CN107983424B

CN107983424B - Liquid drop biological analysis chip and application and use method thereof

Info

Publication number: CN107983424B
Application number: CN201710996579.5A
Authority: CN
Inventors: 刘大渔; 舒博文
Original assignee: Guangzhou First Peoples Hospital
Current assignee: Guangzhou First Peoples Hospital
Priority date: 2017-10-19
Filing date: 2017-10-19
Publication date: 2021-03-12
Anticipated expiration: 2037-10-19
Also published as: CN107983424A

Abstract

The invention discloses a liquid drop biological analysis chip and an application and a using method thereof. The chip comprises a plurality of groups of parallel micro-pools connected in series, wherein each group of micro-pools connected in series consists of a plurality of micro-pool units which are connected in series through slits; part or all of the micro-cell units in the series micro-cells have an open structure; the bottom surfaces of the series microcells and the slits are of smooth thin-wall structures. The chip has the following advantages: the micro-pool adopts a full or partial open structure, so that the loading of reagents and samples is convenient; the volume of the liquid drop is flexible and is particularly suitable for biological samples with the volume of several microlitres to hundreds microlitres; non-contact operation can be adopted, and cross contamination risk is avoided; the parallel operation of certain biological analysis can be realized in a fully integrated mode, and the automation of an analysis instrument is convenient to realize. The chip can be used for extracting and analyzing biological samples such as protein, nucleic acid and the like.

Description

Liquid drop biological analysis chip and application and use method thereof

Technical Field

The invention belongs to the technical field of biomedical analysis, and particularly relates to a liquid drop bioanalysis chip and application thereof, and a use method of the liquid drop bioanalysis chip.

Background

Droplet analysis is a new and new technique for manipulating minute volumes of liquid that has been developed in recent years. Droplet analysis is mostly carried out on-chip, using two immiscible liquids, one as the continuous phase and the other as the dispersed phase. The dispersed phase is dispersed in the continuous phase in minute volumes to form droplets. Depending on the dispersed and continuous phases, the droplets are divided into two categories: W/O (water-in-oil) type droplets and O/W (oil-in-water) type droplets, in which the aqueous phase (water) generally refers to various aqueous solutions and the oil phase (oil) is an organic solvent insoluble with water.

In the W/O type droplet system, due to the wrapping and isolation of the oil phase, the reaction solution can be dispersed and formed into a relatively independent micro-reactor or reservoir, thereby having the following advantages: 1) the volume is very flexible, the reaction volume of the liquid drop can be reduced to nanoliter or even femtoliter magnitude, the method is particularly suitable for high-throughput reaction or reaction of limited source electrode of a sample, and meanwhile, the use of expensive reagents can be reduced, and the test cost is reduced; 2) the samples are not diffused and cross contamination among the samples is avoided, and because each sample solution is wrapped and surrounded by an immiscible oil phase, on one hand, sample molecules are retained in the aqueous solution to be beneficial to keeping the concentration of the samples, and on the other hand, the reaction solution is not in direct contact with the wall of the device, and the adjacent reaction solutions are isolated by the oil phase to be beneficial to avoiding substance exchange among the adjacent liquid drops; 3) the reaction condition is stable, the evaporation of water molecules in the solution is inhibited due to the surrounding of the oil phase, and the reaction condition in the liquid drop is hardly influenced by the outside.

For the developed droplet chip analysis systems, the homogeneity analysis mode, i.e. parallelization of the same type of analysis, is mostly adopted. Although this analysis mode has a great advantage in terms of throughput, the number of analysis samples is limited, and integrated analysis is difficult to achieve.

Disclosure of Invention

The invention provides a liquid drop biological analysis chip, which overcomes the defects and shortcomings of the prior art in the aspects of analysis flux, analysis speed, automation degree and reagent consumption.

Another object of the present invention is to provide an application of the droplet bioanalysis chip.

It is still another object of the present invention to provide a method for using the droplet bioanalysis chip.

The purpose of the invention is realized by the following technical scheme: a liquid drop biological analysis chip comprises a plurality of groups of parallel micro-pools connected in series, wherein each group of micro-pools connected in series consists of a plurality of micro-pool units which are connected in series through slits; part or all of the micro-cell units in the series micro-cells have an open structure; the bottom surfaces of the series microcells and the slits are of smooth thin-wall structures.

