CN108593952A

CN108593952A - The detecting system and detection method of online addition reaction reagent

Info

Publication number: CN108593952A
Application number: CN201810282214.0A
Authority: CN
Inventors: 罗贞; 王陈成; 王茜; 马云林
Original assignee: SHANGHAI AIRUIDE BIOTECHNOLOGY Co Ltd
Current assignee: SHANGHAI AIRUIDE BIOTECHNOLOGY Co Ltd
Priority date: 2018-03-28
Filing date: 2018-03-28
Publication date: 2018-09-28
Anticipated expiration: 2038-03-28
Also published as: CN108593952B

Abstract

The present invention relates to a kind of detecting systems and detection method of online addition reaction reagent.Specifically, detecting system of the invention includes：Micro-fluidic chip generates submodule including micro-fluidic flow-through channel and microlayer model；Online addition subsystem, including driving power module, flow injecting module, microlayer model label processing module and data processing and control signal generation module；And detection subsystem.System and method through the invention, sample microlayer model and reagent microlayer model etc. can be accurately controlled, complete the processes such as fusion, extraction, incubation, division, signal detection, to realize the process for carrying out online addition reaction reagent to sample and completing detection, manual operation when biology sample detection is reduced.

Description

The detecting system and detection method of online addition reaction reagent

Technical field

The invention belongs to the detection and analysis fields of biological sample, more particularly to the detection system of online addition reaction reagent System and detection method.

Background technology

The detection technique for being currently used for biological sample includes biochemistry detection, Molecular Detection and anti-based on antigen and antibody specific The immune detection etc. answered.There are still following problems to need to optimize in above-mentioned detection technique specific implementation：Time-consuming for analytic process, sample This demand is larger, and detection reagent consumption is more, and Compact Detection System flux is not enough and cost is higher, precision is poor；Greatly Type detecting system integration degree is low, take up a large area and equipment failure rate is high, and detection process will produce more biohazard object Deng.

Microfluidic system is widely used to biological and chemical inspection as a kind of platform of set various functional blocks in recent years It in survey, is known as " influencing one of 15 most important inventions of human future ".Detection technique based on microfluidic system has Following advantage：Manual operation is few, and detection reagent consumption is few, generally only needs minimal amount of sample to be checked, reaction efficiency height etc..

However, the degree of automation of microfluidic system is still to be improved, especially online in terms of addition reaction reagent, at present Detecting system cannot achieve satisfactory efficiently accurately automatic on-line addition.

Therefore this field need to develop new achievable efficiently and accurately automatic on-line addition reaction reagent based on micro- The detecting system and its detection method of fluidics.

Invention content

The present invention provides can be achieved efficiently and accurately automatic on-line add reaction reagent based on microflow control technique Detecting system and its detection method.This is a kind of online add-on system of the reaction reagent based on microlayer model chip, and being based on should A kind of specimen inspection system and its detection method of system.

The first aspect of the present invention provides a kind of detecting system of online addition reaction reagent, the system comprises：

(1) micro-fluidic chip, the micro-fluidic chip include：

(1.1) micro-fluidic flow-through channel：The micro-fluidic flow-through channel is used for for being taken in continuous phase and continuous phase The microlayer model of band flows, including：Continuous phase adds section, sample addition section, sample microlayer model and generates section, the generation of reagent microlayer model Section, microlayer model fusion section and signal detection section；With

(1.2) microlayer model generates submodule：Mouth is generated including microlayer model；The microlayer model generates submodule for making point Dephasing forms microlayer model, and the dispersed phase includes：Sample liquid or reagent solution；

Wherein, reagent microlayer model generation mouth is located at reagent microlayer model generation section；

(2) subsystem is added online, for generating the fusion microlayer model containing sample and reagent, the online addition subsystem System includes：

(2.1) power module is driven：The driving power module is used to provide the driving force needed for dispersed phase and continuous phase, from And so that the microlayer model of continuous phase and each dispersed phase is obtained required flow velocity and form reagent microlayer model；

(2.2) flow injecting module：The flow injecting module is connected with continuous phase and each dispersed phase, the company Continuous phase and each dispersed phase are entered by the module in the micro-fluidic chip, and the flow injecting module includes control valve Door, the control valve enter the micro-fluidic flow-through channel for controlling continuous phase and dispersed phase；

(2.3) microlayer model marks processing module：The microlayer model label processing module includes liquid drop sensor；Described Number and number consecutively of the liquid drop sensor for reading microlayer model and the flow velocity for reading microlayer model；With

(2.4) data processing and control signal generation module：The data processing and control signal generation module are according to institute State the predefined parameter of detecting system, the characteristic parameter of continuous phase and each dispersed phase fluid, the flow velocity of the microlayer model and a The flow velocity of number and continuous phase generates signal for controlling the driving power module and for controlling the flow injecting module Signal；And

(3) subsystem is detected：The detection subsystem is used to carry out the fusion microlayer model for having generated readable signal Signal-obtaining, and record feedback signal data.

In another preferred example, described (2.1) drive in power module, needed for the microlayer model acquisition of each dispersed phase Flow velocity is the relative velocity relative to continuous phase.

In another preferred example, described (2.1) drive in power module, and the microlayer model of each dispersed phase is the micro- liquid of sample Drop and/or reagent microlayer model.

In another preferred example, the control valve is selected from the group：Paraffin valve, paraffin hot melt valve, magnet move valve Door, operated pneumatic valve, diaphragm valve, hydrophobic valve, machinery valve or combinations thereof.

In another preferred example, the micro-fluidic flow-through channel is selected from the group：Straight channel, circular passage, folding shape channel, Back-shaped channel, cavity or combinations thereof channel.

In another preferred example, the system is controlled by control centre；Preferably, the control centre is computer, And the system is controlled by computer software.

In another preferred example, the valve is additionally operable to control continuous phase and/or dispersed phase enters the microfluid The volume of circulation passage and/or time.

In another preferred example, the reagent microlayer model that described (1.2) microlayer model generates submodule generates mouth as T-type or cross Type microlayer model generating structure, and generate section positioned at sample or reagent microlayer model.

In another preferred example, the liquid drop sensor of described (2.3) the microlayer model label processing module is additionally operable to read micro- liquid The volume of drop.

In another preferred example, it is generated at micro-fluidic flow-through channel of the mouth to reagent microlayer model generation mouth in sample microlayer model Equipped with the first liquid drop sensor, first liquid drop sensor is used to read the number of the sample microlayer model and compiles successively Number, and read the flow velocity of each sample microlayer model.

In another preferred example, the outlet end of the microlayer model fusion section is equipped with the second liquid drop sensor, for reading warp Cross the volume of the sample microlayer model of the microlayer model fusion section, wherein the volume read by the liquid drop sensor is for true Determine whether microlayer model merges success.

In another preferred example, the driving power module provides forward drive power and reverse actuating force；Wherein, described Reverse actuating force is used to make continuous phase in the micro-fluidic chip and sample microlayer model uniformly to move forward and/or described Forward drive power is for generating the reagent microlayer model merged with sample microlayer model.

In another preferred example, the predefined parameter of the detecting system, including sample microlayer model is from sensor to reagent Microlayer model generates distance, the response time of each valve and liquid drop sensor and the width of micro-fluidic flow-through channel that mouth is passed through Degree and height.

In another preferred example, the characteristic parameter of the fluid includes dynamic viscosity, viscosity and surface tension.

In another preferred example, (2.4) data processing and control signal generation module are generated according to parameters described below Signal,

Including：As system predefined parameter, the distance s of first liquid drop sensor to reagent microlayer model generation mouth, The reagent microlayer model generates reagent microlayer model in the channel width W of mouth, (1.1) micro-fluidic flow-through channel and generates the logical of section Road depth h, the reagent microlayer model generate width w, the response time of each valve and the sound of sensor of the downstream passage of mouth Between seasonable；

As characteristic parameter, the surface tension between the viscosity and dynamic viscosity and liquid of reagent；With

By the flow velocity v for the sample microlayer model that (2.3) microlayer model label processing module is read_{Drop k}；Wherein, k is the micro- liquid of sample The number of drop；

And

(2.4) data processing and the signal for controlling the generation of signal generation module include：For controlling the fluid The signal t of the dispersed phase valve opening time of sample introduction module_g-k, and for controlling needed for the driving power module generation microlayer model The signal of driving force P.

In another preferred example, described to generate the driving force needed for microlayer model for controlling the driving power module as reagent Pressure in storehouse.

In another preferred example, the pressure in the agent bin of different reagents is respective independence or identical.

In another preferred example, microlayer model label processing module further includes that hematocrit compares sensor；Preferably, described Hematocrit is located at sample microlayer model than sensor and generates at mouth to the micro-fluidic flow-through channel of reagent microlayer model generation mouth.

In another preferred example, the hematocrit is used to read the hematocrit ratio of whole blood sample than sensor, or for reading Sample this turbidity.

In another preferred example, the system is judged and is corrected than the data that sensor is read according to hematocrit.

In another preferred example, the flow injecting module is based on formula 10

Determine the opening time t of the control valve of online addition reaction reagent_g-k, then start to generate reagent microlayer model；

In formula, t₁For sensor response time, t₂Response time, v for valve opening_{Drop k}For the stream of sample microlayer model k Speed, wherein k is the number of sample microlayer model and s is that sample microlayer model is given birth to from first liquid drop sensor to reagent microlayer model At the distance of mouth passed through.

