CN107942050B - A kind of detection method of microfluidic chip based on magnetic bead technology - Google Patents
A kind of detection method of microfluidic chip based on magnetic bead technology Download PDFInfo
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Abstract
The invention discloses a kind of detection method of microfluidic chip based on magnetic bead technology, including step are as follows: step 1, fluorescent marker；Step 2, marked by magnetic bead；Step 3, the coating and drying of confining liquid；Step 4, the fixation of immunomagnetic beads；Step 5, the fixation of fluorescent marker；Step 6, micro-fluidic chip assembles；Step 7, sample-adding prepares；Step 8, it is immunoreacted；Step 9, it cleans；Step 10, it develops the color；Step 11, reading data.The present invention chooses highly sensitive time-resolved fluorescence dyestuff as marker, carries out the label of antibody on magnetic bead and fluorescent material respectively, carries out analysis detection, level of the prepared reagent performance up to same chemical illuminating reagent using the immune response of antibody pair.Meanwhile reagent is reacted using homogeneous liquid, realizes the control of liquid flowing, and ultrasonic mixing is used in micro-fluidic chip chamber, avoids the chromatographic flow reaction of single thread, so that reaction is more abundant, reaction efficiency is higher.
Description
Technical field
The present invention relates to microfluidic art, especially a kind of detection method of microfluidic chip based on magnetic bead technology.
Background technique
For microballs of super-paramagnetic polymer as a kind of novel immobilization support agent, the specified chemical group on surface can
With in conjunction with specific biological molecules, resulting immunomagnetic beads can be specifically bound with corresponding target substance, new answer is formed
Object is closed, when passing through magnetic field, this compound can be detained, and be separated with other groups of split-phases.Immunomagnetic beads have super in magnetic field
The characteristic of paramagnetism and nanoparticle.Its superparamagnetism makes separation of solid and liquid, and the operation is more convenient, and it is many and diverse to save centrifugal filtration etc.
Traditional operation；And the immunomagnetic beads particle of nano-scale is small, large specific surface area, and coupling capacity is big, and suspension stability is good, favorably
In going on smoothly for specific reaction, separated in biomedical such as cell, in the analysis fields such as immune response and DNA extraction
It is widely used.
Conventional magnetic bead technology has mature design and method, is widely used in biology sample detection, many to design
It has been commercialized.But routine immunization magnetic bead technology has much room for improvement also there is also following deficiency.
1. containing sample solution using conventional vial in conventional magnetic bead technology, carry out the operation such as cleaning using pipettor etc.,
It is easily introduced xenobiotic pollutants.
2. conventional magnetic bead technology is to need artificial manipulation experimental procedure, such as sample, cleaning etc. is easily introduced human operational error
Factor leads to the failure of an experiment.
3. conventional magnetic bead technology experiment platform is huger, biggish instrument and equipment is needed, it is difficult to it integrates and is miniaturized, it is difficult
To realize portability.
Microfluidic chip technology is using microchannel network as structure feature, using micro-processing technology in several square centimeters of sizes
Chip on etch microchannel network and other functional units, to prepare comprising sample introduction, reaction, separate, be detected on one
Quick, efficient, low consumption the micro-analytical device of body.Micro-fluidic chip has reagent consumption few in detection platform, when reaction
Between it is short, the characteristics of high degree of automation.In current research, the micromation of chip but gives the separation of sample early period to integrate with mating
Many difficulties are brought, while being fixed in sample, there is also many problems for elution etc..
Magnetic bead technology combination microflow control technique realizes a kind of micro- separating chips system, using the characteristic that magnetic bead efficiently separates with
And microfluidic circuit control liquid stream carries out biological respinse, the micro- magnet assembly and microfluidic circuit device made in this way can be used to construct high integration
Immune detection micro-system.Compared with conventional magnetic bead technology, have the advantage that
1) device is small and does not have manual operation, and reagent consumption is seldom.
2) hybrid reaction that the different reagents in same chip are realized by designing different microfluidic circuits, reduces cumbersome life
Object experimental implementation, the time required to shortening detection.
3) micro-fluidic chip can be engaged with circuit realizes automation control.
4) micro-fluidic chip and mating detection device compact, are easy to portable.
Currently, the existing detection technique combined using immunomagnetic beads and microflow control technique, referring specifically to following patent:
(1) a kind of micro-fluidic chip fluorescence immunoassay quick detection kit of CN201710378773.7 and its preparation and inspection
Survey method.
(2) immune reunion detection method, chip and system of the CN201510073649.0 based on micro-fluidic chip.