The open structure means that the top surface of the micro-cell unit is open or open, thereby facilitating the operation of sample loading and sampling.

The material of the drop bioanalysis chip is hydrophobic polymer material or surface hydrophobic treatment material.

The hydrophobic polymer material is Preferably Polypropylene (PP), Cyclic Olefin Copolymer (COC) or Polytetrafluoroethylene (PTFE).

The inner surfaces of the micro-cell unit and the slit are provided with hydrophobic coatings; so that the inner surfaces of the micro-pools and the slits have hydrophobicity.

The material of the hydrophobic coating is preferably Teflon AF 1600.

The length of the slit is 1-20 mm, and the width or height of the slit has at least one dimension of 0.1-1 mm; the length of the slit is preferably 3mm, and the width or height is preferably 0.5 mm.

The slit acts as a trap to prevent aqueous solutions from passing freely.

The slit is preferably a slit with a gradual transition structure of narrowing width, so as to induce the aggregation of magnetic particles and prevent the magnetic particles from being trapped.

The gradual transition structure is characterized in that an included angle between a forward tangent of the transition structure and the direction from the micro pool to the slit is an acute angle. That is, the width of one end of the slit connected to the preceding micro pool is wider than the width of the other end of the slit connected to the subsequent micro pool along the proceeding direction of the magnetic particles.

The smooth thin-wall structure refers to a flat surface with a flat surface without a sharp-pointed sunken apparent structure on the bottom surface, so that high-efficiency and smooth magnetic particle operation is facilitated, and magnetic particle interception loss is prevented.

The thickness of the bottom surface is 0.1-1 mm, preferably 0.25-1 mm, and more preferably 0.3 mm.

The area of the liquid drop biological analysis chip is preferably 10-600 square centimeters.

The application of the liquid drop biological analysis chip in biological sample analysis is particularly suitable for the application in high-flux biological sample analysis.

The biological sample analysis comprises extraction of biological molecules, and qualitative analysis and/or quantitative analysis.

The biomolecules are preferably proteins and nucleic acids.

The use method of the droplet biological analysis chip comprises the following steps:

(1) loading the liquid drop biological analysis chip on a chip platform, injecting an oil phase solution into a chip micro-pool, and automatically filling the micro-pool and the slit with the oil phase liquid;

(2) then respectively adding water phase solution containing samples or reagents into each micro-cell, wherein the water phase solution spontaneously forms water-in-oil droplets in the oil phase solution due to the surface tension effect;

(3) magnetic particles capable of combining with biomolecules are loaded in an aqueous phase solution, and the magnetic particles move under the action of magnetic field force after being combined with the biomolecules, can pass through an oil-water interface and a slit, and move among liquid drops to finish the processes of biomolecule extraction, qualitative analysis and/or quantitative analysis.

The principle of the invention is as follows: the chip micro-pool and the slit have hydrophobic surfaces, so that the oil phase automatically infiltrates the surfaces of the micro-pool and the slit when the oil phase solution is added into the chip micro-pool. To each micro-cell the corresponding reagent/or sample (aqueous solution) is added, and the added reagent/sample solution automatically forms water-in-oil droplets in the oil phase due to surface tension effects. The liquid drops in the communicated micro-pools are still kept in an independent isolation state under the external disturbance (such as inclination, vibration and heating), so that the stability of the component concentration of each liquid drop is ensured. As shown in fig. 1, after a magnetic field force in a certain direction is applied to the region where the micro-pool is located, the corresponding magnetic particles in the droplets in the micro-pool a are collected at the slit channel and separated from the mother liquid droplets, and continue to move along the slit channel under the action of the field force until the magnetic particles are merged with the droplets in the micro-pool B and then are fused, while the mother liquid droplets in the micro-pool a are still retained in the micro-pool a. Therefore, the process can complete the splitting, moving and fusing of the liquid drops. If capture molecules, such as nucleic acid sequences or antibodies, are labeled on the surface of the magnetic particles, the magnetic particles can bind to specific biomolecules and carry these biomolecules between a series of droplets. In a similar manner, the multi-step operations involved in biological analysis can be completed, and finally, the automated analysis of full-integrated batches of biological samples on a chip can be realized.