In another preferred example, the driving power module provides the driving force needed for line addition reaction reagent based on formula 15 P：

In formula, s generated from first liquid drop sensor to reagent microlayer model by sample microlayer model mouth pass through away from It is that reagent microlayer model generates the channel depth of section, W is that channel width, L that reagent microlayer model generates mouth are reagent microlayer model from, h It generates oral fluid drop initial stage and shows as the liquid length of liquid column form, R as microlayer model radius, v_{Drop k}For sample microlayer model k Flow velocity, w be reagent microlayer model generate mouth downstream passage width, t₁For sensor response time, t₂For the sound of valve opening Between seasonable, F_σSurface tension between continuous phase and dispersed phase, a and b are each independently calibration parameter, and n is reagent fluid Viscosity.

In another preferred example, the detection signal that the detection subsystem can be read is selected from：Chemiluminescence signal, fluorescence Be stimulated emitted fluorescence signal, quantum dot microsphere light of group excites emitted visible light signal, turbidity variable signal Or combinations thereof.

In another preferred example, the micro-fluidic chip further includes that (1.3) sample microlayer model is incubated submodule, described Micro-fluidic flow-through channel further includes being incubated section；After reaction reagent has been added for offer in the sample microlayer model incubation submodule Sample microlayer model fully react and/or mixed condition and the incubation section are fully anti-for providing fusion microlayer model It answers and mixed space.

In another preferred example, the system also includes：

(4) automatic sampling subsystem, the automatic sampling subsystem include：Sampling needle, syringe pump, continuous sample introduction frame, Bar code reading module and sample-adding module；Wherein, the bar code reading module is located in continuous sample introduction frame；The sampling needle leads to The syringe pump is crossed to draw sample and the sample-adding module is added in the sample by the syringe pump；Described adds Egf block is equipped with a sample adding mouth, and the sample-adding module is to be in fluid communication with the flow injecting module.

In another preferred example, the automatic sampling subsystem further includes cleaning module, after a collection of sample is added, institute The cleaning module stated is used to clean the sampling needle and sample-adding module.

In another preferred example, the micro-fluidic chip further includes (1.4) waste collection submodule, the waste liquid Collection module is for collecting cleaning solution, discarded sample microlayer model or discarded reagent microlayer model.

In another preferred example, the system further includes (5) reagent storage subsystem, the reagent storage subsystem Including continuous phase bit of storage, cleaning solution storage position and agent bin；The agent bin includes pre- vortex mixer and one or more Reagent position；

Wherein, the pre- vortex mixer is for avoiding agent precipitate or aggregation；The reagent position can store each examination Agent, the reagent are entered by the flow injecting module in the micro-fluidic chip.

In another preferred example, the reagent is the reaction reagent reacted with sample, and is optionally for described Extraction module cleaning reagent, the shearing reagent or post-treatment reagents for exciting detected signal.

In another preferred example, the system further includes (6) temperature control system, and the temperature control subsystem is used In the temperature of the control micro-fluidic chip, to control detection temperature of reaction system.

In another preferred example, the temperature control subsystem is additionally operable to control the temperature of the reagent storage subsystem Degree.

In another preferred example, the system further includes (7) extraction sub-system, the corresponding micro-fluidic flow-through channel It further include extraction section；

The extraction sub-system generates submodule for controlling microlayer model；When the sample microlayer model be moved to it is predetermined Behind position, the work that the microlayer model generates submodule is controlled, generate the microlayer model of cleaning reagent and shears micro- liquid of reagent Drop, and " sample microlayer model-cleaning reagent microlayer model-shearing reagent is micro- for formation on liquid flow direction in the extraction section The microlayer model queue of drop ", wherein sample microlayer model are located at the forefront of flowing.

In another preferred example, the microlayer model queue refers to sequence of each microlayer model by precalculated position.

In another preferred example, the carrying detectable label is captured from sample microlayer model by the extraction sub-system The detection product of object, and the captured detection for carrying detectable marker is produced with the cleaning reagent microlayer model Object is cleaned；And the detection product of cleaned carrying detectable marker is cut with the shearing microlayer model It cuts, to generate the microlayer model for detection.

In another preferred example, the micro-fluidic flow-through channel further includes extraction section, and the extraction section is located at controllable Field regions, the field regions are controlled by the extraction sub-system；Preferably, the controllable magnetic place is by electromagnet or can Mobile permanent magnet provides.

The second aspect of the present invention additionally provides a kind of detection method that microlayer model adds online, includes the following steps：

(1) detecting system of online addition reaction reagent as described in the first aspect of the invention is provided；

(2) sample liquid to be measured is entered into the micro-fluidic chip by the flow injecting module；

(3) the sample liquid to be measured forms the micro- liquid of sample in the microlayer model of micro-fluidic chip generates submodule Drop marks the liquid drop sensor of processing module to be detected (or reading) simultaneously to the sample microlayer model by sample microlayer model Number optionally is marked to carrying out the sample microlayer model, stops when the sample microlayer model quantity of generation reaches predetermined quantity Only generate sample microlayer model；

(4) after each sample microlayer model in the sample microlayer model reaches the precalculated position in micro-fluidic chip, institute The control signal that the driving power module stated is generated according to the data processing and control signal generation module, it is micro- to generate a reagent Drop simultaneously drives the reagent microlayer model to be moved by predetermined speed, and makes a reagent microlayer model and a sample This microlayer model is in contact fusion in flow process, forms fusion microlayer model；

(5) in the flow process of the fusion microlayer model, the fusion microlayer model is post-processed, and is examined It surveys, to obtain the testing result of the sample liquid.

In another preferred example, described " stopping generating sample microlayer model " is the control entered by closing control sample liquid stream Valve processed.

In another preferred example, the reagent microlayer model and the sample microlayer model respectively enter the microlayer model fusion Section, and in microlayer model fusion section fusion, form the fusion microlayer model.

In another preferred example, in step (5), the fusion microlayer model continues with continuous phase in the miniflow Advance in control chip, until the signal detection section, reads signal by the detection subsystem and record.

In another preferred example, in step (3), further include：By hematocrit than sensor, the sample microlayer model is obtained Sample hematocrit than signal or turbid ity signal, wherein the sample hematocrit than signal or turbid ity signal can be used for judge and/or school Just.

In another preferred example, in step (4), successively while or successively by two or more reagent microlayer models and sample This microlayer model is merged.

In another preferred example, in step (4), two kinds of reagent microlayer models are generated, and carry out with the sample microlayer model Fusion.

In another preferred example, in step (4), after sample microlayer model reaches the designated position in micro-fluidic chip, The driving power module provides corresponding pressure according to the signal that data processing and control signal generation module generate, and opens institute The corresponding valve of reagent is needed, formed and discharges reagent microlayer model；And when control reagent microlayer model quantity >=sample microlayer model quantity When, close corresponding valve.

In another preferred example, the volume ratio of the reagent microlayer model and sample microlayer model is (0.5~10):1.

In another preferred example, after multiple microlayer models of the same sample fully enter step (4), it is described it is automatic into The cleaning module of subsystem starts, and sampling needle draws cleaning solution, closes respective valves, and cleaning solution is pushed into the sample-adding mould Block simultaneously cleans the part for causing cross contamination；Preferably, the part of cross contamination that causes includes：In the sample-adding module Adding mouth and valve.

In another preferred example, further include in step (2)

(2.1) sample (or sample liquid) to be measured is placed on continuous sample introduction frame, open system, continuous phase passes through described Flow injecting module enters the micro-fluidic chip and is full of the micro-fluidic flow-through channel；

(2.2) under the control of the system, the sample on continuous sample introduction frame passes sequentially through bar code read area, identifies sample Bar code, sampling needle draw sample to be tested, and sample to be tested is pushed into the adding mouth of sample-adding module by syringe pump, passes through the stream Body sample introduction module enters the micro-fluidic chip.

In another preferred example, further include in step (5)：

(5.1) the fusion microlayer model is fully reacted or is mixed in the reaction submodule of the micro-fluidic chip.

In another preferred example, further include in step (5)：

(5.2) by the extraction sub-system, cleaning shear treatment is carried out to the fusion microlayer model.

In another preferred example, further include in step (5)：

(5.3) reagent of excitation-detection signal is added again to the fusion microlayer model.

In another preferred example, the sample includes biological sample.

In another preferred example, the total volume of the sample liquid is 0.2-20 microlitres, preferably 0.5-10 microlitres.

In another preferred example, the sample liquid (or sample) is selected from the group：Biological initial liquid sample or through processing Biological initial liquid sample.

Preferably, the sample is selected from the group：Blood, blood plasma, serum, tissue fluid, lymph, urine, or combinations thereof.

In another preferred example, the sample comes from people, non-human mammal or birds.

In another preferred example, the substance to be detected (object i.e. to be checked) contained in the sample liquid is selected from the group：Antigen, Haptens, antibody, protein, nucleic acid, liposome, peptide fragment, nucleotide, amino acid, virus, bacterium, parasite, cell, drug, Ion, salt, or combinations thereof.

In another preferred example, the method is in-vitro method.

In another preferred example, the method is the nondiagnostic and non-property controlled.

In another preferred example, the method further includes：Before detecting signal, after being carried out to the fusion microlayer model Reason.

In another preferred example, the post-processing includes that the one or more successively, simultaneously or successively carried out is selected from down The subprocessing of group：It is incubated, extracts and divides.

In another preferred example, include carrying out reaction selected from the group below in the incubation：Double-antibody method reaction, ring are situated between Lead isothermal nucleic acid amplification reaction, BCA methods reaction, or combinations thereof.