Above-mentioned patent application has following insufficient:
1. the structure of chip is excessively complicated, accurate regulation can not be made to reaction, cleaning step and liquid conductive are deposited
In biggish pollution.
2. having used multiple liquid chambers and solid phase carrier (magnetic bead and fluorescent microsphere while using), reagent whole design
It is excessively complicated.
3. immune response uses heterogeneous reaction, mean that solid phase carrier is reacted with the surface of solid phase carrier, reacts
Insufficient, sensitivity and repeatability are restricted.
4. failing to choose with higher sensitivity fluorescence or chemiluminescence reaction, but it is lower than turbid to use sensitivity
Reaction or colloidal gold chromatographic reaction, especially with the reagent of similar chromatography reaction, the root problem release of chromatography is incomplete
And unidirectional response causes the accuracy of detection to be affected.
In addition, General reactions are completed on 96 orifice plates, and one complete in existing enzyme linked immunological (ELISA) detection technique method
It should include an automatic pipettor that whole system, which is prepared, a microplate oscillator, a microwell plate board-washing machine, a couveuse,
One fluorescence immunity analyzer.Detecting step includes sample-adding originally and dilution, dynamic respons about 60min, board-washing cleaning are had the final say
It cleans, fluorescent marker, dynamic respons about 30min is added, developing solution is added in board-washing, vibrates about 5min, detection.All detections
Process executes 4 steps sample-adding, board-washing 2 times, vibrates 3 times, used time about 150~180min.And semi-automatic detection device is used, it needs
Time is longer, more Fei Renli.
Further, current chemiluminescence, one side detection time is longer, and single testing result is reported from being loaded to out
Announcement needs half an hour even more long；On the other hand, chemiluminescence experiment porch is huger, not only needs biggish instrument
Equipment, it is difficult to integrated and be miniaturized, it is difficult to it realizes portability, while also needing higher maintenance cost and maintenance cost,
The cost of single part test is high.
Further, present immuno-chromatographic assay technology generally uses colloidal gold or fluorescence as marker, detects nothing
Method is accomplished completely quantitatively, simultaneously because chromatography is incomplete from the release on film and single thread reacts insufficient etc. and asks
Topic, causes to detect less reproducible.
Summary of the invention
In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to provide one kind to be based on magnetic bead technology
Detection method of microfluidic chip, highly sensitive time resolution should be chosen based on detection method of microfluidic chip of magnetic bead technology
Fluorescent dye carries out the label of antibody on magnetic bead and fluorescent material respectively, utilizes the immune response of antibody pair as marker
Carry out analysis detection, level of the prepared reagent performance up to same chemical illuminating reagent.Meanwhile using homogeneous reaction, In
By ultrasonic mixing in micro-fluidic chip chamber, the chromatographic flow reaction of single thread is avoided, so that reaction efficiency is higher and anti-
It should more sufficiently.After immune response, immunomagnetic beads are fixed by externally-applied magnetic field completion, it is using cleaning solution that extra component is complete
After full removal, adds developing solution and read, solve the problems, such as that portion is difficult to clean or cleans and is halfway in the chip.
In order to solve the above technical problems, the technical solution adopted by the present invention is that:
A kind of detection method of microfluidic chip based on magnetic bead technology, includes the following steps.
Step 1, fluorescent marker: the antibody starting material dialysed is marked using time-resolved fluoroimmunoassay, and is received
Collect fluorescent marker.
Step 2, marked by magnetic bead: magnetic bead is reacted with antibody-solutions, after reaction, close to magnetic bead clear
It washes, and after use magnetic bead preservation liquid redissolution, forms immunomagnetic beads；Wherein, the magnetic bead reacted with antibody-solutions chooses surface
The magnetic microsphere of sulfonyl containing toluene.
Step 3, the coating and drying of confining liquid: being closed to quantitative-reaction chamber addition confining liquid of micro-fluidic chip,
Then, by confining liquid drying and processing.
Step 4, the fixation of immunomagnetic beads: after the confining liquid drying in step 3, exempt from what is formed after step 2 marked by magnetic bead
Epidemic disease magnetic bead is added in the reaction tank cover board on the chip of middle layer, and then, drying fixes immunomagnetic beads with middle layer chip.
Step 5, the fixation of fluorescent marker: after the confining liquid drying in step 3, fluorescent marker that step 1 is collected
It is added in lower layer chip in the intermediate separate space of quantitative-reaction tank, then, drying fixes fluorescent marker with lower layer chip.
Step 6, micro-fluidic chip assembles: by upper layer chip, being fixed with the middle layer chip of immunomagnetic beads and is fixed with glimmering
The lower layer chip of signal object successively carries out assembling merging.