Compared with the prior art, the invention has the following advantages and effects:

(1) the generation and positioning of the droplets on the chip provided by the invention do not need to use a complex fluid control device;

(2) the liquid drop biochip provided by the invention has the advantages of simple manufacturing process and low cost;

(3) the liquid drop biochip provided by the invention has flexible and variable liquid drop volume, and is particularly suitable for biological samples with the volume of several microliters to hundred microliters;

(4) the liquid drop biochip provided by the invention adopts non-contact operation, and has no cross contamination risk;

(5) the liquid drop biochip chip provided by the invention can realize parallel operation of certain bioanalysis in a fully integrated mode, and is convenient for realizing automatic high-throughput analysis.

In conclusion, the analysis method of the liquid drop biochip provided by the invention has the advantages of simple operation, rapid analysis, high automation degree and higher test flux.

Drawings

FIG. 1 is a schematic diagram of a droplet bioanalysis chip according to the present invention.

FIGS. 2.1-2.3 correspond to example 1.

FIG. 2.1 is a schematic diagram of the structural functional region of the droplet bioanalysis chip in example 1.

FIG. 2.2 is a schematic view showing the sample loading in use of the droplet bioanalytical chip in example 1.

FIG. 2.3 is a schematic diagram showing the operation of the droplet bioanalysis chip of example 1.

FIGS. 3.1-3.2 correspond to example 2.

FIG. 3.1 is a schematic diagram of the structural functional region of the droplet bioanalysis chip in example 2.

FIG. 3.2 is a schematic view showing the sample loading in use of the droplet bioanalytical chip in example 2.

FIG. 4 is a schematic diagram of the structural functional regions of the droplet bioanalysis chip of example 3.

FIGS. 5.1-5.2 correspond to example 5.

FIG. 5.1 is a perspective view of a droplet bioanalysis chip of example 5.

Fig. 5.2 is an enlarged view of a portion of the micro-wells and slits of fig. 5.1.

Wherein, 1 is a chip substrate, 2 is a micro-pool, 3 is a slit, 4 is an oil phase, 5 is a water phase, 6 is magnetic particles, 7 is a magnetic field, 8 is a magnet, and 9 is a sealing bottom layer.

1-1 is nucleic acid extract, 1-2 is sample, 1-3 is ethanol-free washing solution, 1-4 is amplification reaction solution, and 1-5 is enzyme solution.

2-1 is a sample, 2-2 is a lysis solution, 2-3 is mineral oil, 2-4 is a binding solution, 2-5 is a magnetic particle suspension, 2-6 is an ethanol-free washing solution, 2-7 is an eluent, and 2-8 is a reaction premix.

A. B, C, D, E are for convenience of description to distinguish the chip regions.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1

As shown in fig. 2.1, a droplet bioanalysis chip integrating RNA extraction, isothermal amplification and detection comprises 15 sets of serial microcells, each set of serial microcells consisting of 5 microcells; the micro-pools of the same group are communicated through a slit.

Specifically, the chip is made of Polydimethylsiloxane (PDMS) material. The chip is composed of 15 groups of micro-pools connected in series, the width and the depth of each micro-pool are 3.2mm, the thickness of the bottom surface of each micro-pool is 0.25mm, and the size of each slit is 3mm (length) multiplied by 0.5mm (width) multiplied by 3.2mm (height). The micro-wells and slit surfaces were treated with a Teflon AF1600 hydrophobic coating. RNA extraction, RNA isothermal amplification and detection are integrated on the chip, the chip is placed on a chip platform, and the basic operation flow is as follows:

(1) oil phase loading: sequentially adding 25 muL, 20 muL, 15 muL and 10 muL of paraffin oil into each micro-pool of the chip A-E area, and ensuring that slits among the micro-pools are soaked by the paraffin oil;

(2) reagent loading: sequentially adding 25 μ L of nucleic acid extract, 100 μ L of washing solution, 16 μ L of amplification reaction solution and 4 μ L of enzyme solution into each micro-cell in the A-E region of the chip to respectively form independent water-in-oil type droplets; the nucleic acid extracting solution comprises a magnetic particle suspension modified with an olig-dT capture probe, the washing solution is an ethanol-free washing solution, the reaction solution comprises a primer, dNTP, a fluorescent probe and a reaction buffer solution, and the enzyme solution comprises M-MLV reverse transcriptase, T7RNA polymerase and an enzyme preservation solution;