The third aspect of the present invention additionally provides side described in system and second aspect described in first aspect present invention The application of method, they be used to detect biological sample.

Preferably, the detection is based on double-antibody method, based on loop-mediated isothermal amplification or based on BCA The detection of method.

In another preferred example, the detection is for detecting object to be checked selected from the group below：Antigen, haptens, antibody, albumen Matter, nucleic acid, liposome, peptide fragment, nucleotide, amino acid, virus, bacterium, parasite, cell, drug, ion, salt or its group It closes；

Preferably, can be used for detecting antigen, nucleic acid or protein.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Description of the drawings

Fig. 1 shows the schematic diagram of the system of the present invention.

Fig. 2 shows the control protocol schematic diagram of subsystems in the present invention.

Fig. 3 shows the user oriented operating process schematic diagram of the device of the invention.

Fig. 4 shows the schematic diagram of the operational process of the device of the invention.

Fig. 5 shows sample of the present invention drop (compared with Dark grey) and reagent droplet (more light grey) fusion process schematic diagram, Wherein scale is 50 μm.

Fig. 6 shows that homogeneous phase time discrimination fluorescence method detects BNP standard curves in the embodiment of the present invention 1.

Fig. 7 shows that the present invention is based on BCA methods to detect urine total protein concentration standard curve.

Fig. 8 shows the part-structure schematic diagram of chip of the present invention；

Wherein, 1 be sample microlayer model production mouth, 2 be the first liquid drop sensor, 3 be reagent microlayer model generate mouth.

Specific implementation mode

The present inventor after extensive and in-depth study, develops a kind of online addition subsystem of unique structure for the first time, And the specimen inspection system and detection method based on microlayer model chip using the online addition subsystem.The present invention Line adds subsystem, not only realizes that sample carries out online addition detection, but also can be to the generation of reaction reagent microlayer model into stroke It sequence, systematization and accurately controls, and then realizes that reagent microlayer model is merged with the accurate of sample microlayer model.In addition, of the invention The detecting system of optimization can be in the flow process of fusion microlayer model, the post-processings such as the subsequent extraction of canbe used on line, incubation, division Automation and the processes such as signal detection, to which manual operation when sample detection be significantly reduced or eliminated and significantly improve inspection Survey accuracy and precision.The present invention is completed on this basis.

Term

As used herein, term " negative pressure power source " refers to providing forward thrust for chip dies fluid in the block, is made Fluid can equably flow forward, avoid because of test value difference caused by different in flow rate.

As used herein, term " positive pressure power source " refers to providing thrust for the reagent in agent bin, makes reagent one Enter chip module under constant current speed.

As used herein, term " continuous phase " sample disperses to form microlayer model in the phase, generally adopts in the present invention Use oil phase.

As used herein, term " dispersed phase " refers to the fluid to form various microlayer models, such as (but not limited to), sample, Reaction reagent (one or more) etc..

As used herein, term " sample microlayer model " refer to need to be added to other reagents containing sample need to be detected Microlayer model, sample microlayer model described in the different disposal stage can also include in addition to comprising sample to be detected reaction reagent, Shine excitation substrate etc..

As used herein, term " reagent " refers to the various reagents used in detection process, including but not limited to respectively A reaction reagent, cleaning reagent, shearing reagent, post-treatment reagents etc., corresponding " reagent microlayer model " refers in detection process The microlayer model formed by various reagents, including but not limited to each reaction reagent microlayer model, cleaning reagent microlayer model, shearing examination Agent microlayer model, post-treatment reagents microlayer model etc..Various microlayer models are necessary to determine whether to need according to detection and sample microlayer model melts It closes.

As used herein, " microlayer model chip " refers to a kind of chip using immiscible two-phase properties of flow, wherein Under the action of hydrodynamic shear and interfacial tension, wherein phase dispersion is in the other phase, extremely with the frequency generation nanoliter of kHz A series of monodispersed independent microlayer models of picoliters volume.In the present invention, the system is utilized so that microlayer model is in micro- liquid It drips in chip, fusion can be in a short time realized under proceduralization and efficient control, incubation, aggregation, division, is divided The processes such as choosing, extraction and analysis.

As used herein, term " microlayer model queue " is only represented by microlayer model by the sequence in precalculated position, " micro- liquid Microlayer model in drop queue " can exist in microchannel simultaneously, and followed by precalculated position；Alternatively, successively generating micro- liquid Drop, and followed by precalculated position.

In order to make it easy to understand, being described below in conjunction with attached drawing.It should be understood that these attached drawings do not limit this hair in any way Bright protection domain.

Reaction reagent adds subsystem online

The system of the present invention can realize that will produce multiple reagent microlayer models to a sample is added online, described The microlayer model that online addition will namely generate each microlayer model of the sample and required reagent to be added carries out one-to-one Fusion.To realize this purpose, the size and generation time that need to be generated to reagent microlayer model are accurately controlled.

Collective effect of the fluid flowing dependent on motive power in microchannel, including interfacial tension, shearing force, viscosity Power, inertia force, gravity etc., but in microchannel, the influence of inertia force and gravity is very little, can ignore.Microlayer model It is formed, refers to thering is sufficiently large shearing force to upset the surface tension between continuous phase and dispersed phase, to form the mistake of microlayer model Journey.Therefore, the viscosity, surface tension, the external force of application of fluid and the width of runner are all the key factors that microlayer model is formed.

Detailed process is as follows, when sensor detects the microlayer model that sample generates, i.e., is marked respectively to microlayer model, successively Be 1,2,3,4 ... k ... n-2, n-1, n, and detect that oil speed is respectively v at this time_{Oil 1}, v_{Oil 2}, v_{Oil 3}, v_{Oil 4}…….v_{Oily k}…… v_{Oily n-2}, v_{Oily n}- 1, v_n, microlayer model movement speed is v_{Drop 1}, v_{Drop 2}, v_{Drop 3}, v_{Drop 4}…….v_{Drop k}……v_{Drop n-2}, v_{Drop n-1}, v_{Drop n}, The distance that sensor distance reagent microlayer model generates mouth is s (as shown in Figure 8), and microlayer model is flowed to by sensing station in microlayer model During generating mouth, controller is used for reagent by the data analysis for microlayer model k, and according to the output control of calculating The valve switch of gas pressure and reagent merges to generate corresponding reagent microlayer model K with sample microlayer model k.It is given below The computational methods of required reagent pressure：

During generating microlayer model, continuous phase and liquid phase flow into runner main channel, dispersion with certain flow velocity respectively The intersection for mutually reaching T-type channel or cross channel forms two-phase interface with continuous phase, and continues to develop to main channel, shape At microlayer model head, as dispersed phase fluid continues to flow into, microlayer model head constantly increases, and main channel is gradually blocked up by microlayer model It fills in, is flowed in the thin layer that continuous phase can only be between tube wall and microlayer model, the reduction for flowing scale makes continuous phase to microlayer model Head generates the extruding force of bigger, and driving microlayer model head is developed to continuous phase export direction, and microlayer model neck, which is stretched, to attenuate, Final fracture forms independent microlayer model.

At this point, when microlayer model is formed, there are three types of force effect microlayer model is formed, as two-phase interface tension (F_σ)、 Viscous shear power (F_τ) and since continuous phase overstocks the resistance (F generated on dispersed phase head_R), F_σIt is fixed for one to be considered as Value, and shearing force and resistance can be expressed as

Wherein, μ_cFor the dynamic viscosity of continuous phase, Q_cFor the flow of continuous phase.ε is between two liquid interfaces and channel wall Film thickness, w be downstream passage width, be approximately equal to the length L on microlayer model head.With the increase of microlayer model volume, Film thickness ε between two liquid interfaces and channel wall constantly reduces, by formula 1 and 2 it is found that viscous shear power (F at this time_τ) and Since continuous phase overstocks the resistance (F generated on dispersed phase head_R) constantly increase, this trend is formed until microlayer model.For Flow Q,

Q=uS=uhw (formula 3)

Wherein S accumulates for fluid cross-section, and u is fluid velocity, and h is the microchannel height of microlayer model formation zone, and by formula 3 It can obtain：

Q_cIt is the flow of continuous phase, Q_dIt is the flow of dispersed phase, u_cIt is continuous phase flow velocity, u_dIt is dispersed phase flow velocity, w is liquid The downstream passage width of continuous phase after drop generates, d is the neck feature width that drop formation mouth punishes dephasing, by formula 4 and public affairs Formula 5, the length L that can obtain being formed microlayer model are

Microlayer model volume is V at this time_Drop, when the fluid column that microlayer model has still just generated, there is the cubature formula to be

V_Drop≈ Lwh (formula 7)

When above-mentioned fluid column, which enters channel, forms microlayer model, the radius of microlayer model is R, then has

And it is t to generate the above-mentioned microlayer model required time_g, then have

Therefore, the whole process of generation reagent microlayer model is：Sensor detects sample microlayer model k, and measures at this time Oil phase (continuous phase) speed and sample microlayer model movement speed be v_{Oily k}And v_{Drop k}, the sensor response time is t₁, microlayer model The distance that mouth is generated from liquid drop sensor to reagent microlayer model is s, and data transfer to the controller of sensor (is set to data processing And control signal generation module), controller calculates the reagent flow rate v needed_Liquid-k, then calculate the corresponding control pressure of needs Then P opens valve, the response time of valve is t₂, calculated since sample droplets flow through liquid drop sensor to opening reagent Valve time be t_g-k。

Realize the online addition to sample microlayer model, i.e., when microlayer model k, which flow to reagent microlayer model, generates mouth, reagent is micro- Drop generates completion, to realize the fusion of microlayer model, therefore has

In conjunction with formula 6-10, the flow velocity v that the reagent reacted with sample is needed for sample microlayer model k can be calculated_Liquid-k For

At this point, v_{Drop k}For the speed for the sample microlayer model k that sensor measures；R is the half of the reagent microlayer model for needing to generate Diameter, w are the width of downstream passage, and h is the height of the microchannel (microlayer model fusion section) of microlayer model integration region, t₁For sensor Response time, t₂Device opens the response time of valve in order to control, and in the system of the present invention, these numerical value are that system fixes ginseng Number.