Step 7, sample-adding prepares: pipettor sample-adding draws the sample-adding that the micro-fluidic chip that step 6 is completed is added in sample
In hole；Then, the micro-fluidic chip that sample-adding is completed is entered in detecting instrument, micro-fluidic chip is in conjunction with chip contact device；
Gas path device in chip contact device is slowly inflated, and sample is pushed to be moved along, and gas is flowed out by ventilation mouth, and sample flows into fixed
In amount-reaction chamber, and it is in contact respectively with the immunomagnetic beads of oven-dried condition and fluorescent marker；When sample continues to move along simultaneously
When touching the conductive rubber of quantitative-reaction chamber end, capacitance variations touch runner valve switch, close runner valve；Together
When, air circuit breaker is closed, pressurization is stopped.
Step 8, it is immunoreacted: opening ultrasonic generator, quantitative-reaction to the micro-fluidic chip that step 7 sample-adding is completed
Chamber is ultrasonically treated, and is redissolved immunomagnetic beads and fluorescent marker, and mixed with sample, is immunoreacted；It is immune
Reaction time controlled at 5-10 minutes.
Step 9, clean: after immune response, kicker magnet carries out magnetically fixed, gas push sample to magnetic bead immune complex
This forward movement, dries up well and runner sample, and cleaning fluid path device starting regulates and controls strong magnetic into quantitative-reaction chamber
Body, opens Vltrasonic device, and ultrasound is mixed 1-3 minutes, cleaned；After cleaning, magnetic force is applied to quantitative-reaction chamber, then
Secondary fixed magnetic bead immune complex, extra sample and fluorescent marker are cleaned to devil liquor recovery area；Then, at adding mouth
Gas push cleaning solution continue to move, drying is quantitative-reaction chamber and runner liquid, and removes strong magnetic force.
Step 10, develop the color: the starting of enhancement solution fluid path device, into runner, into quantitative-reaction chamber, control is quantitative
The addition of enhancement solution, ultrasound mix 3-8 minutes, carry out chromogenic reaction.
Step 11, reading data: the fluorescence intensity on micro-fluidic chip in quantitative-reaction tank is read, calculates and provides knot
Fruit report.
In step 3, confining liquid includes 2% BSA, 0.5% Tween-20,10% trehalose and 0.2% sweet dew
Alcohol.
In step 3, quantitative-reaction chamber is carried out using confining liquid closed method particularly includes: quantitative in lower layer chip-anti-
The confining liquid of 5ul is added in each separate space of Ying Chi, is added in each cover board fission of reaction tank cover board on the chip of middle layer
The confining liquid of 6ul；After confining liquid addition, under 45 DEG C of baking ovens, drying and processing 5min is carried out.
In step 6, by the way of ultrasonic bond, by upper layer chip, it is fixed with the middle layer chip of immunomagnetic beads and consolidates
There is the lower layer chip of fluorescent marker successively to assemble merging surely.
In step 1, the method that the antibody starting material dialysed is marked using time-resolved fluoroimmunoassay, including such as
Lower step.
Step 1a, antibody dialysis: taking CRP antibody, adds the CB buffer of pH9.6, fills bag filter, then with PH9.6's
CB buffer is dialysed 24 hours, changes liquid processing in three times；
Step 1b, EU label: after dialysis treatment, taking EU-DTTA that CB is added to dissolve, and then, the antibody starting material dialysed is added,
Wherein, the mass ratio of EU-DTTA and antibody starting material is 1:1；It mixes well, polytetrafluoroethylene (PTFE) magnetic force is put into brown bottle and is stirred
Son is mixed, 2-8 DEG C is set and is stirred to react no less than 20 hours, forms EU marker.
Step 1c, chromatographic column prepare: Sepharose 6B being filled chromatographic column, is balanced with Tris-HCl buffer.
Step 1d, fluorescent marker are collected: take the EU marker formed in step 1b to cross the chromatographic column prepared in step 1c,
Then it is eluted with Tris-HCl buffer, flow velocity 2ml/ pipe/5min, the first effluent of collection is fluorescent marker.
In step 2, magnetic bead is reacted with antibody-solutions method particularly includes: the Dynal Tosyl magnetic bead of 200ul is taken,
After the cleaning twice of the borate buffer of 50mM pH9.0, the CRP antibody-solutions of 200ul and the borate buffer of 800ul is added
Liquid, hybrid reaction, reaction density 20mg/ml react 24 hours.