(3) sample loading: respectively adding 100 mu L of samples into the nucleic acid extracting solution drops in each micro-pool in the area A of the chip, wherein the samples are samples to be detected, positive controls, negative controls or internal standards; the loading of the droplets in each micro-well of the chip is shown in FIG. 2.2;

(4) release of RNA and binding to magnetic particles: oscillating the chip for 30 seconds in a reciprocating manner to fully mix the sample and the nucleic acid extracting solution, then keeping the micro pool in the area A of the chip at 60 ℃ for 5 minutes, restoring the temperature of the micro pool in the area A of the chip to room temperature, and standing for 3 minutes;

(5) RNA washing and purification: as shown in FIG. 2.3, a magnetic field is applied to the chip A-area micro-pool to drive the magnetic particles to move along the slit to the B-area micro-pool until the magnetic particles enter the liquid drops in the B-area micro-pool, the linear velocity of the movement is 1mm/s, and in the process, the magnetic particles carry RNA to be separated from the liquid drops of the nucleic acid extracting solution, and then the RNA reaches the B-area micro-pool through the slit and is fused with the liquid drops of the washing solution in the B-area micro-pool. Oscillating the chip to fully mix the magnetic particles with the washing solution; applying a magnetic field to the micro pool in the area B of the chip to pull magnetic particles to move to the micro pool in the area C along the slit, wherein the linear speed of the movement is 1mm/s until the magnetic particles are fused with the washing liquid drops in the micro pool in the area C; oscillating the chip to fully mix the magnetic particles with the washing solution;

(6) RNA elution and primer-template annealing: applying a magnetic field to the micro pool in the area C of the chip to pull magnetic particles to move to the micro pool in the area D along the slit with the linear speed of 1mm/s until the magnetic particles are fused with the droplets of the amplification reaction liquid in the micro pool in the area D, then oscillating the chip to fully mix the magnetic particles with the washing liquid and maintaining the temperature of the micro pool in the area D of the chip at 60 ℃ for 5 minutes to enable the eluted RNA template to be combined with the primers in the amplification reaction liquid in an annealing way;

(7) RNA isothermal amplification: keeping the temperature of a micro-pool in the area E of the chip at 42 ℃, driving magnetic particles to drive the liquid drops in the area D to move close to the liquid drops of the enzyme solution in the area E, wherein the linear velocity of the movement is 2 mm/s; when the two liquid drops are contacted, the magnetic particles are driven to reciprocate between the D, E-area micro-pools for 30 seconds, the linear velocity of the motion is 10mm/s, the fusion of the two liquid drops is promoted, and the liquid drops of the amplification reaction liquid and the enzyme liquid are fully mixed. The temperature of the zone E microcell was maintained at 42 ℃ for 40 minutes.

Example 2

As shown in fig. 3.1, a droplet bioanalysis chip integrating DNA extraction, loop-mediated isothermal amplification (LAMP) and real-time fluorescence detection comprises 15 sets of serial microcells, each set of serial microcells consisting of 5 microcells; the micro-pools of the same group are communicated through a slit; the micro-cells have an open structure in whole or in part.

Specifically, the chip is made of polytetrafluoroethylene. The droplet analysis chip had a size of 85.5mm (length) × 54mm (width) and a thickness of 4.2 mm. The chip is composed of 15 groups of micro pools connected in series, the depths of the micro pools and the slits are both 3.2mm, the sizes of the slits are 3mm (length) multiplied by 0.5mm (width) multiplied by 3.2mm (height), and the thickness of the bottom layer of the micro pool is 1 mm. DNA extraction, LAMP constant temperature amplification and detection are integrated on the chip, the chip is placed on a chip platform, and the basic operation flow is as follows:

(2) reagent loading: the following reagents or samples, zone a, were added to each microwell of the chip in sequence: 50 mu L of lysis binding buffer solution and 2 mu L of magnetic particle suspension to form a nucleic acid extracting solution droplet; and a B region: 100 μ L of washing solution; and a C region: 100 μ L of washing solution; and (3) region D: 16 μ L of eluate; and a region E: 4 mu L of 5 times LAMP reaction premixed solution to respectively form independent water-in-oil type droplets;

(3) sample loading: and respectively adding 20 mu L of samples into the nucleic acid extracting solution drops in each micro-pool in the area A of the chip, wherein the samples are samples to be detected and sample lysates of positive control or negative control. The loading of the droplets in each micro-well of the chip is now shown in fig. 3.2.