Liquid in microchannel is generated for reagent, mainly by the power from three aspects, i.e., from can control Gas-powered power (F₁), the surface tension (F between dispersed phase and continuous phase (water oil)_σ) and microchannel internal pressure loss (F₂), when When liquid velocity is stablized, these three power can reach balance, wherein

F₁=PS=PWh (formula 12)

Wherein P is the air pressure that can be adjusted by controller, and S is the sectional area of reagent droplet formation zone microchannel, and W is Reagent generates the width of mouth, and h is the height of reagent droplet formation zone microchannel, and L is drop formation initial stage liquid in reagent pipeline The length of column, n are the viscosity (related to temperature, viscosity is definite value under fixed temperature) of reagent fluid, and v is the flowing of reagent fluid Speed.During air pressure adjustment, surface tension is held essentially constant.

By formula 12 and formula 13, when finally reaching dynamic balance, required pressure P and reagent flow rate relationship can be calculated For

In the controls, the v in formula 11_Liquid-kV as in formula 14, therefore just have,

In formula 14,15, a, b are calibration parameter, related from the testing result of different detection architecture quality-control products.By formula 15 can obtain, and when sample microlayer model flows to sensor, sensor detects to obtain microlayer model speed to be v_{Drop k}, this information is transmitted To controller, the pressure P of reagent is applied to required for being calculated according to formula 15, pressure is adjusted to by adjustment driving force controller P opens reagent driving force control valve, generates reagent microlayer model K and is merged with microlayer model sample k.Complete microlayer model reagent Online adding procedure.

Add the detecting system of subsystem online based on reaction reagent

The present invention also provides a kind of detecting system, which adds subsystem online based on reaction reagent above-mentioned System realizes the fusion process of sample microlayer model and reagent merged etc. between various microlayer models in microlayer model chip.

Fig. 1 is the detecting system schematic diagram of one embodiment in the present invention.

As shown in Figure 1, a kind of biological sample quantitative detection system based on microlayer model chip, including subsystem is added online System, temperature control subsystem, the driving power module of online addition subsystem, automatic sampling subsystem, reagent storage subsystem, inspection Subsystem, micro-fluidic chip, waste collection module etc. are surveyed, wherein：

A) automatic sampling subsystem：The module includes sampling needle, syringe pump, continuous sample introduction frame, bar code reading module etc..

The sample to be tested for posting sample information bar code is temporarily stored into continuous sample introduction frame, under the control of present system, according to Sequence is advanced forward successively, after obtaining sample essential information and project to be detected by bar code read area, reaches specimen sample area Domain, sampling needle draw sample using syringe pump, then utilize syringe pump that sample is pushed into the adding mouth of sample-adding module again, are flowing Enter chip channel in body sample introduction module under the control of valve 1 (as shown in Figure 1).

B) the driving power module of subsystem is added online：The module includes positive pressure power source, negative pressure power source etc., difference It refer to the driving force system of forward and reverse.

The negative pressure power source, is connected with waste collection module, is indirectly connected with chip dies waste liquid pool in the block, is core Piece mould fluid in the block provides forward thrust, makes the uniform flow forward of fluid, avoids because of test caused by different in flow rate Value difference is different.The power source can be provided by air compressor or negative pressure pump.

The positive pressure power source, is connected with the agent bin of reagent storage subsystem, and thrust is provided for the reagent in agent bin, Reagent is set to enter chip module under certain flow rate.The power source can be provided by air compressor or compressed gas source.

C) reagent storage subsystem：The subsystem includes agent bin, cleaning liquid level, continuous phase (oil phase) etc..

The oil phase is connected with chip dies continuous phase addition section in the block, and chip channel is entered under the control of valve 2, is Continuous phase in microlayer model generation.

The cleaning solution is used to clean all places for being contacted with test sample, being possible to generation cross contamination, including adopts Sample needle, injection port, valve 1,3 etc., as shown in Figure 1.

The agent bin includes pre- vortex mixer, reagent 1,2,3 ... the reagent position that M, N ... are waited.Pre- vortex mixer refers to reagent Reagent 1 in storehouse, 2,3 ... M, N ... wait carry out mixing, avoid agent precipitate or aggregation.Reagent 1,2,3 ... M, N ... wait be with The various reagents of sample reaction, are pre-stored in kit, take out being fixed on corresponding reagent position when needing from kit, It is connected with online addition subsystem by conduit, and then is in fluid communication with micro-fluidic chip.

It is that can including but not limited to be wrapped with the substance that the object to be checked of the target in sample to be tested is combined that reagent 1,2 ..., which waits, By on nano particle (magnetic bead, fluorescent microsphere) antibody 1, be marked with fluorescent dye antibody 1, be marked with the anti-of fluorescent dye Body 2 is marked with catalyzing enzyme or the antibody 2 of other tracers etc..Reagent 3 is to carry out pretreated cleaning before the assay to microlayer model Reagent, such as added with proper amount of surfactant and adjusted the salt buffer solution of pH.Reagent M be to microlayer model before the assay into The pretreated eluent of row, such as adjusted the glycine solution of pH.It is after being carried out before the assay to microlayer model that reagent N ..., which waits, The reagents such as the reagent of reason, such as chemiluminescent substrate.

D) temperature control subsystem：The module controls agent bin, agent bin and chip module associated conduit, chip module Deng temperature.

The temperature control storehouse temperature of the agent bin is 2-8 DEG C, convenient for reagent 1,2,3 ... M, N ... wait can it is long-term, stablize and protect It deposits, does not influence the quality of reagent and the sensitivity of detection.

The temperature control storehouse temperature of the agent bin and chip module associated conduit is 25-37 DEG C, to entering the examination of chip module Agent is preheated, and is made to reach required reaction temperature in advance into chip dies reagent in the block, is further increased reaction efficiency, is shortened Reaction time.

The temperature in the temperature control storehouse of the chip module ensures that the reagent in chip can fully react at 25-65 DEG C.

E) subsystem is detected：It may include CCD/COMS imaging systems, exciting light sources, photomultiplier transit in the subsystem Pipe, photoelectric converter etc..Directly or indirectly it is connected with the signal-obtaining region in chip by modes such as optical fiber, in microlayer model Useful signal recorded and analyzed.

F) waste collection module：All waste liquids that the module collection generates in sample continuous mode, with chip module In waste liquid pool be connected.

G) micro-fluidic chip：The chip include microlayer model generate submodule, microlayer model fusion section, signal detection section, And optionally it is incubated the structural regions such as section, extraction section, post-processing section, waste liquid pool.And the liquid drop sensor on chip Optionally hematocrit ratio sensor, flow injecting module being connected to chip fluid (and each valve in the module) etc..

The sample introduction module is the channel that dispersed phase (i.e. sample and reagent etc.) and continuous phase (i.e. oil phase) etc. enter chip Preceding storage room.Sample to be detected can be biological initial liquid sample or other treated biological initial liquids Sample.The including but not limited to blood, blood plasma, serum, tissue fluid, lymph of people or other animals or urine etc..It is to be measured In sample mainly for detection of target object to be checked include but is not limited to antigen, haptens, antibody, protein, nucleic acid, fat Plastid, peptide fragment, nucleotide, amino acid, virus, bacterium, parasite, cell, drug, ion, salt and combinations thereof and other one A little unimolecules or complex etc..

The valve is a kind of controllable switch, accurately controls time and volume that fluid enters chip channel.Valve 1,3 Dispersed phase sample introduction is controlled, valve 2 controls continuous phase sample introduction, and valve 1a, 2a ... wait control reagent injection port, valve 3a control cleanings Reagent sample introduction, valve Ma control eluent sample introduction, and valve Na ... waits control post-treatment reagents sample introduction.Valve includes but is not limited to Paraffin valve, paraffin hot melt valve, magnet mobile valve, operated pneumatic valve, diaphragm valve, hydrophobic valve, machinery valve and its group It closes.

The hematocrit refers to detecting the microlayer model of generated whole blood sample when coping with blood whole blood sample than sensor Hematocrit ratio, or have the sample of corresponding turbidity in loading to the initial judgement of its turbidity.

The liquid drop sensor refers to judging microlayer model, while being digitized mark to qualified microlayer model, with Realize the microlayer model communication of real-time status and control flow anticipation in the chip module.

Microlayer model fusion refers to being added and being combined with object to be detected in the microlayer model that detection sample is formed Labeled substance or the substance that specific reaction occurs therewith merge shape after realizing reagent microlayer model and the demulsification of sample microlayer model At mixing microlayer model.

Incubation refers to providing mixing and parking space for microlayer model, so that solution fully reacts in microlayer model.

Extraction refers to the pretreatment before being measured to the reactant in microlayer model, and removing potentially interferes with testing result Non-targeted reaction product or other substances make it easy to signal detection with enrichment and purification of target object to be checked.

