure starting, and micro-droplets of the cleaning solution are generated at 12, and the micro-droplets of the cleaning solution wash away the unreacted aztine ester labeled BNP antibody II and other unbound substances in the chamber, or substances which are subjected to nonspecific binding. Subsequently, a shearing solution containing a treating agent is introduced into the same structure, and the treating agent can separate the magnetic beads from the antibodies to form new droplets containing the target analyte (i.e., the shearing solution after the shearing reaction), as shown in fig. 4E.

11. Post-treatment of the droplets: the droplet is detected by the sensor 20, information on the droplet is captured, and whether or not the droplet is an effective droplet (containing an antigen-antibody sandwich complex formed by the target analyte) is determined. If the droplet is a valid droplet, chemiluminescent substrate is added through the addition port 24 and control valve 23. If the droplets are waste liquid, the droplets are discharged from the outlet 28 following the droplet train without performing the following steps.

12. Reading of droplet signals: the signal reading detection module comprises a columnar structure area 26 and a sensor 27 for detecting signals. After the module reads the chemiluminescence signal, the droplet enters the outlet 28 and is discharged.

13. Discharging waste liquid: the sample outlet 28 is connected to the waste reservoir module. For collecting drops and other waste fluids that have been tested.

Secondly, the result is:

through the steps. The microfluidic chip realizes the chemiluminescence detection of BNP, and the detection speed, the detection result, the amount of the required antibody and the current mainstream detection method are shown in the following table 1:

TABLE 1

As can be seen from table 1, the detection method based on the microfluidic chip of the present invention is equivalent to the current detection method in terms of detection effect, but the method of the present invention has very significant advantages in terms of detection repeatability and antibody dosage. In particular, for a single test, the amount of antibody required for the method of the invention is greatly reduced, only 0.45-2.8pg, which is about 1/100-1/1000 of the amount used in conventional methods.

In addition, the micro-fluidic chip can directly complete the cleaning operation, thereby reducing additional manual operation, improving the integration level of the whole detection process, simplifying the operation of the detection process, and greatly improving the detection precision through the extraction process of the method.

Example 2:

detection of type B Natriuretic Peptide (BNP) by double antibody sandwich method

In this example, the microfluidic chip shown in fig. 1 was used to perform BNP detection on human blood samples by a double antibody sandwich method.

First, experiment method

1. As in example 1, first, pretreatment of the blood sample and the reagent was performed: a human blood sample A was taken in an EDTA anticoagulation tube to prevent coagulation.

2. Two antibodies for the double antibody sandwich method were treated: the fluorescent labeling antibody I and the magnetic bead coupling antibody II.

3. Sample introduction: a sample A is added into a disperse phase sample inlet 6 shown in figure 1, continuous phase mineral oil is added into a continuous phase sample inlet 1 shown in figure 1, a control valve 5 is closed at the moment, a control valve 7 is opened, negative pressure is given to a sample outlet 28 and a waste liquid port 4 shown in figure 1 to serve as a power source, and the design of the sample inlet is shown in figure 2.

4. Droplet generation: the droplet generation process as shown in fig. 4A, mineral oil and sample form micro droplets at the droplet generation interface 8 under the driving force.

5. Cleaning a sample inlet: and after the micro-droplets are formed, closing the control valve 7 and adding a sample inlet cleaning solution. And opening the control valve 5, cleaning the sample inlet and quickly discharging waste liquid. After the sample inlet is cleaned, the control valve 5 is closed, cleaning liquid is added again, the control valve 7 is opened, the sample flow channel is cleaned, the cleaning liquid can form liquid drops at the position 8, the liquid drops are identified by the sensor and the control system, reagents cannot be added or other treatments cannot be carried out, and a waste liquid drop queue is directly formed and is discharged through the sample outlet 28. And adding the sample B to start the sample introduction of a new sample after the sample introduction module is cleaned.

6. Drop counting and identification: the generated droplets are subjected to detection of identification and droplet number by the sensor 9, so as to determine whether the generated droplets are valid sample droplets, and subsequent reagent addition is guided according to the identification and the number. The following steps are performed for valid droplets (sample), and if invalid droplets (waste liquid) are detected, the invalid droplets are discharged directly from the outlet 28 following the droplet queue.

