CN103207169B - Microfluidic time-resolved fluorescence immunoassay device and application thereof - Google Patents
Microfluidic time-resolved fluorescence immunoassay device and application thereof Download PDFInfo
- Publication number
- CN103207169B CN103207169B CN201310081065.9A CN201310081065A CN103207169B CN 103207169 B CN103207169 B CN 103207169B CN 201310081065 A CN201310081065 A CN 201310081065A CN 103207169 B CN103207169 B CN 103207169B
- Authority
- CN
- China
- Prior art keywords
- sample
- pool
- valve
- liquid pool
- pump
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Automatic Analysis And Handling Materials Therefor (AREA)
Abstract
The invention provides a microfluidic time-resolved fluorescence immunoassay device. The device comprises a sample injection unit (101), a microfluidic control unit (103), a detection unit (104) and a microfluidic chip (102) adopting a membrane pump drive mode, and the microfluidic chip (102) is positioned in a microfluidic chip carrier on an analysis workbench of the microfluidic control unit (103). The microfluidic time-resolved fluorescence immunoassay device has the advantages that by adopting the microfluidic technology, a detection system is simplified; and a microplate oscillator and a microplate washer are omitted, a microfluidic control system is simple in structure, optimized, free of mechanical movements and structures of cleaning, vibration, transmission and the like, and automation is achieved conveniently.
Description
Technical field
The invention belongs to field of immunology, particularly Time-resolved fluorescence assay device and application thereof.
Background technology
Time-resolved fluorescence assay technology has the lanthanide series of unique fluorescent characteristic and sequestrant thereof as tracer, a kind of novel on-radiation trace analysis of foundation.TRFIA methodological study and clinical practice development are rapidly, become a new milestone of labelling immunoassay development, TRFIA technology have highly sensitive, standard curve range is wide, easy and simple to handle, the advantage such as no radioactivity pollute, multiple labeling, be widely used in the clinical trial diagnosis of tumour, infectious disease, endocrine system disease, autoimmune disease, genetic disease, become one of analysis means commonly used in biomedical research and clinical ultramicron biochemical investigation.
TRFIA(time resolved fluoro-immunoassay) make use of the 3 valency rare earth ions with unique fluorescent characteristic and chelate is that tracer replaces fluorescent material, enzyme, isotope, chemiluminescent substance, labelled antibody, antigen, hormone, polypeptide, protein, nucleic acid probe and biological cell, question response system (as: antigen-antibody reaction, nucleic acid probe hybridization, biotin-labeled pentylamine reaction and target cell pairing effect cell kill and wound reaction etc.) occur after, by the fluorescence intensity in TRFIA detector assaying reaction product.According to the ratio of product fluorescence intensity and relative intensity of fluorescence, judge the concentration analyzing thing in reaction system, thus reach quantitative test.
In the time-resolved fluoroimmunoassay detection method of prior art, general detection completes on 96 microwell plates, a complete optimization system preparation should comprise an automatic pipettor, a microwell plate oscillator, a microwell plate washes trigger, a couveuse, a time-resolved fluorescence immunoassay instrument; Or the full-automatic time-resolved fluorescence immunoassay instrument of an integrated above-mentioned functions.Detecting step is generally: 1) → add sample, add sample-A 50 μ l; 2) → add dilution-B 100 μ l; 3) → vibration, antigen-antibody (bag quilt) reaction bonded, about 60min; 4) → and wash plate, add cleaning fluid-suction empty, then it is empty to add cleaning fluid-suction, 2 ~ 6 times; Or manual cleaning, buckle is cleaned; 5) → add europium marking fluid-C 150 μ l; 6) → vibration, " tape label antibody "+" antigen-antibody (bag quilt) reaction bonded body, is formed sandwich ", about 30min; 7) → wash plate, with step 4; 8) → add the enhancing liquid-D 150 μ l that dissociates; 9) → vibration, dissociate europium, about 5min; 10) → detect.Whole testing process, performs 4 step application of samples, and (altogether 96*4=376 time), wash plate 2 times, vibrate 3 times, the used time is 150 ~ 180min about.And perform the basic support equipment of semi-automatic detection and be: a microwell plate autopipette, microwell plate oscillator, microwell plate washes trigger.Prior art uses semi-automatic checkout equipment, and the time of needs is longer, more take manpower.