Post-processing refer to be added in microlayer model can with object to be detected in conjunction with and show the substance of signal.

Detection subsystem is read out and analyzes to the signal of object to be detected.

The waste liquid pool is connected with waste collection module, collects waste liquid all in chip, including oil phase waste liquid, cleaning solution Waste liquid, eluent waste liquid, the microlayer model waste liquid etc. for being unsatisfactory for requirement.There are many places waste liquid pool, predominantly dispersed phase sample introduction in chip Waste liquid pool and chip channel end waste liquid pool at mouthful.Waste liquid pool mainly collects cleaning solution waste liquid, chip at dispersed phase injection port Waste liquid pool collects all waste liquids at channel end.

Chip module can be that single layer of chips can also be multilayer chiop, and the material of chip module includes but is not limited to glass Glass, silicon chip, ceramics, plastics, paper and rubber and combinations thereof.Optionally, these materials can be through overdoping, modification, modification Material.Optionally, the micro-fluidic chip made of the material, while suitable for disposable and reuse.

Reagent 1 in the agent bin of present system, 2,3 ... M, N ... are waited and micro-fluidic chip can all be replaced, And the position being connected in replacement process can realize seamless connection by bayonet or other modes, without causing leakage etc. to ask Topic.

As shown in Fig. 2, the present invention system in, online add subsystem (driving power module, flow injecting module it is each Valve, hematocrit are than sensor and liquid drop sensor etc.), temperature control subsystem, signal detection module, automatic sampling subsystem, Waste collection module and reagent storage subsystem carry out signal transmission by mster-control centre, and monitoring, operation, anticipation, receiving is respectively The signal of a module judge and to each module control element, sensor passes signal, is precisely accomplished and is planning to each module Interior control.

Detection method

The present invention also provides a kind of detection methods based on online addition subsystem, as shown in Figure 3, Figure 4, the side Method includes：

1) reagent in the estimated detection project kit examined is placed on corresponding reagent position, system is whole to module Body state includes oil phase (continuous phase) state, cleaning liquid status, driving force module status, valve state, temperature controller state Equal self-tests adjust system according to self-detection result, until self-test is by ensureing that system being capable of normal operation；

2) sample is collected, all samples to be tested are deposited on continuous sample introduction frame, the start button of master controller is clicked, from The dynamic switch for opening negative pressure controller, opens valve 2 (as shown in Figure 1), and oil phase enters core by the continuous phase injection port of chip Piece channel is simultaneously full of all channels in a short time, under the detection of each sensor of chip module, completes self-test, process of self-test master To include whether to have bubble, whether oil phase flowing continuous etc.；

3) under the control of this system, the sample on continuous sample introduction frame passes sequentially through bar code read area, identifies sample bar code, Switch under total system to corresponding detection project control logic, sampling needle draws sample to be tested, and sample to be tested is pushed into core The dispersed phase injection port of piece module opens valve 1；

4) sample to be tested generates the microlayer model of sample to be tested in microlayer model generates submodule；Optionally, sample microlayer model By hematocrit than sensor, system obtains sample hematocrit and than signal and is judged and corrected；Sample microlayer model is passed by drop Sensor judged and marked to it, and system obtains sample microlayer model digital signal, when qualified sample microlayer model quantity reaches inspection It surveys and requires to close valve 1；

5) system automatically turns on positive power source, when the micro- liquid for the sample microlayer model arrival chip for detecting first mark Drop fusion section, Open valve 1a, 2a etc. will belong in the push-in chip channel such as reagent 1,2 under this project logic to be checked and generate 2 microlayer model of 1 microlayer model of reagent and reagent etc., the quantity such as control reagent microlayer model 1,2 be slightly more than or with sample microlayer model quantity Unanimously, close valve 1a, 2a etc., reagent microlayer model contact with each other with sample microlayer model permeate it is a mix microlayer model, pass through Sensor is marked to mixing successfully mixing microlayer model, and system obtains the digital signal of mixing microlayer model；

6) optionally, when detect first mark mixing microlayer model reach be incubated section (or leave microlayer model fusion Section), mixing microlayer model starts to be incubated；Meanwhile sampling needle draws cleaning solution, is pushed into and is full of in the state that valve 1,3 is closed Dispersed phase injection port in chip opens valve 3, and cleaning solution waste liquid flows into waste liquid pool at dispersed phase injection port by waste fluid channel, It is multiple to repeat this operation, valve 3 is closed after cleaning up；

7) optionally, system automatically turn on magnetic field, when detect first mark mixing microlayer model be incubated completely into Region is extracted, the magnetic bead mixed in microlayer model is fixed in chip channel, still keeps small microlayer model state, opens valve 3a, cleaning reagent enter chip channel generate cleaning reagent microlayer model, control cleaning reagent microlayer model quantity be slightly more than or with Sample microlayer model amount is consistent, closes valve 3a, and cleaning reagent microlayer model is in contact with the sample microlayer model for resting on extraction region It merges and then is split into two microlayer models, respectively still fixed microlayer model containing magnetic bead and target object to be checked and do not include The microlayer model containing non-targeted object to be checked of magnetic bead, it is logical that the microlayer model containing non-targeted object to be checked not comprising magnetic bead flows directly into chip The waste liquid pool of road end；

8) optionally, Open valve Ma, the reagent M (eluent) belonged under this project logic to be checked give birth into chip channel At reagent M (eluent) microlayer model, control reagent M (eluent) microlayer model amounts be slightly more than or with containing magnetic bead and target object to be checked Microlayer model amount it is consistent, close valve Ma, reagent M (eluent) microlayer models with it is fixed containing magnetic bead include target object to be checked Microlayer model contact fusion then be split into two microlayer models, do not include the microlayer model for including target object to be checked of magnetic bead respectively The still fixed microlayer model for including magnetic bead, sensor carry out the microlayer model comprising target object to be checked not comprising magnetic bead Label, system obtain the digital signal of the microlayer model comprising target object to be checked not comprising magnetic bead；

9) optionally, after detecting that the microlayer model comprising target object to be checked for not including magnetic bead of first mark enters Processing region, Open valve Na, the reagent N (post-treatment reagents) belonged under this project logic to be checked enter chip channel and generate examination Agent N (post-treatment reagents) microlayer model, control reagent N (post-treatment reagents) microlayer model quantity be slightly more than or with not comprising magnetic bead Including the microlayer model quantity of target object to be checked is consistent, close valve Na, reagent N (post-treatment reagents) microlayer model with do not include magnetic bead The microlayer model comprising target object to be checked be in contact and be fused to the microlayer model that finishes of post-processing, sensor finishes post-processing micro- Drop is marked, and system obtains the digital signal for the microlayer model that post-processing finishes, and post-processes the microlayer model finished and starts again at It is incubated；

10) switch of open detection subsystem, when detect first mark it is detectable or post-processing finish it is micro- Drop reads the detection signal that microlayer model directly or indirectly provides completely into signal detection section, by detection subsystem, synchronous to pass It is sent to progress numerical value processing in master controller and provides visual information；

11) simultaneously, when detecting first mark microlayer model entering signal detection section, sampling needle draws cleaning solution, in valve Door 1,3 is pushed into the state of closing is full of chip dispersed phase injection port, opening valve 1, cleaning solution flushing valve 1, subsequently into Chip interior channel finally enters chip channel end waste liquid pool in the form of microlayer model waste liquid, synchronizes and starts to repeat step 3；

12) detection finishes, and optionally closes magnetic field, and all microlayer models flow into chip channel end waste liquid pool.

The detection method of the present invention selects some or all of step therein according to specific detection project.

The test sample amount volume is 0.5-10 μ L.The single sample testing time be 5-15min, but the present invention detection Method, which can synchronize, carries out multiple sample tests, greatly reduces the population sample testing time.

Main advantages of the present invention include：

(a) microlayer model fusion is accurate, it can be achieved that adding reagent etc., high degree of automation online.

(b) the detection sensitivity height to biological sample can be achieved, reproducible, strong antijamming capability, detection time is small, leads to The purpose of amount is big, and sample consumption is minimum.

(c) microlayer model microfluidic chip technology is used, sample is generated, is mixed, is reacted, is collected and detection is integrated in chip On, all reagent components for reacting required can be accurately controlled by valve in addition chip, and easy to operate, system integration is automatic Change degree height is, it can be achieved that accurately control the behavior of microlayer model.

(d) test sample amount volume described in the device of the invention is 0.5-10 μ L.The single sample testing time is 5- 15min, but the test process can synchronize and carry out multiple sample tests, greatly reduce the population sample testing time.

To sum up, the present invention is by unique online addition subsystem, to the generation of reaction reagent microlayer model carry out it is procedural, The control of systematization is generated and is merged with the accurate of sample microlayer model to realize stablizing for reagent microlayer model, to carry For a kind of extremely efficient and controllable minisize reaction kettle.For the detection process of the biological sample based on differential responses principle, originally Invention can greatly improve biochemical reaction efficiency and detection flux, greatly reduce the consumption of the sample and reagent needed for detection.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are weight percent and weight Number.