7. Droplet fusion: the sensor 10, the reagent fusion port 11, the reagent fusion sample inlets 12 and 13, the control valve 14 and the pipeline connected with the control valve form a reagent fusion module together. When multiple markers in a sample are detected simultaneously, a reagent fusion module can be added to add corresponding reagents as shown in the figure. In this embodiment, a first antibody coated with magnetic beads is added into the sample inlet 12, a second antibody labeled with fluorescence is added into the sample inlet 13, and under the response and feedback of the sensor 10, the two antibodies are controlled by the control valve 14 to generate droplets, and the droplets coated with the sample are fused one-to-one, and the fusion process is as shown in fig. 4B, and then the droplets flow forward to the incubation module.

8. Sample incubation: the incubation module consists of an incubation pipe 15 and an external temperature controller, and the module enables liquid in the liquid drop to be fully mixed and complete reaction at a proper temperature through the turbulent motion of the liquid drop. The process is shown in figure 4C and for this experiment refers to a sufficient reaction of BNP with antibody one and antibody two. And the liquid drops after full reaction enter a liquid drop treatment module.

9. Liquid drop treatment: the droplet processing module is divided into a droplet processing chamber and an input of droplet cleaning/treating agents. The liquid drop treatment chamber is composed of a cavity 18 with a distributed columnar structure and a magnetic field 19 for wrapping the cavity, and the whole cavity is positioned in the magnetic field 19. The input of the cleaning liquid is composed of a sample inlet 16, a valve 17 and a cleaning liquid droplet generating port 12, and the number of the structures can be increased as required. The liquid drop moves to the liquid drop processing chamber, the columnar structure can slow down the speed of the liquid drop, magnetic beads in the liquid drop are subjected to the action of a magnetic field, substances with the magnetic beads are reserved in the liquid drop processing chamber, and the micro liquid drop is cleaned to wash away the unreacted fluorescently-labeled BNP antibody II and other unbound substances in the chamber, as shown in FIG. 4D. Subsequently, a treating agent is introduced into the same structure, and the treating agent can cut off the connection between the magnetic beads and the first antibody to form a new droplet containing the target analyte, as shown in FIG. 4E.

10. Signal reading-the signal reading detection module comprises a columnar structure area 26 and a sensor 27 for detecting signals. After the excitation light with the proper wavelength of the module excites the fluorescent substance to emit fluorescence, and the light-excited fluorescence sensor finishes signal reading, liquid drops enter the sample outlet 28 to be discharged.

11. Discharging waste liquid: the sample outlet 28 is connected to the waste reservoir module. For collecting drops and other waste fluids that have been tested.

Secondly, the result is:

there are several fluorescence detection methods for BNP, such as magnetic bead + fluorescence, lateral flow chromatography, etc., and the comparison of the detection effect of the chip is shown in table 2:

TABLE 2

In the table, it can be seen that the detection method based on the microfluidic chip of the present invention has comparable or similar effect to the current detection method in terms of detection effect, but the method of the present invention has significant advantages in terms of single detection duration and detection throughput. In addition, since it is a droplet homogeneous reaction, it is excellent in the stability of detection, and the amount of antibody required for a single detection is greatly reduced. In particular, for a single test, the amount of antibody required for the method of the invention is greatly reduced, only 0.45-2.8pg, which is about 1/100-1/1000 of the amount used in conventional methods. The micro-droplet chip and the detection method based on the micro-droplet chip can directly finish the cleaning operation, reduce additional manual operation, improve the integration level of the whole detection process, simplify the operation of the detection process, and greatly improve the detection precision through the extraction process of the method.

Example 3

Preparation of standard curve for detecting B-type natriuretic peptide (BNP) by double antibody sandwich method

In this example, for the detection of type B Natriuretic Peptide (BNP) by the double antibody sandwich method based on immune response, the results in different extraction processes are compared and a standard curve is prepared. Further, based on the detection results, the cleaning effects are compared.

The method comprises the following steps: preparing a series of BNP calibrators with concentration; adopting the same calibrator, and cleaning one time of micro-droplets in the extraction process (a) as the sample droplets; and (b) respectively measuring the number of the cleaning micro-droplets in the extraction process which is twice that of the sample droplets, comparing the results of different extraction processes, and making a standard curve.