In step 2, after reaction, magnetic bead is closed using the PB buffer containing 1%BSA and pH=7.4, is sealed
After closing 24 hours, magnetic bead is cleaned, and saves liquid using magnetic bead and redissolves, is configured to the solution of 0.4mg/ml；Wherein, magnetic bead
Preservation liquid is the PBS buffer solution containing 1%BSA.
Quantitative-reaction in step 9, after cleaning, by from the enhancement solution inlet on microcontroller chip to microcontroller chip
Chamber injects increased response liquid, removes magnetic attracting device, and is aided with ultrasonic mixing processing.
Increased response liquid is using glacial acetic acid and Potassium Hydrogen Phthalate as buffer ions pair, using β-NTA as complexing agent
Liquid.
In step 9, after cleaning, air is injected to quantitative-reaction chamber by gas path device, excludes quantitative-reaction chamber
Interior cleaning solution.
The invention has the following beneficial effects: the present invention compared with routine immunization detection technique, and sample-adding measures big first
Big reduction, and due to the addition of hemofiltration pad, leading to sample type includes multiple types such as serum, blood plasma and whole blood, sample
It interferes lower.Detection time also greatly shortens simultaneously, and single testing result is completed between 10-15min.Furthermore due to miniflow
The integration degree for controlling chip is higher, and required manual operation is less, only load procedure, therefore matched testing result can
Reliability is higher.Compared with many immune equipment, in addition, detection method of the invention not only reaches in detection accuracy and accuracy
To chemiluminescent level, while micro-fluidic chip detection device volume greatly reduces, and testing cost is lower, is detection reagent
Provide portability.
More importantly detection method of the invention chooses highly sensitive time-resolved fluorescence object as marker,
The label for carrying out antibody on magnetic bead and fluorescent material respectively carries out analysis detection using the immune response of antibody pair, prepared
Reagent performance up to same chemical illuminating reagent level.Meanwhile using homogeneous reaction, pass through in micro-fluidic chip chamber
Ultrasonic mixing, the chromatographic flow reaction for avoiding single thread are reacted more abundant so that reaction efficiency is higher.After immune response, lead to
It crosses externally-applied magnetic field completion immunomagnetic beads are fixed, after completely removing extra component using cleaning solution, adds developing solution
It is read, solves the problems, such as that portion is difficult to clean or cleans and is halfway in the chip.Due to being liquid-phase reaction system, reaction
It is more abundant, in addition the rare-earth europium fluorescent tracing object and magnetic bead amplification system that use, so that detection sensitivity and detection repeatability
It is significantly larger than chromatography method.
Further, the present invention also provides certain basic platform simultaneously for high-throughput and multi-channel detection, conventional
For sample when cooperating method of the invention, more parts of samples can be detected simultaneously by being able to achieve the multiple project indicators of detection or one chip
This effect, there is very big potential advantages.
Detailed description of the invention
Fig. 1 shows the structure schematic diagram of micro-fluidic chip mesonexine chip of the present invention.
Fig. 2 shows the positive structure schematic of lower layer chip in micro-fluidic chip of the present invention.
Fig. 3 shows structural schematic diagram when detecting using micro-fluidic chip.
The structural schematic diagram of Fig. 4 is shown when being detected using micro-fluidic chip chip contact device.
Wherein have:
10. immunomagnetic beads；
20. fluorescent marker；
30. micro-fluidic chip；
2. middle layer chip；2-10. reaction tank cover board；
3. lower layer chip；3-2. quantifies-reaction tank；
40. chip contacts device；
41. controllable kicker magnet；42. enhancement solution inlet；43. cleaning solution inlet；44. air pump inlet；
50. chip carrier；60. ultrasonic generator.
Specific embodiment
Xia Mianjiehefutuhejuti compare Jia Shishifangshiduibenfamingzuojinyibuxiangxishuoming.
Micro-fluidic chip uses the China of Patent No. 201710531301.0 filed in applicant's on July 3rd, 2017
Chip structure in patent of invention.
Micro-fluidic chip 30 is three-decker, is followed successively by upper layer chip, middle layer chip 2 and lower layer chip 3 from top to bottom.
As shown in Figure 1, the back side of middle layer chip 2 is provided with reaction tank cover board 2-10, it is provided in the middle part of reaction tank cover board
Reaction tank cover board is divided into two cover board fissions by partition b, partition b.
As shown in Fig. 2, being provided with quantitative-reaction tank 3-2 in lower layer chip 3 ,-reaction tank will be quantified by two pieces of partition a
It is divided into three separate spaces.
After micro-fluidic chip assembling, reaction tank cover board can close quantitative-reaction tank sealing cover, and form quantitative-reaction
Chamber.
In addition, further including hemofiltration pad in micro-fluidic chip, it is loaded gasket, conductive rubber, blotting paper and sealing ring etc..