Embodiment 1：The double antibody sandwich method detection b-type natriuretic peptide (BNP) based on immune response is realized using the system

Prepare before operation：Reagent 1, reagent 2, reagent 3, reagent M are taken out from the kit of BNP double antibody sandwich methods, are tried Agent 1 is the anti-BNP antibody 1 being coated in 100nm-3um super-paramagnetic bead, and reagent 2 is the anti-BNP antibody 2 for being marked with fluorescent dye, Reagent 3 is cleaning solution, usually the adjustment containing surfactant pH salting liquid, reagent M is eluent, usually pH value Lower solution can be the glycine solution for adjusting pH.Reagent 1,2,3, M are stuck in the corresponding reagent position of agent bin.System System starts self-test, such as：

System self checks oil phase state.The oil phase is the mineral oil of formulated.Such as need to change new oil phase, only need by The mineral oil that the system is provided is placed in the system oil screens, and connects corresponding connector, waits for that system self-check passes through.

System self-check cleaning liquid status, cleaning solution be adjusted added with proper amount of surfactant pH salt buffer it is molten Liquid.Such as the cleaning solution that system prompt more renews, the cleaning solution that need to be only provided the system is placed in the cleaning systems liquid screens, and Corresponding connector is connected, waits for that system self-check passes through.

System self checks power plant module, i.e., positive and negative die block, and positive and negative die block can provide and accurately maintain 1000- A certain setting pressure under 10000Pa atmospheric pressure waits for that system voluntarily detects and passes through.

System is adjusted according to self-detection result, self-test starts continuous mode after passing through.It is as follows to detect detailed process：

1) it posts the whole blood sample comprising sample or patient information and project bar code to be checked to be placed on continuous sample introduction frame, click Start button, this system open the controller switches (1000Pa) as negative pressure, and Open valve 2, continuous phase enters chip module Inside, and all channels of chip module are filled in a short time, under the detection of each sensor of chip module, self-test is completed, from Inspection process includes mainly whether to have bubble, and whether oil phase flowing is continuous etc..After oil phase self-test passes through, this system voluntarily controls as examination Each reagent provides positive pressure in agent storehouse, which is more than atmospheric pressure.This system continues self-test, waits for all chip module channels Start sample process after the exceptions such as interior bubble-free；

2) after the completion of System self-test, this system continuous sample introduction frame controls sample by bar code read area, identifies on sample Bar code information, and automatically switched under corresponding detection control logic according to sample type and project to be checked, sampling needle is inhaled at this time This 10 μ l of sampling, are pushed into the dispersed phase injection port of chip module, open valve 1, and it is qualified micro- enough to wait for that chip module detects After amount of droplets close valve 1, this sample microlayer model generated time usually in 10s, in the detection project shown in this example, sample This generation frequency is 120/s, and microlayer model continuously generates 20-22, the volume of each microlayer model can be estimated by formula (7), Such as 1000 ± 50pL；

3) for whole blood sample, the hematocrit ratio of different samples will have a direct impact on accuracy in detection, but can pass through acquisition Detection was met after sample hematocrit ratio and was corrected, whole blood sample described in this example detects BNP antigens, needs to detect hematocrit ratio To be corrected to its result.At this point, the sample microlayer model generated is sensed respectively by hematocrit than sensor by hematocrit ratio Device obtains sample hematocrit ratio and is transferred to control centre.Sample microlayer model by liquid drop sensor, to the microlayer model that passes sequentially through into Row judges and label, respectively " sample A project BNP microlayer models 1 ", " sample A project BNP microlayer models 2 " ... " sample A projects Digital signal is returned control centre by BNP microlayer models 22 ", and control centre identifies each microlayer model information respectively, and determines each micro- The subsequent control logic of drop；

4) control centre judges the reagent needed for project BNP detections, the response time t of liquid drop sensor₁For 12ms, valve The response time t of unlatching₂It is 8000 μm that for 18ms, in chip, liquid drop sensor generates distance s at mouth with reagent droplet, at this time " sample A project BNP microlayer models 1 " microlayer model volume V_DropFor 1000pl, when mark " sample A project BNP microlayer models 1 " After microlayer model is detected by liquid drop sensor, movement speed v_DropFor 350 μm/s, 22.8s (t_g-k) afterwards control Open valve 1a and 2a, at this time reagent 1,22 microlayer model of 1 microlayer model of reagent and reagent is generated under the driving of chip module negative pressure and agent bin positive pressure, In this chip, the radius R of 2 microlayer model of 1 microlayer model of reagent and reagent is 50 μm, and reagent 1 and the viscosity n phases of reagent 2 are all 2.1N·s/m², it is 35 μm that reagent, which generates mouth width degree W, and downstream passage width w generates the length of fluid column in microchannel with reagent droplet It is all 150 μm to spend L phases, and channel depth h is 100 μm, and the interfacial tension F σ of oil phase and reagent are 28mN/m, and calibration parameter a is 10^-8, calibration parameter b is 10^-6m^-1.Reagent 1 is calculated according to formula (15) through system and the positive pressure driving pressure (P) of reagent 2 is 353.9mbar.After obtaining the above-mentioned data of union, 1 microlayer model of reagent and 2 microlayer model of reagent can be accurately controlled simultaneously Online addition enters the System on Chip/SoC microlayer model integration region, before entering microlayer model integration region, by liquid drop sensor Identification, and control addition and be slightly more than or the microlayer model quantity consistent with sample microlayer model, control herein generates 20-22 micro- liquid Drop.Hereafter, sample microlayer model 1-22 marks magnetic bead reagent microlayer model 1-22 to merge generation mixed with anti-BNP antibody respectively Close microlayer model 1-22.Because of the fusion of no reagent microlayer model, volume do not change extra sample microlayer model, continues to chip end Hold waste liquid pool flowing；

5) it when detecting No. 1 arrival incubation region of mixing microlayer model, mixes microlayer model and starts incubation process, simultaneity factor control Sampling needle processed draws cleaning solution, is pushed into and is full of dispersed phase injection port in the state that valve 1,3 is closed, and opens valve 3, cleaning Liquid waste liquid flows into waste liquid pool at dispersed phase injection port by waste fluid channel, repeats this operation 3 times, valve 3 is closed after cleaning up；

6) system control automatically turns on magnetic field, extracts region when detecting that mixing microlayer model 1 reaches, mixes in microlayer model Magnetic bead be fixed in chip channel, still keep small microlayer model state, open valve 3a, cleaning solution enters chip channel life At cleaning solution microlayer model, control cleaning solution microlayer model quantity is slightly more than or, closing valve 3a consistent with mixing microlayer model quantity, Cleaning solution microlayer model is in contact with mixing microlayer model and merges and then be split into two microlayer models, respectively still fixed to contain magnetic The microlayer model of pearl and target object to be checked and the microlayer model containing non-targeted object to be checked not comprising magnetic bead, do not include magnetic bead contains non-mesh The microlayer model for marking object to be checked flows directly into the waste liquid pool of chip channel end；

7) Open valve Ma, the reagent M (eluent) belonged under this project logic to be checked enter chip channel and generate reagent M Microlayer model, control eluent microlayer model quantity be slightly more than or with the microlayer model quantity one for including target object to be checked containing magnetic bead It causes, closes valve Ma, it is right that eluent microlayer model contacts fusion with the fixed microlayer model comprising target object to be checked containing magnetic bead After be split into two microlayer models, do not include the microlayer model comprising target object to be checked of magnetic bead respectively and still fixed include The microlayer model comprising target object to be checked not comprising magnetic bead is marked in the microlayer model of magnetic bead, sensor, and system is not wrapped The digital signal of the microlayer model 1-22 comprising target object to be checked containing magnetic bead.

8) automatically turn on signal detection module controller switch, when detect not comprising magnetic bead include target object to be checked No. 1 entering signal reading area of microlayer model, read the fluorescent assay signal that directly gives of microlayer model, synchronous driving to master control Numerical value processing is carried out in device provides visual information；

9) simultaneously, when detecting No. 1 entering signal reading area of microlayer model comprising target object to be checked not comprising magnetic bead, Sampling needle draws cleaning solution, and chip dispersed phase injection port is pushed into and be full of in the state that valve 1,3 is closed, and opens valve 1, clearly It is useless finally to enter chip channel end subsequently into chip interior channel in the form of microlayer model waste liquid for washing lotion flushing valve 1 Liquid pool synchronizes and starts to repeat step 3；

10) detection finishes, and closes magnetic field, and all microlayer models flow into chip channel end waste liquid pool.

11) for the chemiluminescence detection system that marker is acridinium ester etc., corresponding kit only need to be placed, wherein Reagent 1 is the anti-BNP antibody 1 being coated in 100nm-3um super-paramagnetic bead, and reagent 2 is the anti-BNP antibody 2 for being marked with a word used for translation and stining ester, Reagent 3 is cleaning solution, and reagent N is chemiluminescent substrate reagent.It additionally needs and above-mentioned microlayer model is recycled to melt after extraction process The mode of conjunction adds the chemiluminescent substrate reagent of reagent N position, and the reading of subsequent detection position can be entered by carrying out being incubated again Useful signal.

12) for homogeneous phase time discrimination fluorescence method detection architecture, corresponding kit only need to be placed, wherein reagent 1 is The anti-BNP antibody 1 of Terbium dye markers, reagent 2 are the anti-BNP antibody of d2 dye markers 2.When micro- liquid of reagent 1 and reagent 2 After drop is merged with sample microlayer model, without cleaning and elution, subsequent detection position can be directly entered and read useful signal.

Experimental result：

Herein by taking homogeneous phase time discrimination fluorescence method detection architecture as an example.

Fig. 5 is sample droplets and reagent droplet fusion process schematic diagram；

A-e as shown in Figure 5) it is sample droplets (compared with Dark grey) and reagent droplet (more light grey) fusion process schematic diagram, Wherein scale is 50 μm.