Full-automatic time-resolved fluorescence immunoassay instrument refers to that time-resolved fluoroimmunoassay experiment (comprise application of sample, hatch, wash plate, add operations such as strengthening liquid) and the detection of fluorescence successively all complete on whole instrument, needs artificial participation hardly; Although eliminate the loaded down with trivial details of artificial application of sample and inaccuracy, especially when the detection of great amount of samples, but there is following major defect: apparatus structure is complicated, expensive equipment; Instrument uses, maintenance requirement is high, and comprehensive use cost is high, cannot popularize use.
And semi-automatic detection system configuration needs 4 kinds of equipment: one " microwell plate autopipette ", one " microwell plate oscillator ", one " microwell plate rinsing maching ", a time-resolved fluorescence immunoassay instrument.It also has following defect: detection system is complicated, uses 4 kinds of equipment; Instrument uses, maintenance requirement is high, and comprehensive use cost is high, cannot popularize use.
Summary of the invention
In order to solve the problem, the object of the invention is to adopt Time-resolved fluorescence assay technology and film to move the application of microfluidic chip technology in biological chemistry and molecular biology application, a kind of combination using film to move microfluidic chip technology and time-resolved fluoroimmunoassay detection technique is provided especially, realizes full-automatic micro-fluidic time-resolved fluoroimmunoassay detection method.
In order to realize the object of the invention, concrete technical scheme is:
A kind of micro-fluidic time resolved fluoro-immunoassay device, comprise application of sample unit, micro-fluidic control module, detecting unit, it also comprises the micro-fluidic chip adopting membrane pump type of drive, and described micro-fluidic chip is arranged in the micro-fluidic chip seat on micro-fluidic control module analytical work platform.
Wherein, described micro-fluidic chip comprises: sample pool, dilute liquid pool, mark liquid pool, the liquid pool that dissociates, cleaning liquid pool, waste liquid pool;
Sample valve and sample through hole is provided with in wherein said sample pool; Dilution valve and dilution through hole is provided with in dilution liquid pool; Be provided with marking fluid valve and marking fluid through hole in described mark liquid pool, described in dissociate in liquid pool and be provided with dissociation solution valve and the fluid through-hole that dissociates, described cleaning liquid pool is provided with cleaning fluid valve and cleaning fluid through hole; Described waste liquid pool is provided with waist valve and waste liquid through hole;
Main valve is arranged at the centre of each valve, and each valve is connected with main valve respectively by passage.
Wherein, described sample valve, main valve and dilution liquid pool valve and passage, form sample pool-dilution liquid pool two-way pump Sample Dilution pump; Described sample valve, main valve and waist valve and passage, form sample pool-waste liquid pool one-way pump sample waste drains pump; Described sample valve, main valve and cleaning fluid valve and passage, form sample pool-cleaning liquid pool one-way pump sample scavenging pump; Described sample valve, main valve and marking fluid valve and passage, form sample pool-mark liquid pool two-way pump sample labeling pump; Described sample valve, main valve and dissociation solution valve and passage, form the sample pool-liquid pool two-way pump sample solution that dissociates from pump.
Wherein, described sample pool inwall is coated with antibody or antigen.
Wherein, described micro-fluidic chip is that light tight polymeric material is made.Be preferably polystyrene (PS) or acrylonitrile-butadiene-styrene (ABS) plastics (ABS).
Wherein, described application of sample unit includes three-dimensional motion mechanism, and three-dimensional motion mechanism has feed head and sample-adding pump; Three-dimensional motion mechanism is at analytical work platform range of motion; Described application of sample unit also includes reagent cup, and described reagent cup comprises dilution cup, marking fluid cup, dissociation solution cup, and cleaning fluid cup is all placed on device analysis work top.
Wherein, described micro-fluidic control module, comprises single-chip microcomputer, control circuit system, controls finishing equipment operation and the communication with external unit; Chip carrier and chip match, and the position corresponding with micro-fluidic chip membrane pump are furnished with drives structure, for the driving to micro-fluidic chip membrane pump.
Membrane pump mechanical work principle as shown in Figure 9.The position of departing from its script when barrier film stress deformation just defines diaphragm seal chamber.This film chamber makes two passages be linked up, and makes liquid in passage be inhaled into film chamber.By continuous print six step cycle work, realize liquid transmission, just define pump.Inverted order operation the left side and the right valve film can alter flowing direction, realize the two-way operation of pump.In Fig. 9, down arrows represents the direction of the power acting on barrier film; Right arrow represents pumping direction.Before runtime, pump is in a state, and all valves are all in closed condition.When b state, liquid feed valve is opened, and liquid is inhaled into liquid feed valve from liquid inlet channel.Next be c state, pumping diaphragm is opened, and is drawn into more liquid and enters pumping system.D, e, f state is respectively: liquid feed valve is closed, and liquid valve is opened, and pumping diaphragm is closed to pump liquid.