As shown in Figure 3 and Figure 4, flexible biological probe includes chip contact device 40, chip carrier 50 and ultrasound
Generator 60.
The bottom of chip contact device 40 is provided with controllable kicker magnet 41, enhancement solution inlet 42, cleaning solution inlet
43 gentle pump injection ports 44.
The underface of chip contact device is arranged in chip carrier, for loading micro-fluidic chip.
Supersonic generator is located at the lower section of chip carrier, and the top of supersonic generator can be micro- on chip carrier to being located at
Quantitative-reaction chamber of fluidic chip is ultrasonically treated.
A kind of detection method of microfluidic chip based on magnetic bead technology, includes the following steps.
Step 1, fluorescent marker.
The antibody starting material dialysed is marked using time-resolved fluoroimmunoassay, and collects fluorescent marker.
In this step 1, the method that the antibody starting material dialysed is marked using time-resolved fluoroimmunoassay, preferably
Include the following steps.
Antibody dialysis: step 1a takes CRP antibody, preferably takes CRP antibody 1331(Meridian) 1mg；Add 50mM
The CB buffer of pH9.6 fills bag filter, is dialysed, 24 hours, changed in three times with 50mM PH9.6 CB buffer 500ml to 1ml
Liquid processing.
Step 1b, EU label: after dialysis treatment, taking 1mg EU-DTTA that 50mM CB is added to dissolve, and then, it is saturating that 1mg is added
The antibody starting material of analysis, wherein the mass ratio of EU-DTTA and antibody starting material is 1:1；It mixes well, is put into brown bottle poly-
Tetrafluoroethene magnetic stir bar is put into polytetrafluoroethylene (PTFE) magnetic stir bar in brown bottle, sets 2-8 DEG C and is stirred to react no less than
20 hours, preferably 24 hours form EU marker.
Step 1c, chromatographic column prepare: Sepharose 6B is filled into chromatographic column (1.6 × 80cm), it is slow with 50mM Tris-HCl
Fliud flushing balance.
Step 1d, fluorescent marker are collected: take the EU marker formed in step 1b to cross the chromatographic column prepared in step 1c,
Then it is eluted with 50mM Tris-HCl buffer, flow velocity 2ml/ pipe/5min, the first effluent of collection is fluorescent marker.
Above-mentioned fluorescent marker is the antibody for being marked with rare-earth europium fluorescence, and rare-earth europium ion is different by DTTA(isophorone
Thiocyanates diethylenetriamine) complex formation complex compound, wherein isothiocyanate group can be carried out with the amino in protein
Addition reaction, so that antibody or antigenic substance be marked.Marked product is purified by chromatographic column, and it is extra to remove
Rare earth europium complex.Fluorescent marker is liquid solution, can be disintegrated down from chip after the drying, and keeps original biology
Activity.
The present invention creatively uses time-resolved fluorescence as fluorescent marker, detects signal and compares traditional immune inspection
Test agent is greatly improved, and the design of quantitative-reaction chamber uses transparent optical device, can effective detection light letter
Number, and fluorescent molecule is stabilized, and is not influenced by time and excitation number.
Step 2, marked by magnetic bead: magnetic bead is reacted with antibody-solutions, after reaction, close to magnetic bead clear
It washes, and after use magnetic bead preservation liquid redissolution, forms immunomagnetic beads.
Wherein, the magnetic bead that is reacted with antibody-solutions chooses the magnetic microsphere of surface sulfonyl containing toluene, by with
Amino in protein structure is reacted.After reaction, enclosed cleaning is carried out to magnetic bead, removes extra albumen.Immune magnetic
Pearl is liquid suspension, no particle agglomeration, and without phenomenon is separated by solid-liquid separation in the short time, behind portion, magnetic is immunized in the chip in coating drying
Pearl keeps original bioactivity, does not move with packaging, transport and storage.
Above-mentioned magnetic bead is reacted preferred with antibody-solutions method particularly includes:
The Dynal Tosyl magnetic bead for first taking 200ul is added after the cleaning twice of the borate buffer of 50mM pH9.0
The CRP antibody-solutions of 200ul and the borate buffer of 800ul, hybrid reaction, reaction density 20mg/ml react 24 hours.
After reaction, magnetic bead is closed using the PB buffer containing 1%BSA and pH=7.4, is closed 24 hours
Afterwards, magnetic bead is cleaned, and saves liquid using magnetic bead and redissolves, be configured to the solution of 0.4mg/ml；Wherein, magnetic bead preservation liquid is
PBS buffer solution containing 1%BSA.