The results of the comparison are shown in FIG. 6, FIG. 7, and Table 3.

Fig. 6 is a picture obtained after injecting BNP solutions of different concentrations into the chip and completing an experiment, wherein the three left pictures are original result pictures obtained without performing extraction cleaning, once performing extraction cleaning (twice cleaning micro-droplets) and twice performing extraction cleaning (twice cleaning micro-droplets) after adding the same reagent into a BNP antigen of 400pg/mL for reaction. The right three pictures are the result pictures obtained after the BNP solution of 0pg/mL is added with the same reagent for reaction, and the BNP solution is not washed, is extracted and washed once and is extracted and washed twice.

Table 3 shows the corresponding data obtained after the pictures are processed by the image processing software.

TABLE 3

Fig. 7 is a visual analysis result obtained by using the above data.

From FIG. 6, FIG. 7 and Table 3, in the case of non-extraction, the difference between the BNP detection results at concentrations of 400pg/mL and 0pg/mL was very small, and the target analyte (BNP) could not be detected. Under the condition of one-time extraction, the difference between the BNP detection results of 400pg/mL and 0pg/mL is obvious and can be obviously distinguished, and the difference between the two-time extraction results is larger than that between the two-time extraction results, so that higher sensitivity can be achieved. It can therefore be seen that reliable detection results cannot be obtained without extraction, and that a higher sensitivity can be obtained with two extractions.

In FIG. 8, the comparative detection of different degrees of washing (different numbers of washing micro-droplets) during the extraction process was performed using the calibrator, and the results of the comparison were compared in both cases, and it was found that the signal detection result was slightly higher when the number of washing micro-droplets was small (one time that of the sample droplets) than when the number of washing micro-droplets was large (two times that of the sample droplets), but R was lower (one time that of the sample droplets) when the number of washing micro-droplets after curve fitting was small (one time that of the sample droplets)²Much smaller than R when the number of washing micro-droplets is larger (twice that of the sample droplet)²It is shown that the degree of washing directly affects the detection accuracy, and the higher the removal degree of the substance interfering with the signal detection, the higher the detection sensitivity. Although the extraction times are not more and better, the process is indispensable, and the chip can flexibly control the extraction times according to the needs to achieve the optimal signal intensity and signal to noise ratio. Thereby improving the detection sensitivity, repeatability and detection accuracy of the sample to be detected.

Discussion of the related Art

In the prior art, the micro-fluidic chip based on micro-droplets is often complex in operation and insufficient in accuracy. For example, in conventional methods, the substances to be reacted (e.g., addition of IDE libraries, target molecules, and fluorescent enzyme substrates) are typically formed into droplets, which are then collected after all incubation and sorted (e.g., by FACS sorting out the desired droplets, e.g., fluorescent droplets containing aptamers), followed by fragmentation, dilution, and partition repackaging in an off-chip (e.g., EP tube). Then, droplets containing the reactants (such as the selected aptamer, the target molecule and the fluorescent enzyme substrate) are generated again on another chip, and then the droplets are incubated and collected for further detection. The whole process is complicated, the required function integration can not be completed on a single chip, additional manual operation is required to be carried out outside the chip, and the automation degree is low.

Accordingly, many conventional methods for biological detection diagnosis include immunoblotting, chemiluminescence immunoassay, and enzyme-linked immunosorbent assay (ELISA), among the existing in vitro diagnostic methods. These detection-based methods require relatively long analysis times, relatively low throughput, inadequate accuracy, and large equipment. In the droplet chip of the present invention, on one hand, a new extraction module is provided, and other functional modules are integrated, so that the extraction process described above can be performed on the generated droplets on the same chip, substances that have non-specific binding or interfere with detection are removed, only substances for signal detection remain in the droplets, and the whole process is more integrated and automated. In addition, the generation of sample liquid drops, the addition and incubation of required reagents, the cleaning and extraction processes and the final detection process can be realized on the same chip, no additional operation outside the chip is needed, the whole process is a liquid phase reaction, the full reaction of a reaction system is facilitated, and the required reagent dosage is very small, so that the microfluidic chip analysis and detection with the advantages of high flux, high accuracy, high sensitivity, low cost and the like is realized.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (30)