(4) Release of DNA and binding to magnetic particles: oscillating the chip for 30 seconds to fully and uniformly mix the sample and the lysis binding buffer solution, then maintaining the microcell in the area A of the chip at 56 ℃ for 6 minutes, and controlling the chip to move back and forth in the process to promote the magnetic particles to oscillate in the liquid drops in the area A so as to promote mixing;

(5) DNA washing: and applying a magnetic field to the micro pool in the area A of the chip to pull the magnetic particles to move to the micro pool in the area B along the slit until the magnetic particles enter washing liquid drops in the micro pool in the area B (the linear speed is 2mm/s), separating the DNA carried by the magnetic particles from the cracking-combined liquid drops in the process, and then reaching the micro pool in the area B through the slit and fusing with the washing liquid drops in the micro pool in the area B. The chip is shaken to fully mix the magnetic particles with the washing solution. And applying a magnetic field to the micro pool in the area B of the chip to pull the magnetic particles to move to the micro pool in the area C along the slit (the linear speed is 2mm/s) until the magnetic particles are fused with the washing liquid drops in the micro pool in the area C. Oscillating the chip to fully mix the magnetic particles with the washing solution;

(6) DNA elution: and applying a magnetic field to the micro pool in the area C of the chip to pull the magnetic particles to move to the micro pool in the area D along the slit until the magnetic particles enter the eluent in the micro pool in the area D (the linear velocity is 1mm/s), and slightly oscillating the chip to ensure that the DNA on the magnetic particles is fully decomposed and dissociated into the eluent. On the chip structure design, a slit-free structure is arranged between the D area and the E area, and the D/E area liquid drops can move along with the magnetic beads under certain conditions, so that one liquid drop can be dragged to approach to the other liquid drop by the magnetic beads until fusion. Unlike example 1, since the magnetic beads need to be removed in the DNA amplification reaction, the magnetic beads need to be separated and removed immediately after the DNA eluate is fused with the amplification reaction solution. Therefore, in the operation process, the magnetic beads combined with the DNA firstly enter the D-area eluent liquid drops, the chip is oscillated to ensure that the DNA on the magnetic particles is fully decomposed and dissociated into the eluent, then the D-area eluent liquid drops are dragged by the magnetic particles to be close to and fused with the E-area amplification premix liquid drops, the magnetic beads are immediately and rapidly dragged to the C-area to be separated from the amplification reaction liquid, and the amplification reaction liquid is retained in the E-area to execute DNA amplification.

(7) Isothermal amplification and real-time fluorescence detection of LAMP: and applying a magnetic field to the micro pool in the D area of the chip to pull the magnetic particles to move towards the micro pool in the E area until the magnetic particles enter the micro pool in the E area and the droplets of the amplification premix liquid are fused (linear velocity is 1mm/s), and then rapidly driving the magnetic particles to move in the reverse direction (linear velocity is 5mm/s) to separate from the amplification reaction liquid and reach the C area through a slit in the D area-C area. And (3) fully mixing the oscillation chip eluent with the LAMP amplification premixed solution, and maintaining the temperature of the micro-pool in the D-E area of the chip at 65 ℃ for 50 minutes. During the period, the fluorescence signals of the liquid drops in each micro-pool in the E area are collected at certain time intervals, so that a curve of the change of the fluorescence intensity of the liquid drops along with the amplification time, namely an amplification curve, is drawn, and the amplification time threshold Tt of the sample corresponding to each micro-pool in the D-E area is determined.

(8) And (3) judging the DNA detection result: and determining the test result by combining the negative control and the positive control according to the time threshold Tt of the real-time fluorescent signal.

Example 3

As shown in fig. 4, a droplet bioanalysis chip integrating DNA extraction and fluorescence real-time quantitative PCR detection comprises 15 sets of serial microcells, each set of serial microcells consisting of 5 microcells; the micro-pools of the same group are communicated through a slit; the micro-cells have an open structure in whole or in part.