Step 3, the coating and drying of confining liquid: being closed to quantitative-reaction chamber addition confining liquid of micro-fluidic chip,
Then, by confining liquid drying and processing.
Above-mentioned confining liquid preferably comprises 2% BSA, 0.5% Tween-20,10% trehalose and 0.2% sweet dew
Alcohol.
Above-mentioned confining liquid is not involved in reaction, and can place fluorescent marker and immunomagnetic beads, during the drying process, still
It is able to maintain bioactivity, and avoids that non-specific binding occurs with microcontroller chip.
Closed specific preferred method is carried out to quantitative-reaction chamber using confining liquid are as follows: quantitative-reaction tank in lower layer chip
Each separate space in add the confining liquid of 5ul, add 6ul's in each cover board fission of reaction tank cover board on the chip of middle layer
Confining liquid；After confining liquid addition, under 45 DEG C of baking ovens, drying and processing 5min is carried out, the surface layer of microcontroller chip is made to form one layer of guarantor
Cuticula.
Step 4, the fixation of immunomagnetic beads: after the confining liquid drying in step 3, exempt from what is formed after step 2 marked by magnetic bead
Epidemic disease magnetic bead is added in the reaction tank cover board on the chip of middle layer, and then, drying fixes immunomagnetic beads with middle layer chip.
Step 5, the fixation of fluorescent marker: after the confining liquid drying in step 3, fluorescent marker that step 1 is collected
It is added in lower layer chip in the intermediate separate space of quantitative-reaction tank, then, drying fixes fluorescent marker with lower layer chip.
Creatively storage is dried in immunomagnetic beads and fluorescent marker by the present invention in microcontroller chip, in addition,
The use of confining liquid then effectively separates microcontroller chip and liquid reagent, so that immunomagnetic beads and fluorescent marker are capable of fixing
The nonvoluntary flowing inside microcontroller chip, while can be dissolved rapidly when sample and dilution is added and keep original life
Object activity.Confining liquid also acts as the effect of the absorption for preventing fluorescent marker in microcontroller chip simultaneously, fluorescent marker due to
It is not direct to be contacted with microcontroller chip, therefore reduce nonspecific absorption.
In addition, the present invention creatively by magnetic bead technical application into micro-fluidic chip, in limited microfluidic chip structure
It is middle that magnetic bead is used to greatly improve the specific surface area of reaction as solid phase carrier, greatly improve the signal of reaction.Furthermore
Antibody label is carried out using magnetic bead, facilitates immunomagnetic beads and is fixed by Magneto separate, it is only necessary to it is simple to carry out Magneto separate
Cleaning, so that it may enormously simplify the process step of immune response.The micro-fluidic chip based on magnetic bead technology can be significantly simultaneously
The fluid path structure for simplifying micro-fluidic chip, avoids cleaning separating step complicated in chip, and it is anti-to realize liquid in chip
The unicity answered avoids the cross contamination between different liquids.
Step 6, micro-fluidic chip assembles: by upper layer chip, being fixed with the middle layer chip of immunomagnetic beads and is fixed with glimmering
The lower layer chip of signal object, it is preferred to use the mode of ultrasonic bond successively carries out assembling merging.
Step 7, sample-adding prepares:
Pipettor sample-adding is drawn sample and is added in the well for the micro-fluidic chip that step 6 is completed.Then, will add
The micro-fluidic chip that sample is completed enters in detecting instrument, namely is placed on chip carrier 50.Then, chip contacts device height
Decline, micro-fluidic chip are combined with chip contact device 40；Chip contacts the gas path device in device, namely is injected by air pump
Mouthfuls 44 slowly inflations, push sample to be moved along, and gas is flowed out by ventilation mouth, and sample flows into quantitative-reaction chamber, and respectively with
The immunomagnetic beads and fluorescent marker of oven-dried condition be in contact (immunomagnetic beads of contact oven-dried condition in advance, afterwards and fluorescent marker
Object contact), when sample continues to move along and touches the conductive rubber of quantitative-reaction chamber end, capacitance variations touch stream
Road valve switch closes runner valve；Meanwhile air circuit breaker is closed, stop pressurization.Namely it is controlled and is reacted by conductive rubber
The indoor total amount of liquid of chamber.
Step 8, it is immunoreacted: opening ultrasonic generator 60, the quantitative-anti-of the micro-fluidic chip completed is loaded to step 7
It answers chamber to be ultrasonically treated, redissolves immunomagnetic beads and fluorescent marker, and mixed with sample, be immunoreacted；Exempt from
The epidemic disease reaction time controlled at 5-10 minutes.