Wherein, described micro-fluidic control module, also comprises analytical work platform, platform has micro-fluidic chip seat, and chip fixes fastener, and chip is stably fixed on chip carrier.
Wherein, described fastener is mechanical type fastener or vapour-pressure type fastener.
Wherein, described detecting unit comprises excitation source and excitation light path unit, receiving light path unit, signal processing unit.
Using device provided by the invention to carry out the method for micro-fluidic time resolved fluoro-immunoassay, is on micro-fluidic chip, and by the control to microfluidic valve pump, the institute of deadline resolved fluorometric immune detection in steps.
Particularly, micro-fluidic time-resolved fluorescence immunoassay method, comprises the following steps:
(1) sample is added sample pool, dilution adds dilution liquid pool, and cleaning fluid adds cleaning liquid pool, and marking fluid adds mark liquid pool, and the enhancing liquid that dissociates adds the enhancing liquid pool that dissociates;
(2) start micro-fluidic control module work: antigen-antibody combines, effluent discharge, cleaning, effluent discharge, mark combines, effluent discharge, cleaning, effluent discharge, and enhancing of dissociating detects.
Wherein, described in step (2), antigen-antibody in conjunction with detailed process is: sample pool-dilution liquid pool two-way pump starts two-way operation, sample is mixed with dilution; During stopping, mixed liquor is all stored in sample pool.
Described effluent discharge detailed process is: sample pool-waste liquid pool one-way pump one-way only operation, enters waste liquid pool by mixed liquor.
Described cleaning detailed process is: sample pool-cleaning liquid pool one-way pump one-way only operation, sucks sample pool by cleaning fluid.
Or described cleaning detailed process is: to dilute liquid pool as cleaning Buffer Pool, sample cell first gets rid of waste liquid, sucks cleaning fluid, then closes one-way pump sample scavenging pump and one-way pump sample waste drains pump; Start two-way pump, cleaning fluid is flowed between sample pool and dilution liquid pool, cleans; During stopping, cleaning fluid is in sample pool.
Described mark in conjunction with detailed process is: two-way pump starts, and marking fluid is flowed between sample pool and mark pond, carries out mark and combine, stop tense marker liquid in sample cell.
Described dissociating strengthens detailed process and is: two-way pump starts, and makes to dissociate to strengthen liquid at sample pool with dissociate to strengthen between liquid pool and flow, and dissociates, and dissociates to strengthen liquid dissociating and strengthen in liquid pool during stopping.
Described detection detailed process is: the detection position that detecting unit moves to micro-fluidic detection chip is detected.
The application of method in biomedical research or clinical ultramicron biochemical investigation that the present invention proposes.
Beneficial effect of the present invention is:
1. adopt microflow control technique, detection system is simplified; Eliminate microwell plate oscillator, microwell plate washes trigger, and micro-fluidic Control system architecture is simple, optimizes, and without mechanical motion and structures such as cleaning, vibration, transmissions, is convenient to realize robotization;
2. microflow control technique improves reaction efficiency: antigen-antibody combines, in sample pool, antibody is coated on sample pool, membrane pump works, solution is ceaselessly back and forth flowed between two ponds, wash away sample pool inwall bag quilt cover, make the antibody of the antigen in solution or antibody and bag quilt cover or antigen effectively contact more abundant;
3. improve cleaning efficiency: micro-fluidic pump valve drives liquid flow, realize flowing cleaning;
4. detection speed improves;
5. the preparation of sample and reagent, before the reaction, by sample and various reagent is disposable adds in container respective in chip, completes sample, reagent prepares; Undertaken by micro-fluidic the operation that sample, reagent carry out afterwards, in the sample with a collection of detection, all operations control, and the reaction time is on all four, avoids the deviation between sample on the reaction time;
6. sample and reagent are communicated with by the valve pump of micro-fluidic inside and mix, clean, and avoid the sputtering using pipettor, sample injector may cause at chip upper direction and pollute;
7. mark combination, dissociation process and antigen-antibody cohesive process are similar, equally because membrane pump drives solution flowing, improve reaction velocity;
8. operating process is closed, and avoids the pollution to personnel and environment, improves bioorganism security.