1. A microfluidic chip, comprising:

(a) a microfluidic flow-through channel for the flow of the continuous phase and microdroplets carried therein;

(b) a reaction microdroplet generation module for generating microdroplets for reaction, the reaction microdroplets comprising: the kit comprises a sample to be detected and a reaction reagent which reacts with the sample to be detected, wherein a substance to be detected in the sample to be detected reacts with the reaction reagent to form a detection product carrying a detectable marker;

(c) the cleaning micro-droplet generation module is used for generating cleaning liquid micro-droplets for cleaning;

(d) the shearing micro-droplet generation module is used for generating shearing liquid micro-droplets for shearing;

(e) a capture-wash-shear module, also referred to as an extraction module, for capturing the detectable label-bearing detection product from the incubated reaction microdroplets and washing the captured detectable label-bearing detection product with the wash solution microdroplets; shearing the washed detection product carrying the detectable marker by using the sheared micro-droplets so as to generate micro-droplets for detection;

(f) the signal reading and detecting module is used for reading the signal of the micro liquid drop for detection; and

(g) the liquid drop queue control module is connected with the cleaning micro liquid drop generation module and the shearing micro liquid drop generation module and is used for controlling the cleaning micro liquid drop generation module and the shearing micro liquid drop generation module to work after the reaction micro liquid drops move to a preset position, so that a micro liquid drop queue of 'reaction micro liquid drops, cleaning micro liquid drops and shearing micro liquid drops' is formed in the microfluid flow channel in the liquid flow direction, wherein the reaction micro liquid drops are positioned at the forefront of the flow.

2. The microfluidic chip of claim 1, wherein said microfluidic flow channel is a capillary channel.

3. The microfluidic chip of claim 1, wherein said microfluidic flow channel is a microchannel having a depth of 10 to 1000 μm and/or a width of 10 μm to 1000 μm.

4. The microfluidic chip of claim 1, wherein the reaction micro-droplet generation module further comprises a droplet count identification submodule for counting and numbering reaction micro-droplets.

5. The microfluidic chip according to claim 1, wherein the reaction microdroplet generation module further comprises a microdroplet fusion submodule, wherein a sample microdroplet and reaction reagent microdroplets are generated in the reaction microdroplet generation module, and the reaction reagent microdroplets and the sample microdroplets are fused into the reaction microdroplet through the microdroplet fusion submodule.

6. The microfluidic chip of claim 1, wherein the reaction microdroplet generation module further comprises a valve assembly for controlling the generation of the reaction reagent microdroplets and the sample microdroplets or for controlling the generation of the reaction microdroplets.

7. The microfluidic chip according to claim 1, wherein the microfluidic chip further comprises a dispersed phase sample injection submodule, the dispersed phase sample injection submodule comprising a dispersed phase sample injection port, a waste liquid outlet and two valves; the dispersed phase sample inlet is used for adding a sample, the valve and the waste liquid outlet are used for rapidly cleaning the dispersed phase sample inlet module, and the waste liquid outlet is connected with the waste liquid collecting module.

8. The microfluidic chip of claim 1, wherein the reaction microdroplet generation module, the cleaning microdroplet generation module, and the shearing microdroplet generation module have a T-junction structure, a cross-shaped droplet generation structure, or a combination thereof, and the structures are configured to generate microdroplets.

9. The microfluidic chip of claim 1, wherein the reaction microdroplet generation module, the cleaning microdroplet generation module, and the shearing microdroplet generation module have a flow confocal structure, a coaxial flow structure, or a combination thereof, and the structures are configured to generate microdroplets.

10. The microfluidic chip of claim 1, wherein said signal is selected from the group consisting of: a chemiluminescent signal, a light signal emitted by excitation of a fluorophore, an absorbance change signal, a magnetic field strength signal, or a combination thereof.

11. The microfluidic chip according to claim 1, wherein the signal is a visible light signal emitted by the quantum dot microsphere after being excited by light.

12. The microfluidic chip of claim 1, wherein said microfluidic flow channel comprises the following sections: a continuous phase sample injection section, a detection sample injection section, a reaction reagent addition section, an incubation reaction section, a cleaning micro-droplet addition section, a shearing micro-droplet addition section, a capturing-cleaning-shearing treatment section, a signal detection section and an optional waste liquid channel section.