The present invention has creatively used mixing method of the Probe Ultrasonic Searching as micro-fluidic chip, and ultrasonication is in micro-control core
The left side side of piece, in the optical property premise for not influencing the finish of microcontroller chip, ultrasonic energy transmission again will it is quantitative-
Immunomagnetic beads and fluorescent marker are redissolved and reacted in reaction chamber, reaction is controlled to liquid in single chamber and is guaranteed
No cross contamination.The effect of ultrasonic treatment can greatly improve the efficiency of immune response, need 30 minutes by popular response
Or more, ultrasonic reaction can be reduced to 5-10 parts of minutes to reach the plateau of reaction.
Step 9, it cleans: after immune response, strong magnetic force being applied to quantitative-reaction chamber by controllable kicker magnet 41,
While fixed magnetic bead immune complex, gas push sample is moved forward, and dries up well and runner sample, cleans fluid path
Device starting injects cleaning solution into quantitative-reaction chamber by cleaning solution inlet 43, removes strong magnetic force, and ultrasound mixes 1-3 points
Clock is cleaned.
After cleaning, kicker magnet fixes magnetic bead immune complex again, and extra sample and fluorescent marker is clear
It is washed till devil liquor recovery area；Then, the gas push cleaning solution at adding mouth continues to move, and dries up quantitative-reaction chamber and stream
Road liquid.
After cleaning ,-reaction chamber is preferably quantified to microcontroller chip by the enhancement solution inlet 42 on microcontroller chip
Increased response liquid is injected, magnetic attracting device is removed, and is aided with ultrasonic mixing processing.
Increased response liquid is preferably using glacial acetic acid and Potassium Hydrogen Phthalate as buffer ions pair, using β-NTA as complexing
The liquid of agent.This increased response liquid can combine complexing europium ion, europium ion after dissociation in DTTA originally in acid condition
Fluorescence intensity can greatly be promoted, and in immune complex in the concentration of europium ion and sample the concentration of determinand in just
Correlation, therefore can analyze the concentration for calculating determinand in sample.
Finally, excluding in quantitative-reaction chamber it is preferred that continue to inject one section of air to quantitative-reaction chamber by gas path device
Cleaning solution and increased response liquid.
The present invention has creatively used dypass to clean and add the sample-adding mode of enhancement solution on micro-fluidic chip, makes
Cleaning solution or enhancement solution are injected by micro-control core by external pressure under the premise of guaranteeing that leakage does not occur for side port with seal washer
Method inside quantitative-reaction chamber of piece, on the one hand simplifies the structure of microcontroller chip, avoids occurring multiple chambers in microcontroller chip
Room, on the other hand realize plays the role of sufficiently cleaning to the reaction chamber in micro-fluidic chip, avoid the reflux of liquid with
And the pollution of sample.
Step 10, develop the color: the starting of enhancement solution liquid fluid path device, into runner, into quantitative-reaction chamber, ultrasound is mixed
It is 3-8 minutes even, carry out chromogenic reaction.
Step 11, reading data: the fluorescence intensity on micro-fluidic chip in quantitative-reaction tank is read, calculates and provides knot
Fruit report.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail a variety of equivalents can be carried out to technical solution of the present invention within the scope of the technical concept of the present invention, this
A little equivalents all belong to the scope of protection of the present invention.