In a word, micro-fluidic time resolved fluoro-immunoassay device of the present invention, adopts microflow control technique, detection system is simplified; Eliminate microwell plate oscillator, microwell plate washes trigger, and micro-fluidic Control system architecture is simple, optimizes, and without mechanical motion and structures such as cleaning, vibration, transmissions, is convenient to realize robotization; Micro-fluidic time-resolved fluorescence immunoassay method of the present invention substantially increases the detection efficiency of sample.
Accompanying drawing explanation
Fig. 1 is the micro-fluidic time resolved fluoro-immunoassay structure drawing of device of the present invention;
Fig. 2 is micro-fluidic chip vertical view of the present invention;
Fig. 3 is micro-fluidic chip bottom plan view of the present invention;
Fig. 4 is sample pool of the present invention-dilution liquid pool two-way pump workflow diagram;
Fig. 5 is sample pool of the present invention-waste liquid pool one-way pump and sample pool-cleaning liquid pool one-way pump workflow diagram;
Fig. 6 is that the present invention dilutes liquid pool and cleaning Buffer Pool workflow diagram;
Fig. 7 is sample pool of the present invention-mark liquid pool two-way pump workflow diagram;
Fig. 8 is sample pool-dissociation solution (detection) pond of the present invention two-way pump workflow diagram;
Fig. 9 is membrane pump mechanical work principle figure.
Wherein, each component serial numbers of device is as follows:
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to protection scope of the present invention.
If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art.
Embodiment 1: micro-fluidic time resolved fluoro-immunoassay device
As shown in Figure 1, Figure 2 and Figure 3, a kind of micro-fluidic time resolved fluoro-immunoassay device, comprises application of sample unit 101, micro-fluidic control module 103, detecting unit 104, and it also comprises the micro-fluidic chip 102 adopting membrane pump type of drive.Micro-fluidic chip 102 is arranged in the micro-fluidic chip seat on micro-fluidic control module 103 analytical work platform.
Micro-fluidic core 102 comprises following 6 ponds: sample pool 201, dilution liquid pool 202, mark liquid pool 203, dissociate liquid pool 204, cleaning liquid pool 205, waste liquid pool 206, wherein be provided with sample valve 301 and sample through hole 211 in sample pool 201, dilution valve 302 and dilution through hole 212 is provided with in dilution liquid pool 202, marking fluid valve 303 and marking fluid through hole 213 is provided with in mark liquid pool 203, the liquid pool 204 that dissociates is provided with dissociation solution valve 304 and the fluid through-hole 214 that dissociates, cleaning liquid pool 205 is provided with cleaning fluid valve 305 and cleaning fluid through hole 215, waste liquid pool 206 is provided with waist valve 306 and waste liquid through hole 216, each valve is connected with main valve respectively by passage.
Sample pool valve 301, main valve 307 and dilution liquid pool valve 302 and passage, form sample pool 201-and dilute liquid pool 202 two-way pump Sample Dilution pump 402; Sample valve 301, main valve 307 and waist valve 306 and passage, form sample pool 201-waste liquid pool 206 one-way pump sample waste drains pump 406; Sample valve 301, main valve 307 and cleaning fluid valve 305 and passage, form sample pool 201-and clean liquid pool 205 one-way pump sample scavenging pump 405; Sample pool valve 301, main valve 307 and marking fluid valve 303 and passage, form sample pool 201-and mark liquid pool 203 two-way pump sample labeling pump 403; Sample valve 301, main valve 307 and dissociate liquid pool valve 304 and passage, form sample pool 201-and dissociate liquid pool 204 two-way pump sample solution from pump 404.
Wherein, sample pool 201 inwall is coated with antibody or antigen.
This micro-fluidic chip 102 adopts light tight polymeric material polystyrene (PS) to prepare.
Embodiment 2: with the measurement device hepatitis b virus s antigen of embodiment 1
1, coated antibody: antibody is anti-hepatitis B virus surface antigen (Anti-HBsAg antibody) monoclonal antibody
1) with the sodium carbonate-bicarbonate damping fluid of 50mM pH9.6, the antibody 1:6000 being used for wrapping quilt is doubly diluted, for subsequent use;
2) antibody of dilution is added in sample pool by 100 μ l/ holes;
3) sample pool is placed in 4 DEG C of environment, wraps by 20 hours;
4) bag is moved to end, and by 300 μ l/ holes, with wash liquid 2 times, washing lotion composition is that the PBS of 10mMpH7.4 is containing 5% Tween-20 (volume ratio);
5) washing terminates, and is placed in by sample pool on absorbent filter and blots surplus solution, add confining liquid afterwards in sample pool by the amount in 150 μ l/ holes, closes 2 hours under room temperature, and confining liquid composition is that the PBS of 10mM pH7.4 is containing 1%BSA(bovine serum albumin);
6) close end, confining liquid is thrown away, sample pool is placed on absorbent filter and blots surplus solution, be then placed in 28 DEG C of constant incubator inner dryings 20 hours.