What is provided in Fig. 5 is the microphoto of the one-to-one fusion process of sample droplets and reagent droplet, as can be seen from Figure 5 two Kind drop is mixed into a kind of drop then progress next step operation under passive control.Wherein, continuous phase of the invention flows to the right Dynamic, sample microlayer model (such as sample microlayer model 1) and reagent microlayer model (such as reagent microlayer model 1) enter microlayer model with continuous phase and merge Section is simultaneously merged in fusion section contact, and continues to flow with continuous phase, ultimately forms the mixing drop of sample and reagent.

BNP is detected based on this system, the standard curve for measuring gradient concentration first is needed, is listed in table 1 The concrete numerical value of standard curve.What is provided in Fig. 6 is the standard curve of homogeneous phase time discrimination fluorescence method detection BNP.

1 homogeneous phase time discrimination fluorescence method of table detects BNP standard curve numerical value

The concentration listed in table 1 is detected and is then back-calculated according to standard curve, obtains being back-calculated result such as table 2 It is shown.It is found that detection linearly ranging from 25-25600pg/ml of this detecting system for BNP, linearly dependent coefficient r >=0.99, Functional Sensitivity reaches 25pg/mL, repeated coefficient of variation CV≤5%, relative deviation RD≤10%.

2 homogeneous phase time discrimination fluorescence method of table detection BNP standard curves are back-calculated result

It extracts the sample that 5 are detected determining various concentration value through goldstandard and carries out Preliminary detection, result in the present system In table 3.

Table 3 detects the pattern detection result for determining various concentration value through goldstandard

Sample number	Concentration (pg/ml)	Ratio (665/620)	Result of calculation	Relative deviation
					1	15.4	536.7232	16.82391	9%
2	55.67	565.0035	57.09397	3%
					3	126.8	609.9557	121.1042	- 4%
4	245.4	694.2445	241.1282	- 2%
					5	846.53	1156.398	899.2169	6%

Experiment conclusion：Thus provable this system is by accurately calculating and manipulating the online addition, it can be achieved that reaction reagent, Complete the accurate quantification detection of BNP.

Embodiment 2：Realize that the influenza A virus based on loop-mediated isothermal amplification detects using the system

Prepare before operation：It include reagent 1 in influenza A virus detection kit.Wherein reagent 1 is containing for A type The specificity amplification primer of influenza virus design is to, the reaction buffer etc. of dNTPs, BstDNA polymerase, certain ion concentration. The process of self-test of system is same as Example 1.

Specifically detection process is：

1) post the sample of nucleic acid after purified comprising sample or patient information and project bar code to be checked be placed on continuously into In sample rack, start button is clicked.

2) read bar code information post-sampling needle draw sample push-in chip module dispersed phase injection port at, remaining process with Embodiment 1 is identical, wherein it is 80-120/s that sample, which generates frequency, and microlayer model continuously generates 5-10, can by formula (7) Estimate the volume of each microlayer model, such as 1000 ± 50pL.

3) sample microlayer model is judged and is marked to the microlayer model passed sequentially through, respectively " sample by liquid drop sensor This A project first streams microlayer model 1 ", " sample A project first streams microlayer model 2 " ... " sample A project first streams microlayer model 10 ", remaining mistake Journey is same as Example 1.

4) control centre judges the reagent needed for the detection of project first stream, the response time t of liquid drop sensor₁For 12ms, valve The response time t that door is opened₂It is 10000 μm that for 18ms, in chip, liquid drop sensor generates distance s at mouth with reagent, at this time " sample A project first streams microlayer model 1 " microlayer model volume V_DropFor 1000pl, when mark " sample A project first streams microlayer model 1 " Microlayer model detected by liquid drop sensor after, movement speed v_DropFor 400 μm/s, 25s (t_g-k) Open valve 3a is controlled afterwards, this When 1 premixed liquid of reagent premixed liquid microlayer model is generated under the driving of chip module negative pressure and agent bin positive pressure, in this chip, in advance Mixed liquid microlayer model radius R is 100 μm, and the viscosity n phases of premixed liquid are all 5.1Ns/m², it is 80 μm that premixed liquid, which generates mouth width degree W, Downstream passage width w and the length L phases of fluid column in premixed liquid drop formation microchannel are all 200 μm, and channel depth h is 300 μm, The interfacial tension F of oil phase and reagent_σIt is 10 for 34mN/m, calibration parameter a^-5, calibration parameter b is 10^-5m^-1.It is pre- through system-computed The positive pressure driving pressure (P) of mixed liquid is 640.2mbar.After obtaining the above-mentioned data of union, premixed liquid drop can be by It accurately controls and adds online and enter the System on Chip/SoC microlayer model integration region, before entering microlayer model integration region, by Liquid drop sensor identifies, and controls addition and be slightly more than or the microlayer model quantity consistent with sample microlayer model, and control herein generates 10 microlayer models.Hereafter, sample microlayer model 1-10 merges generation with premixed liquid microlayer model 1-10 and mixes micro- liquid respectively Drop 1-10.

5) when detecting No. 1 arrival incubation region of mixing microlayer model, the temperature control storehouse temperature of chip module is 60-65 at this time DEG C, mixing microlayer model starts incubation process, and completes specific amplification process in the process.Start simultaneously at cleaning dispersed phase into Sample mouth, detailed process are same as Example 1.

6) after ten minutes, microlayer model flow to detection zone via area is incubated, and No. 1 arriving signal of microlayer model is mixed when detecting Reading area automatically turns on the switch of signal detection module controller, reads the turbidity variable signal of microlayer model, synchronous driving is arrived Numerical value processing is carried out in master controller provides visual information；Wash-out valve 1 simultaneously, process is same as Example 1.

7) detection finishes, and all microlayer models flow into chip channel end waste liquid pool, synchronizes and starts to repeat step 2.

Embodiment 3：It is realized using the system and urine total protein concentration is detected based on BCA methods

Prepare before operation：Include reagent 1, reagent 2 in the kit of BCA methods detection urine total protein concentration.Its pilot scale Agent 1 is the 1%BCA disodium salts of pH11.25,2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% sodium hydroxide, 0.95% The mixed solution of sodium bicarbonate, reagent 2 are 4% copper-baths.All reagents are placed at corresponding reagent position.System Process of self-test is same as Example 1.

Specifically detection process is：

1) it posts the urine comprising sample or patient information and project bar code to be checked to be placed on continuous sample introduction frame, click starts Button.

2) read bar code information post-sampling needle draw sample push-in chip module dispersed phase injection port at, remaining process with Embodiment 1 is identical, wherein it is 200/s that sample, which generates frequency, and microlayer model continuously generates 30-32, can be estimated by formula (7) Calculate the volume of each microlayer model, such as 500 ± 25pL.

3) sample microlayer model is judged and is marked to the microlayer model passed sequentially through, respectively " sample by liquid drop sensor This A project BCA microlayer models 1 ", " sample A project BCA microlayer models 2 " ... " sample A project BCA microlayer models 32 ", remaining process with Embodiment 1 is identical.

4) control centre judges the reagent needed for project BCA detections, the response time t of liquid drop sensor₁For 12ms, valve The response time t of unlatching₂It is 6000 μm that for 18ms, in chip, liquid drop sensor generates distance s at mouth with reagent, at this time The microlayer model volume V of " sample A project first streams microlayer model 1 "_DropIt is micro- when mark " sample A project first streams microlayer model 1 " for 500pl After drop is detected by liquid drop sensor, movement speed v_DropFor 300 μm/s, 20.0s (t_g-k) Open valve 1a and 2a are controlled afterwards, Reagent 1,2 generates 2 microlayer model of 1 microlayer model of reagent and reagent under the driving of chip module negative pressure and agent bin positive pressure at this time, In this chip, the radius R of 2 microlayer model of 1 microlayer model of reagent and reagent is 75 μm, and reagent 1 and the viscosity n phases of reagent 2 are all 1.8N·s/m², it is 40 μm that reagent, which generates mouth width degree W, and downstream passage width w generates the length of fluid column in microchannel with reagent droplet It is all 150 μm to spend L phases, and channel depth h is 200 μm, the interfacial tension F of oil phase and reagent_σIt is 10 for 30mN/m, calibration parameter a^-6, calibration parameter b is 10^-6m^-1.Positive pressure driving pressure P through system-computed reagent 1 and reagent 2 is 305.4mbar.It is obtaining simultaneously After the above-mentioned data of operation, 1 microlayer model of reagent and 2 microlayer model of reagent can be accurately controlled and add online and enter this and be Chip microlayer model integration region of uniting is identified before entering microlayer model integration region by liquid drop sensor, and controls addition slightly It is more than or the microlayer model quantity consistent with sample microlayer model, herein control generates 30-32 microlayer model.Hereafter, sample microlayer model No. 1-32 merges generation mixing microlayer model 1-32 with 1 microlayer model of reagent and 2 microlayer model of reagent respectively.The extra micro- liquid of sample The fusion because of no reagent microlayer model is dripped, volume does not change, continues to flow to chip end waste liquid pool.

5) it when detecting No. 1 arrival incubation region of mixing microlayer model, mixes microlayer model and starts incubation process, start simultaneously at clear Dispersed phase injection port is washed, detailed process is same as Example 1.

6) when detecting mixing No. 1 arriving signal reading area of microlayer model, signal detection module controller is automatically turned on Switch, read the optical absorption intensity signal of microlayer model, in synchronous driving to master controller according to the standard curve of measured in advance into The processing of line number value provides testing result；Wash-out valve 1 simultaneously, process is same as Example 1, synchronizes and starts to repeat step 2.