13. The microfluidic chip according to claim 1, wherein the capture-wash-shear module further comprises a magnetic field region, and the magnetic field region is a magnetic field region that can be controlled to be turned on or off.

14. The microfluidic chip of claim 1, wherein said cleaning micro-droplet generation module and said shearing micro-droplet generation module further comprise: a control valve assembly controlled by a drop train control module.

15. The microfluidic chip according to claim 1, wherein said control valve assembly is selected from the group consisting of: an electromagnetic valve, a paraffin valve, a magnet moving valve, a pneumatic valve, a diaphragm valve, or a combination thereof.

16. The microfluidic chip according to claim 1, wherein the control valve assembly is a paraffin hot melt valve.

17. The microfluidic chip of claim 1, wherein the control valve assembly is a hydrophobic valve.

18. The microfluidic chip of claim 1, wherein the control valve assembly is a mechanical valve.

19. The microfluidic chip of claim 1, further comprising a driving force component, wherein the driving force component is a pressure driving component.

20. The microfluidic chip of claim 1, wherein said microfluidic chip further comprises: (i) an incubation control module for controlling incubation parameters of the microfluidic flow-through channel of the incubation section.

21. The microfluidic chip of claim 1, wherein said microfluidic chip further comprises: (j) the waste liquid collecting module is used for collecting waste liquid generated in the detection process.

22. The microfluidic chip according to claim 1, wherein the signal reading detection module further comprises a post-processing sub-module, wherein a substance for exciting a detection signal is added to the micro-droplets for detection in the post-processing sub-module.

23. The microfluidic chip of claim 22, wherein the post-processing sub-module is further provided with a sensor for identifying whether the microdroplets to be detected are valid, and a substance for generating a detection signal is added to the valid microdroplets to be detected in the post-processing sub-module.

24. The microfluidic chip according to claim 1, wherein the material of the microfluidic chip is selected from glass, silicon-based materials, polydimethylsiloxane, acrylic plastics, cyclic olefin copolymer materials, polypropylene plastics, polystyrene plastics, or a combination thereof.

25. The detection method based on the microfluidic chip as claimed in claim 1, comprising the following steps:

a. adding a mobile phase and filling the microfluidic flow channel with the mobile phase;

b. pretreating the reagent;

c. preparing a biological sample to be tested, said sample containing a target substance to be tested;

d. adding a sample and a pretreated reagent, generating a sample micro-droplet and a reagent micro-droplet through the reaction micro-droplet generation module, and fusing the sample micro-droplet and the reagent micro-droplet in the reaction micro-droplet generation module to form a reaction micro-droplet; or the sample and the pretreated reagent are mixed in advance to form reaction liquid, and the reaction liquid is added to generate reaction micro-droplets through the reaction micro-droplet generation module;

e. under the control of the liquid drop queue control module, a cleaning micro liquid drop generation module generates cleaning micro liquid drops;

f. under the control of the liquid drop queue control module, a shearing micro-liquid drop generation module generates shearing micro-liquid drops;

g. e, forming a micro-droplet queue by the micro-droplets generated in the step f and the reaction micro-droplets;

h. the micro liquid drop queue enters a capture-cleaning-shearing module and forms micro liquid drops for detection;

i. the micro liquid drops for detection pass through a signal reading and detecting module, and the reading and detecting module detects and reads signals.

26. The method for detecting a microfluidic chip according to claim 25, wherein in the step b, the size of the magnetic beads is 0.01 to 10 μm.

27. The method for detecting a microfluidic chip according to claim 26, wherein in the step b, the size of the magnetic beads is 1 to 2.8 μm.

28. The method for detecting a microfluidic chip according to claim 25, wherein the volume ratio of the sample micro-droplet to the reagent micro-droplet is 1:0.5 to 1: 2.

29. The method for detecting a microfluidic chip according to claim 25, wherein in the step b, the pretreatment of the reagent is coating or coupling magnetic beads.

30. The method for detecting a microfluidic chip according to claim 25, wherein the method is a detection method based on a double antibody sandwich method or a competitive binding method.

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