2, labelled antibody:
1) Eu
3+the preparation of-DTPA marking fluid
By purified water (containing Eu
3+10
-6mol/L) 1-(4-isothiocycmatobenzyl is diluted) diethylene triamine pentacetic acid (DTPA) (being called for short DTPA), the solution after dilution is placed in 37 DEG C of waters bath with thermostatic control and adds thermal response 2 hours;
2) Eu
3+-DTPA labelled antibody
By 1mg antibody to 0.1M carbonate buffer solution (pH9.3) 4 DEG C dialysis 16 hours, after dialysis, antibody-solutions is transferred to EP pipe, gets Eu
3+-DTPA 0.2mg, adds in antibody-solutions, and room temperature lucifuge stirs 14 hours;
3) purifying
By in Superdex 200 filler mixing loading 1 × 30cm post, after filler sinks, carry out compression leg by purified water, flow control, at 3ml/min, flows 2 column volumes.After pillar presses, with 0.1mmol/L NaOH, pillar is processed, flow control at 3ml/min, 2 column volumes.Then wash with water flat, then use column equilibration liquid (0.1% high-purity BSA aqueous solution) to balance 1 hour to purification column.Draw with pipettor the antibody marked slowly to join in pillar, carry out wash-out with eluent (50mMTris-HCl, containing 0.9%NaCl and 0.05% Sodium azide pH7.8) to sample, flow control is at 1ml/min.
4) collect
The sample 1ml/ collected after wash-out manages, merge according to five pipes that the absorbance of Protein Detection instrument 280nm albumen selects absorbance high, and by degerming through 0.22 μm of membrane filtration for the sample merged, be placed in 4 DEG C of environment and preserve, obtain the antibody-solutions of europium mark, be called for short europium marking fluid.
3, preparation is dissociated and is strengthened liquid, dilution, cleaning fluid
Dissociate enhancing liquid: the Potassium Hydrogen Phthalate adjust pH 3.2 of 6ml glacial acetic acid 0.1M, add 15 μm of ol β-NTA(β-naphthoyltrifluoroacetones), 50 μm of ol TOPO(trioctyl-phosphine oxide), 1ml Triton X-100, purified water is settled to 1L, stirs and evenly mixs.
Sample dilution: containing 1% bovine serum albumin(BSA), Tris-HCl damping fluid containing 0.02% disodium ethylene diamine tetraacetate;
Cleaning fluid: containing the 0.2MTris-HCl damping fluid of 5%Tween20 (polysorbas20);
4, detection method:
1) see Fig. 1, Fig. 2.Joined in micro-fluidic detection chip 102 sample pool 201 by 100 μ l samples (clinical sample), micro-fluidic detection chip 102 be placed on micro-fluidic control module 103, place dilution, cleaning fluid, marking fluid, dissociate enhancing liquid;
2) start detection, application of sample unit 101(makes by oneself) 200 μ l dilutions are added dilution liquid pool 202,2.0ml cleaning fluid and add cleaning liquid pool 205,200 μ l europium marking fluid and add mark liquid pool 203,200 μ l and dissociate and strengthen liquid and add to dissociate and strengthen liquid pool 204;
3) antigen-antibody (bag quilt) reaction bonded; Sample pool 201-dilutes liquid pool 202 two-way pump 402 and starts two-way operation (Fig. 4, Fig. 6), and sample is mixed with dilution, continues 50min; During stopping, mixed liquor is all stored in sample pool 201;
4) effluent discharge, the one-way only operation of sample pool 201-waste liquid pool 206 one-way pump 406, enters waste liquid pool 206 by mixed liquor;
5) suck cleaning fluid, sample pool 201-cleans liquid pool 205 one-way pump 405 one-way only operation (Fig. 5), cleaning fluid is sucked sample pool 201;
6) clean, repeated execution of steps 4) effluent discharge, 5) suck cleaning fluid, carry out 4 times;
7) europium mark combines, and two-way pump 403 starts, and make the flowing (Fig. 7) between sample pool 201 and mark pond 203 of 150 μ l europium marking fluids, " tape label antibody "+" antigen-antibody (bag quilt) reaction bonded body, is formed sandwich ", continues about 30min; Stop tense marker liquid in sample cell 201, then perform step 4) and get rid of waste liquid in waste liquid pool 206;
8 wash plate, perform step 6) cleaning;
9) dissociate enhancing, two-way pump 404 starts, and 150 μ l is dissociated strengthen liquid at sample pool 201 and dissociates and strengthen flowing between liquid pool 204, dissociating, continue 5min, dissociates to strengthen liquid and strengthen in liquid pool 204 (Fig. 8) dissociating during stopping;
Above-mentioned steps needs time 110-120min.