7) detection finishes, and all microlayer models flow into chip channel end waste liquid pool.

Experimental result：

Urine total protein concentration is detected based on this system, needs the standard for measuring gradient concentration standard items first Curve lists the concrete numerical value of standard curve in table 4, and Fig. 7 is standard curve.

Table 4 is based on BCA methods and detects urine total protein concentration standard curve numerical value

The sample for extracting 5 various concentration values is detected, and result is in table 5.

5 pattern detection result of table

Sample number	OD₅₆₂	It is back-calculated result (ug/ml)
			1	1.8567	1262.357
2	0.8233	524.2143
			3	0.3064	155
4	0.2356	104.4286
			5	0.1527	45.21429

Experiment conclusion：Thus provable this system by accurately calculate and manipulate, it can be achieved that reaction reagent it is accurate online The accurate quantification detection that urine total protein concentration is detected based on BCA methods is realized in addition.

Compared with lateral flow chromatographs platform method, by taking the BNP of embodiment 1 detections as an example, the microfluidic platform in the present invention Method has required sample size and amount of reagent few, and detection flux is big, and the detection range of linearity is wide, and detection CV values are small etc. main excellent Gesture.

The demand that lateral flow chromatographs plasma sample in platform method is 50-250 μ l, and 10 μ l are only needed in the present invention；Side It is 0.3 μ g to be analysed to fluid layer and each test required 1 dosage of antibody in platform method, and 2 dosage of antibody is 0.625 μ g, and of the invention In each test required 1 dosage of antibody be 0.0023 μ g, 2 dosage of antibody be 0.04 μ g；Lateral flow, which chromatographs in platform method, leads to The smaller each test of amount is required to 15-30min, and there was only first test needs~15min in the present invention, later every 2min A sample is can detect, flux greatly improves；Lateral flow chromatographs the linear ranging from 25pg/ of detection of BNP in platform method Ml-5000pg/ml, and the linear ranging from 50pg/ml-25600pg/ml of the detection of BNP in the present invention；Lateral flow chromatographs platform side The detection CV values of BNP are less than 15% in method, and the detection CV values of BNP can ensure that less than 10% in the present invention.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Claims

1. a kind of detecting system of online addition reaction reagent, which is characterized in that the system comprises：

(1) micro-fluidic chip, the micro-fluidic chip include：

(1.1) micro-fluidic flow-through channel：The micro-fluidic flow-through channel is used for for entrained in continuous phase and continuous phase Microlayer model flows, including：Continuous phase adds section, sample adds section, sample microlayer model generates section, reagent microlayer model generates section, micro- Droplet coalescence section and signal detection section；With

(1.2) microlayer model generates submodule：Mouth is generated including microlayer model；The microlayer model generates submodule for making dispersed phase Microlayer model is formed, the dispersed phase includes：Sample liquid or reagent solution；

(2) subsystem is added online, for generating the fusion microlayer model containing sample and reagent, the online addition subsystem packet It includes：

(2.1) power module is driven：The driving power module is used to provide the driving force needed for dispersed phase and continuous phase, to make The microlayer model of continuous phase and each dispersed phase obtains required flow velocity and forms reagent microlayer model；

(2.2) flow injecting module：The flow injecting module is connected with continuous phase and each dispersed phase, the continuous phase And each dispersed phase is entered by the module in the micro-fluidic chip, the flow injecting module includes control valve, The control valve enters the micro-fluidic flow-through channel for controlling continuous phase and dispersed phase；

(2.3) microlayer model marks processing module：The microlayer model label processing module includes liquid drop sensor；The drop Number and number consecutively of the sensor for reading microlayer model and the flow velocity for reading microlayer model；With

(2.4) data processing and control signal generation module：The data processing and control signal generation module are according to the inspection The predefined parameter of examining system, the characteristic parameter of continuous phase and each dispersed phase fluid, the flow velocity of the microlayer model and number and The flow velocity of continuous phase generates the signal for controlling the driving power module and the letter for controlling the flow injecting module Number；And

(3) subsystem is detected：The detection subsystem is used to carry out signal to the fusion microlayer model for having generated readable signal It reads, and records feedback signal data.

2. the system as claimed in claim 1, which is characterized in that generate mouth to reagent microlayer model in sample microlayer model and generate mouth The first liquid drop sensor is equipped at micro-fluidic flow-through channel, first liquid drop sensor is for reading the sample microlayer model Number and number consecutively, and read the flow velocity of each sample microlayer model.

3. the system as claimed in claim 1, which is characterized in that (2.4) data processing and control signal generation module Signal is generated according to parameters described below,

Including：As system predefined parameter, first liquid drop sensor to reagent microlayer model generates the distance s, described of mouth Reagent microlayer model generates the channel depth that reagent microlayer model in the channel width W of mouth, (1.1) micro-fluidic flow-through channel generates section When spending the response of h, the width w of downstream passage of reagent microlayer model generation mouth, the response time of each valve and sensor Between；

By the flow velocity v for the sample microlayer model that (2.3) microlayer model label processing module is read_{Drop k}；Wherein, k is the volume of sample microlayer model Number；

And

(2.4) data processing and the signal for controlling the generation of signal generation module include：For controlling the flow injecting The signal t of the dispersed phase valve opening time of module_g-k, and generate for controlling the driving power module and driven needed for microlayer model The signal of power P.

4. the system as claimed in claim 1, which is characterized in that the flow injecting module is based on formula 10

In formula, t₁For sensor response time, t₂Response time, v for valve opening_{Drop k}For the flow velocity of sample microlayer model k, In, k is the number of sample microlayer model and s is that sample microlayer model generates mouth from first liquid drop sensor to reagent microlayer model The distance passed through.

5. the system as claimed in claim 1, which is characterized in that the driving power module is based on formula 15 and provides line addition reaction Driving force P needed for reagent：

In formula, s is generated the distance passed through, the h of mouth by sample microlayer model from first liquid drop sensor to reagent microlayer model The channel depth of section is generated for reagent microlayer model, W is that channel width, L that reagent microlayer model generates mouth are that reagent microlayer model generates Oral fluid drop initial stage shows as the liquid length of liquid column form, R is microlayer model radius, v_{Drop k}For the stream of sample microlayer model k Speed, width, the t that w is the downstream passage that reagent microlayer model generates mouth₁For sensor response time, t₂For valve opening response when Between, F_σSurface tension between continuous phase and dispersed phase, a and b are each independently calibration parameter, and n is the viscous of reagent fluid Degree.

6. system described in claim 1, which is characterized in that the micro-fluidic chip further includes that (1.3) sample microlayer model is incubated Submodule is educated, the micro-fluidic flow-through channel further includes being incubated section；The sample microlayer model is incubated submodule for providing The sample microlayer model being added after reaction reagent fully reacts and/or mixed condition and the incubation section are for providing Fusion microlayer model fully reacts and mixed space.

7. the system as claimed in claim 1, which is characterized in that further include：

(4) automatic sampling subsystem, the automatic sampling subsystem include：Sampling needle, syringe pump, continuous sample introduction frame, bar code Read module and sample-adding module；Wherein, the bar code reading module is located in continuous sample introduction frame；The sampling needle passes through institute The syringe pump stated draws sample and the sample-adding module is added in the sample by the syringe pump；The sample-adding mould Block is equipped with a sample adding mouth, and the sample-adding module is to be in fluid communication with the flow injecting module.

8. the system as claimed in claim 1, which is characterized in that the system further includes (5) reagent storage subsystem, described Reagent storage subsystem include continuous phase bit of storage, cleaning solution storage position and agent bin；The agent bin includes premix Even device and one or more reagents position；

Wherein, the pre- vortex mixer is for avoiding agent precipitate or aggregation；The reagent position can store each reagent, institute The reagent stated is entered by the flow injecting module in the micro-fluidic chip.

9. a kind of detection method that microlayer model adds online, which is characterized in that include the following steps：

(1) detecting system of online addition reaction reagent as described in claim 1 is provided；

(3) the sample liquid to be measured forms sample microlayer model in the microlayer model of micro-fluidic chip generates submodule, It marks the liquid drop sensor of processing module to be detected (or reading) to the sample microlayer model by sample microlayer model and appoints Number is marked to carrying out the sample microlayer model in selection of land, stops when the sample microlayer model quantity of generation reaches predetermined quantity Generate sample microlayer model；

(4) described after each sample microlayer model in the sample microlayer model reaches the precalculated position in micro-fluidic chip The control signal for driving power module to be generated according to the data processing and control signal generation module, generates a reagent microlayer model And the reagent microlayer model is driven to be moved by predetermined speed, and make a reagent microlayer model and a sample micro- Drop is in contact fusion in flow process, forms fusion microlayer model；

(5) in the flow process of the fusion microlayer model, the fusion microlayer model is post-processed, and is detected, from And obtain the testing result of the sample liquid.

10. method as claimed in claim 9, which is characterized in that step further includes in (2)

(2.1) sample (or sample liquid) to be measured is placed on continuous sample introduction frame, open system, continuous phase passes through the fluid Sample introduction module enters the micro-fluidic chip and is full of the micro-fluidic flow-through channel；

(2.2) under the control of the system, the sample on continuous sample introduction frame passes sequentially through bar code read area, identifies sample strip Code, sampling needle draw sample to be tested, and sample to be tested is pushed into the adding mouth of sample-adding module by syringe pump, passes through the fluid Sample introduction module enters the micro-fluidic chip.