Specifically, the chip is made of polycarbonate. The droplet analysis chip had a size of 85.5mm (length) × 54mm (width) and a thickness of 4.2 mm. The chip is composed of 15 groups of series micro-cells, the depth of the micro-cells and the depth of the slits are both 3.7mm, the size of the slits is 3mm (length) multiplied by 0.5mm (width) multiplied by 3.7mm (height), the thickness of the bottom layer of the micro-cells is 0.5mm, and the surface is treated by a Teflon AF1600 hydrophobic coating. DNA extraction, LAMP constant temperature amplification and detection are integrated on the chip, the chip is placed on a chip platform, and the basic operation flow is as follows:

(1) oil phase loading: sequentially adding 25 muL, 20 muL, 15 muL and 10 muL of paraffin oil (NO. A630217, biological engineering (Shanghai) Co., Ltd.) into each micro pool of the chip A-E area, and ensuring that slits between the micro pools are soaked by the paraffin oil;

(2) reagent loading: and sequentially adding 20 mu L of lysis buffer solution, 50 mu L of binding solution, 2 mu L of magnetic particle suspension, 100 mu L of washing solution, 10 mu L of eluent and 5 mu L of LAMP premix into each micro-cell in the A-E area of the chip to respectively form independent water-in-oil type droplets. Wherein, the lysis buffer solution, the binding solution and the magnetic bead suspension added into each micro-pool of the chip A area are ensured to form mutually isolated liquid drops (the reagent is a MagaBio plus universal genome DNA extraction and purification kit of Bori science and technology, Inc. in Hangzhou), the washing solution is ethanol-free washing solution, and the eluent is deionized double distilled water;

(3) sample loading: and respectively adding 20 mu L of samples into the nucleic acid extracting solution drops in each micro-pool of the chip A area, wherein the samples are samples to be detected, positive controls or negative controls. The loading of the droplets in each micro-well of the chip is now shown in fig. 3.2.

(4) Release of DNA and binding to magnetic particles: the chip was shaken for 30 seconds to mix the sample with lysis binding buffer and then the chip A zone micro-cell was maintained at 56 ℃ for 5 minutes. And applying a magnetic field to the micro pool in the area A of the chip to pull the magnetic particle liquid drops to enter the combined buffer liquid drops, and after the magnetic particle liquid drops and the combined buffer liquid drops are fused, continuously pulling the magnetic particles to carry the combined buffer liquid drops to enter the lysis buffer liquid until the magnetic particles are fused. The chip was shaken to cause the magnetic particles-binding solution-lysis solution to mix thoroughly and to maintain gentle shaking for 5 minutes.

(5) DNA washing: and applying a magnetic field to the micro pool in the area A of the chip to pull the magnetic particles to move towards the micro pool in the area B along the slit until the magnetic particles enter washing liquid drops in the micro pool in the area B, wherein the linear speed of the movement is 5mm/s, and in the process, the magnetic particles carry DNA to be separated from the cracking-combined liquid drops, and then the magnetic particles reach the micro pool in the area B through the slit and are fused with the washing liquid drops in the micro pool in the area B. The chip is shaken to fully mix the magnetic particles with the washing solution. And applying a magnetic field to the micro pool in the area B of the chip to pull the magnetic particles to move to the micro pool in the area C along the slit (the linear speed is 5mm/s) until the magnetic particles are fused with the washing liquid drops in the micro pool in the area C. The chip is shaken to fully mix the magnetic particles with the washing solution.

(6) DNA elution: and applying a magnetic field to the micro pool in the area C of the chip to pull the magnetic particles to move to the micro pool in the area D along the slit until the magnetic particles enter the eluent liquid drops in the micro pool in the area D (the linear speed range is 3mm/s), and slightly oscillating the chip to ensure that the DNA on the magnetic particles is fully decomposed and dissociated into the eluent.