After adding dissociation solution, europium ion can disintegrate down from forming sandwich labelled antibody with coated antibody, produces fluorescence, according to the light intensity exciting rear generation, can determine the concentration of hepatitis B surface antigen after exciting.The hepatitis B surface antibody standard items (15ng/ml) of same method test concentration known, Parallel testing 20 hole, its precision CV<7%, with comparing of classic method, can improve detection precision, reduce the coefficient of variation.According to " Products in China code 2000 " version regulation, hepatitis B surface antibody diagnostic kit precision CV≤15%.
Table 1: Parallel testing 20 hole sample results
Embodiment 3: with the measurement device hepatitis b virus s antigen of embodiment 1
In step 4, cleaning process is: sample pool-cleaning liquid pool one-way pump 405 one-way only operation, sucks sample pool 201 by cleaning fluid.
To dilute liquid pool 202 as cleaning Buffer Pool, sample cell 201 first gets rid of waste liquid, sucks cleaning fluid, then closes one-way pump sample scavenging pump 405 and one-way pump sample waste drains pump 406; Start two-way pump 402, make cleaning fluid flowing between sample pool 201 and dilution liquid pool 202, clean; During stopping, cleaning fluid is in sample pool 201.
Other step is identical with embodiment 2.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a micro-fluidic time resolved fluoro-immunoassay device, comprise application of sample unit (101), micro-fluidic control module (103), detecting unit (104), it is characterized in that, it also comprises the micro-fluidic chip (102) adopting membrane pump type of drive, and described micro-fluidic chip (102) is arranged in the micro-fluidic chip seat on micro-fluidic control module (103) analytical work platform;
Wherein, described micro-fluidic chip (102) comprising: sample pool (201), dilution liquid pool (202), mark liquid pool (203), the liquid pool that dissociates (204), cleaning liquid pool (205), waste liquid pool (206);
Wherein, sample valve (301) and sample through hole (211) is provided with in described sample pool (201); Dilution valve (302) and dilution through hole (212) is provided with in dilution liquid pool (202); Marking fluid valve (303) and marking fluid through hole (213) is provided with in described mark liquid pool (203), be provided with dissociation solution valve (304) and the fluid through-hole that dissociates (214) in the described liquid pool that dissociates (204), described cleaning liquid pool (205) is provided with cleaning fluid valve (305) and cleaning fluid through hole (215); Described waste liquid pool (206) is provided with waist valve (306) and waste liquid through hole (216);
Main valve (307) is arranged at the centre of each valve, and each valve is connected with main valve (307) respectively by passage.
2. device as claimed in claim 1, it is characterized in that, described sample valve (301), main valve (307) and dilution liquid pool valve (302) and passage, form sample pool (201)-dilution liquid pool (202) two-way pump Sample Dilution pump (402); Described sample valve (301), main valve (307) and waist valve (306) and passage, form sample pool (201)-waste liquid pool (206) one-way pump sample waste drains pump (406); Described sample valve (301), main valve (307) and cleaning fluid valve (305) and passage, form sample pool (201)-cleaning liquid pool (205) one-way pump sample scavenging pump (405); Described sample valve (301), main valve (307) and marking fluid valve (303) and passage, form sample pool (201)-mark liquid pool (203) two-way pump sample labeling pump (403); Described sample valve (301), main valve (307) and dissociation solution valve (304) and passage, form sample pool (the 201)-liquid pool that dissociates (204) two-way pump sample solution from pump (404).
3. device as claimed in claim 1 or 2, it is characterized in that, described sample pool (201) inwall is coated with antibody or antigen.
4. device as claimed in claim 1 or 2, is characterized in that, described micro-fluidic chip (102) is made for light tight polymeric material.
5. application rights requires that the arbitrary described device of 1-4 carries out the method for micro-fluidic time resolved fluoro-immunoassay, it is characterized in that, comprises the following steps:
1) sample is added sample pool (201), dilution adds dilution liquid pool (202), cleaning fluid adds cleaning liquid pool (205), and marking fluid adds mark liquid pool (203), and the enhancing liquid that dissociates adds enhancing liquid pool (204) of dissociating;
2) start micro-fluidic control module (103) work: antigen-antibody combines, effluent discharge, cleaning, effluent discharge, mark combines, effluent discharge, cleaning, effluent discharge, and enhancing of dissociating detects.
6. method as claimed in claim 5, it is characterized in that, step 2) described in antigen-antibody in conjunction with detailed process be: (202) two-way pump Sample Dilution pump (402) two-way operation of sample pool (201)-dilution liquid pool composition, makes sample mix with dilution;
Step 2) described in effluent discharge detailed process be: one-way pump sample waste drains pump (406) one-way only operation that sample pool (201)-waste liquid pool (206) forms, enters waste liquid pool (206) by mixed liquor.
7. method as claimed in claim 5, it is characterized in that, step 2) described in clean detailed process and be: one-way pump sample scavenging pump (405) one-way only operation that sample pool (201)-cleaning liquid pool (205) forms, sucks sample pool (201) by cleaning fluid.
8. method as claimed in claim 5, it is characterized in that, step 2) described in clean detailed process and be: to dilute liquid pool (202) as cleaning Buffer Pool, sample cell (201) first gets rid of waste liquid, suck cleaning fluid, then close one-way pump sample scavenging pump (405) and one-way pump sample waste drains pump (406); Start two-way pump (402), make cleaning fluid flowing between sample pool (201) and dilution liquid pool (202), clean; During stopping, cleaning fluid is in sample pool (201).
9. method as claimed in claim 5, it is characterized in that, step 2) described in mark in conjunction with detailed process be: two-way pump (403) start, make marking fluid flowing between sample pool (201) and mark pond (203), carry out mark to combine, stop tense marker liquid in sample cell (201).
10. method as claimed in claim 6, it is characterized in that, step 2) described in dissociate strengthen detailed process be: two-way pump (404) start, make to dissociate and strengthen liquid in sample pool (201) and flowing between enhancing liquid pool (204) of dissociating, dissociate, dissociating during stopping strengthens liquid dissociating and strengthens in liquid pool (204).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310081065.9A CN103207169B (en) | 2012-03-30 | 2013-03-14 | Microfluidic time-resolved fluorescence immunoassay device and application thereof |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100918391 | 2012-03-30 | ||
| CN201210091839 | 2012-03-30 | ||
| CN201210091839.1 | 2012-03-30 | ||
| CN201310081065.9A CN103207169B (en) | 2012-03-30 | 2013-03-14 | Microfluidic time-resolved fluorescence immunoassay device and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103207169A CN103207169A (en) | 2013-07-17 |
| CN103207169B true CN103207169B (en) | 2015-04-22 |
Family
ID=48754457
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310081065.9A Active CN103207169B (en) | 2012-03-30 | 2013-03-14 | Microfluidic time-resolved fluorescence immunoassay device and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103207169B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104897635B (en) * | 2015-06-19 | 2017-06-16 | 上海凯创生物技术有限公司 | Immunofluorescence analysis instrument |
| CN105381827A (en) * | 2015-12-17 | 2016-03-09 | 北京博晖创新光电技术股份有限公司 | Microfluidic chip magnetic bead even mixing apparatus and method |
| CN106338601B (en) * | 2016-08-26 | 2019-04-05 | 广东普洛宇飞生物科技有限公司 | A kind of AsAb and anti-Miao Leguan hormons quantitative test card and its production, application method |
| CN107321397B (en) * | 2017-05-31 | 2019-08-09 | 深圳市海拓华擎生物科技有限公司 | A kind of micro-fluidic chip and its application |
| CN108080042A (en) * | 2017-11-13 | 2018-05-29 | 成都微康生物科技有限公司 | Micro-fluidic chip of binding time resolved fluorometric technology and its preparation method and application |