(7) Fluorescence real-time quantitative PCR detection: and applying a magnetic field to the micro pool in the D area of the chip to pull the magnetic particles to move towards the micro pool in the E area until the droplets of the PCR premix liquid in the micro pool in the E area are fused, wherein the linear velocity of the movement is 1mm/s, and then rapidly driving the magnetic particles to move in the reverse direction (the linear velocity is 5mm/s) so as to separate from the amplification reaction liquid and reach the C area through a slit in the D area-C area. The chip eluent was shaken and the PCR premix was mixed thoroughly. Conventional PCR thermal cycling was performed on the chip D-E zone micro-wells. During the period, the fluorescence signals of the liquid drops in each micro-pool in the E area are collected once in the process of each thermal cycle, so that the change curve of the fluorescence intensity of the liquid drops along with the cycle number, namely an amplification curve, is drawn, and the cycle threshold Ct of the corresponding sample of each micro-pool in the D-E area is determined.

(8) And (3) judging the DNA detection result: and (4) according to the cycle threshold value of the real-time fluorescent signal, combining a negative control and a positive control to judge the test result.

Example 4

A liquid drop biological analysis chip comprises 5 groups of series-connected micro-pools, wherein each group of series-connected micro-pools consists of 5 micro-pools; the micro-pools in the same group are communicated through a slit; the micro-cells have an open structure in whole or in part.

Specifically, the chip is made of black polytetrafluoroethylene. The droplet analysis chip had a size of 40mm (length) × 25mm (width). The width of the micro-pool is 2mm, the depth is 4mm, and the series connection area of the micro-pools is a slit with the length of 3mm and the width of 0.3 mm. The thickness of the bottom layer of the micro-pool is 0.5 mm.

The analysis flow of the heart disease marker detected on the chip based on chemiluminescence is as follows:

(1) the sampling needle sequentially aspirates 5. mu.L of mineral oil and 5. mu.L of the labeled capture antibody magnetic bead suspension. And repeating the operation for 5 times, and sequentially loading the micro pools in the first row. The capture antibodies labeled with magnetic beads in each droplet in the five groups were directed against aspartate Aminotransferase (AST), creatine kinase isoenzyme (MMB), phosphocreatine kinase (CK), Lactate Dehydrogenase (LDH), and troponin, respectively;

(2) the sampling needle sequentially aspirates 5. mu.L of mineral oil and 10. mu.L of wash solution. Repeating the operation for 5 times, and sequentially loading the micro pools in the second row;

(3) the sampling needle sequentially aspirates 5. mu.L of mineral oil and 10. mu.L of wash solution. Repeating the operation for 5 times, and sequentially loading the micro pools in the third row;

(4) mu.L of mineral oil and 5. mu.L of a solution containing a detection antibody labeled with Horseradish Peroxidase (HRP) were sequentially aspirated by a sampling needle, and the solution was sequentially loaded into each of the micro wells in the fourth row. Detection antibodies labeled by magnetic beads in each droplet in the five groups respectively aim at aspartate Aminotransferase (AST), creatine kinase isoenzyme (MMB), phosphocreatine kinase (CK), Lactate Dehydrogenase (LDH) and troponin;

(5) sequentially sucking 5 mu L of mineral oil and 5 mu L of luminol solution by a sampling needle, repeating the operation for 5 times, and sequentially loading the mineral oil and the luminol solution into each micro-pool in the fifth row;

(6) the sampling needle sequentially aspirates 10. mu.L of mineral oil and 5. mu.L of serum. Repeating the operation for 5 times, sequentially loading the micro pools in the first row, and preserving for 5 minutes;

(7) transferring the chip to the upper part of a magnet, dragging the chip back and forth for 1 minute to enable the magnetic beads to oscillate in the first row of micro-cells;

(8) driving the chip to move above the magnet, dragging the magnetic beads to enter the liquid drops containing the washing liquid from the first row of micro-cells to the second row of micro-cells, and staying at the liquid drops for 30 seconds;

(9) driving the chip to move above the magnet, so that the magnetic beads enter the liquid drops containing the washing liquid from the second row of the micro-cells into the third row of the micro-cells, and stay at the liquid drops for 30 seconds;

(10) driving the chip to move above the magnet, so that the magnetic beads enter the liquid drops containing the detection antibodies in the third row of the micro-pools from the third row of the micro-pools, and keeping for 5 min;

(11) the driving chip moves above the magnet to enable the magnetic beads to enter the liquid drops containing ramono from the fourth row of micro-pools to the fifth row of micro-pools;

(12) the droplet chip is moved into a dark chamber to detect the chemiluminescent signal in the droplet.