| CN110672829B (en) * | 2019-09-10 | 2022-12-30 | 中国科学院上海技术物理研究所 | Microfluidic laser immunity time domain spectrum detection method |
| CN110672828B (en) * | 2019-09-10 | 2022-12-30 | 中国科学院上海技术物理研究所 | Rare animal blood detector of micro-fluidic laser immunity time domain spectrum |
| CN117471105B (en) * | 2023-12-27 | 2024-03-26 | 北京芯迈微生物技术有限公司 | Microfluidic chip for bidirectional double-drive sequential platelet HLA antibody detection and application |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2438154A1 (en) * | 2009-06-02 | 2012-04-11 | Integenx Inc. | Fluidic devices with diaphragm valves |
| CN102166537B (en) * | 2011-01-30 | 2013-05-01 | 南京大学 | Hydrophilic, multifunctional and integrated miniflow control chip easy to optical detection, manufacture method thereof and use thereof |
| CN102851199A (en) * | 2012-10-09 | 2013-01-02 | 重庆大学 | Micro-fluidic cell sorting and acquiring device |
-
2013
- 2013-03-14 CN CN201310081065.9A patent/CN103207169B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103207169A (en) | 2013-07-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103207169B (en) | Microfluidic time-resolved fluorescence immunoassay device and application thereof | |
| CN107942050B (en) | A kind of detection method of microfluidic chip based on magnetic bead technology | |
| EP3779439B1 (en) | Micro-fluidic chip and analysis instrument having same | |
| JP3505689B2 (en) | Capsule chemical sample liquid analyzer and analysis method | |
| CN108519373B (en) | Chemiluminescence micro-fluidic chip and analysis instrument comprising same | |
| CN109870582A (en) | A kind of more target magnetic immunochemiluminescence micro-fluidic chip detection platforms and method | |
| CN107044972A (en) | A kind of micro-fluidic chip fluorescence immunoassay quick detection kit and its preparation and detection method | |
| CN102243234B (en) | Automatic bacterial sorting and labeling method based on immune method, and apparatus thereof | |
| KR20070104347A (en) | Unitary cartridge for particle processing | |
| CN1650162A (en) | Flow-controlled magnetic particle manipulation | |
| WO2019205780A1 (en) | Microfluidic chip and analytical instrument provided with microfluidic chip | |
| EP4012410A1 (en) | Magnetic particle luminescence micro-fluidic chip for multi-marker detection, and detection device | |
| CN105195243A (en) | Magnetic particulate chemiluminescent micro-fluidic chip for quantitatively detecting myohemoglobin | |
| US20220184619A1 (en) | Magnetic particle luminescent micro-fluidic chip for multi-marker detection and detection apparatus | |
| CN104535776A (en) | Immune scatter turbidimetry based full-automatic detection device and method thereof | |
| CN109211867A (en) | A kind of micro-fluidic fluorescence immunoassay chip of rapid quantitative detection BNP | |
| CN110170344A (en) | Micro-fluidic chip and detection device | |
| CN203164120U (en) | Microfluidic time-resolved fluoroimmune assay device | |
| CN209549516U (en) | A kind of micro-fluidic chip and the analysis instrument containing it | |
| CN208795661U (en) | A kind of chemiluminescence micro-fluidic chip and the analysis instrument containing it | |
| CN109211869A (en) | A kind of micro-fluidic fluorescence immunoassay chip of rapid quantitative detection d-dimer | |
| CN106226540A (en) | Full-automatic protein chip analyzer | |
| JP2005069997A (en) | Method for detecting allergen protein, detecting chip and detecting apparatus | |
| CN108414522B (en) | The all-in-one machine detected for micro-fluidic chip and NC film multi-pass amount | |
| CN102879565B (en) | Microorganism sample rapid detection method and detection device thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address |
Address after: 102206 9 life Garden Road, Changping District, Beijing Patentee after: BEIJING BOHUI INNOVATION BIOTECHNOLOGY Co.,Ltd. Address before: 100195 G, block 25, Lu Jia, 25, Bei Wu Village, Beijing. Patentee before: BEIJING BOHUI INNOVATION TECHNOLOGY Co.,Ltd. |
|
| CP03 | Change of name, title or address | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 102206 9 life Garden Road, Changping District, Beijing Patentee after: Beijing Bohui innovative biotechnology group Co.,Ltd. Address before: 102206 9 life Garden Road, Changping District, Beijing Patentee before: BEIJING BOHUI INNOVATION BIOTECHNOLOGY Co.,Ltd. |
|
| CP01 | Change in the name or title of a patent holder |