Example 5

A liquid drop analysis chip comprises series of micro-pools connected in series, wherein each series of micro-pools consists of a plurality of micro-pools; the micro pools connected in series are communicated through a slit; the micro-cells have an open structure in whole or in part.

One of the droplet analysis chip structure designs satisfying the above characteristics, as shown in fig. 2.2, is formed by one step using male die casting or injection molding.

The second structure design of the droplet analysis chip satisfying the above features is composed of a bottom sealing layer and a micro-pool layer as shown in fig. 5.1. As shown in figure 5.2, the micro-pool layer is provided with a micro-pool through hole array, the micro-pools connected in series on one side connected with the bottom sealing layer are communicated by a slit, and the width and the height of the slit are respectively 0.8mm and 0.5 mm. The slit has a transition structure with gradually changed width in the direction from the micro pool to the slit, and an included angle between a forward tangent of the transition structure and the direction from the micro pool to the slit is an acute angle. And after the micro-pool layer is formed, sealing the micro-pool layer with the bottom sealing layer to form the droplet chip with the characteristics. One of the material combinations for forming the bottom sealing layer and the micro-pool layer is respectively a glass slide and PDMS with the thickness not more than 0.5mm, and the corresponding sealing method is plasma bonding; two material combinations for forming the bottom sealing layer and the micro-cell layer are respectively aluminum foil and Cyclic Olefin Copolymer (COC) with the thickness of 0.1mm, and the corresponding method is thermal compression bonding.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A droplet bioanalysis chip, comprising: the device comprises a plurality of groups of parallel micro-pools connected in series, wherein each group of micro-pools connected in series consists of a plurality of micro-pool units which are connected in series through slits; part or all of the micro-cell units in the series micro-cells have an open structure; the bottom surfaces of the series microcells and the slits are of smooth thin-wall structures;

the micro-pool unit and the slit are provided with hydrophobic surfaces and are constructed in a way that an oil phase solution and an oil phase are added into the micro-pool unit to automatically infiltrate the surfaces of the micro-pool unit and the slit; adding corresponding aqueous solution reagents/or samples into each micro-cell unit, wherein the added reagent/sample solution automatically forms water-in-oil droplets in the oil phase due to the surface tension effect; the liquid drops in the communicated micro-cell units are still kept in mutually independent isolation states under the external disturbance, so that the stability of the component concentration of each liquid drop is ensured;

the length of the slit is 1-20 mm, and the width or height has at least one dimension of 0.1-1 mm.

2. The droplet bioanalysis chip of claim 1, wherein: the material of the drop bioanalysis chip is hydrophobic polymer material or surface hydrophobic treatment material.

3. The droplet bioanalysis chip of claim 2, wherein: the hydrophobic polymer material is polypropylene, cycloolefin copolymer or polytetrafluoroethylene.

4. The droplet bioanalysis chip of claim 1, wherein: and hydrophobic coatings are arranged on the inner surfaces of the micro-cell unit and the slit.

5. The droplet bioanalysis chip of claim 1, wherein:

the thickness of the bottom surface is 0.1-1 mm;

the area of the liquid drop biological analysis chip is 10-600 square centimeters.

6. The droplet bioanalysis chip of claim 1, wherein: the slit is a slit with a gradual transition structure formed by narrowing and widening.

7. Use of the droplet bioanalysis chip according to any one of claims 1 to 6, wherein: for biological sample analysis.

8. Use of the droplet bioanalysis chip according to claim 7, wherein: the biological sample analysis comprises extraction of biological molecules, and qualitative analysis and/or quantitative analysis.

9. Use of the droplet bioanalysis chip according to claim 8, wherein: the biological molecules are proteins and nucleic acids.

10. The method for using a droplet bioanalysis chip according to any one of claims 1 to 6, comprising the steps of:

(2) then respectively adding water phase solution containing samples or reagents into each micro-cell, wherein the water phase solution spontaneously forms water-in-oil droplets in the oil phase solution;

(3) magnetic particles capable of combining with biomolecules are loaded in an aqueous phase solution, and the magnetic particles move under the action of magnetic field force after being combined with the biomolecules, pass through an oil-water interface and a slit, and move among liquid drops to finish the processes of biomolecule extraction, qualitative analysis and/or quantitative analysis.