Claims (8)
1. a kind of detection method of microfluidic chip based on magnetic bead technology, characterized by the following steps:
Step 1, fluorescent marker: the antibody starting material dialysed is marked using time-resolved fluoroimmunoassay, and is collected glimmering
Signal object；
Step 2, marked by magnetic bead: magnetic bead is reacted with antibody-solutions, after reaction, carries out enclosed cleaning to magnetic bead, and
After saving liquid redissolution using magnetic bead, immunomagnetic beads are formed；Wherein, the magnetic bead reacted with antibody-solutions chooses surface and contains first
The magnetic microsphere of benzenesulfonyl；
Step 3, it the coating and drying of confining liquid: is closed to quantitative-reaction chamber addition confining liquid of micro-fluidic chip, so
Afterwards, by confining liquid drying and processing；Confining liquid include 2% BSA, 0.5% Tween-20,10% trehalose and 0.2%
Mannitol；Wherein, quantitative-reaction chamber is carried out using confining liquid closed method particularly includes: quantitative-reaction tank in lower layer chip
Each separate space in add the confining liquid of 5 μ L, add 6 μ L's in each cover board fission of reaction tank cover board on the chip of middle layer
Confining liquid；After confining liquid addition, under 45 DEG C of baking ovens, drying and processing 5min is carried out；Quantitative-reaction chamber uses transparent optics device
Part can effectively detect optical signal, and fluorescent molecule is stabilized, and not influenced by time and excitation number；
Step 4, the fixation of immunomagnetic beads: after the confining liquid drying in step 3, the immune magnetic that will be formed after step 2 marked by magnetic bead
Pearl is added in the reaction tank cover board on the chip of middle layer, and then, drying fixes immunomagnetic beads with middle layer chip；
Step 5, the fixation of fluorescent marker: after the confining liquid drying in step 3, the fluorescent marker that step 1 is collected is added
On to lower layer chip in the intermediate separate space of quantitative-reaction tank, then, drying fixes fluorescent marker with lower layer chip；Closing
The use of liquid then effectively separates microcontroller chip and liquid reagent, so that immunomagnetic beads and fluorescent marker are capable of fixing micro-
Control the nonvoluntary flowing of chip interior；It can be dissolved rapidly when sample and dilution is added simultaneously and keep original biology living
Property；Confining liquid also acts as the effect for the absorption for preventing fluorescent marker in microcontroller chip simultaneously, and fluorescent marker is not due to straight
It connects and is contacted with microcontroller chip, therefore reduce nonspecific absorption；
Step 6, micro-fluidic chip assembles: by upper layer chip, being fixed with the middle layer chip of immunomagnetic beads and is fixed with fluorescence mark
The lower layer chip for remembering object, successively carries out assembling merging；
Step 7, sample-adding prepares: pipettor sample-adding draws the well that the micro-fluidic chip that step 6 is completed is added in sample
It is interior；Then, the micro-fluidic chip that sample-adding is completed is entered in detecting instrument, micro-fluidic chip is in conjunction with chip contact device；Core
Gas path device in piece contact device is slowly inflated, and sample is pushed to be moved along, and gas is flowed out by ventilation mouth, and sample flows into fixed
In amount-reaction chamber, and it is in contact respectively with the immunomagnetic beads of oven-dried condition and fluorescent marker；When sample continues to move along simultaneously
When touching the conductive rubber of quantitative-reaction chamber end, capacitance variations touch runner valve switch, close runner valve；Together
When, air circuit breaker is closed, pressurization is stopped；
Step 8, be immunoreacted: opening ultrasonic generator, quantitative-reaction chamber of the micro-fluidic chip that step 7 sample-adding is completed into
Row ultrasonic treatment is redissolved immunomagnetic beads and fluorescent marker, and is mixed with sample, is immunoreacted；Immune response
Time controlled at 5-10 minutes；
Step 9, clean: after immune response, kicker magnet to magnetic bead immune complex carry out it is magnetically fixed, gas push sample to
Preceding movement, dries up well and runner sample, and cleaning fluid path device starting regulates and controls kicker magnet, open into quantitative-reaction chamber
Vltrasonic device is opened, ultrasound is mixed 1-3 minutes, cleaned；After cleaning, magnetic force is applied to quantitative-reaction chamber, is fixed again
Magnetic bead immune complex cleans extra sample and fluorescent marker to devil liquor recovery area；Then, the gas at adding mouth
It pushes cleaning solution to continue to move, dries up quantitative-reaction chamber and runner liquid, and remove strong magnetic force；
Step 10, develop the color: the starting of enhancement solution fluid path device into quantitative-reaction chamber, controls quantitative enhancing into runner
The addition of liquid, ultrasound mix 3-8 minutes, carry out chromogenic reaction；
Step 11, reading data: the fluorescence intensity on micro-fluidic chip in quantitative-reaction tank is read, calculates and provides result report
It accuses.
2. the detection method of microfluidic chip according to claim 1 based on magnetic bead technology, it is characterised in that: in step 6,
By the way of ultrasonic bond, by upper layer chip, it is fixed with the middle layer chip of immunomagnetic beads and is fixed with fluorescent marker
Lower layer chip successively assembles merging.
3. the detection method of microfluidic chip according to claim 1 based on magnetic bead technology, it is characterised in that: in step 1,
The method that the antibody starting material dialysed is marked using time-resolved fluoroimmunoassay, is included the following steps:
Step 1a, antibody dialysis: taking CRP antibody, adds the CB buffer of pH9.6, fills bag filter, then slow with the CB of PH9.6
Fliud flushing is dialysed 24 hours, changes liquid processing in three times；
Step 1b, EU label: after dialysis treatment, taking EU-DTTA that CB is added to dissolve, and then, the antibody starting material dialysed is added,
In, the mass ratio of EU-DTTA and antibody starting material is 1:1；It mixes well, polytetrafluoroethylene (PTFE) magnetic agitation is put into brown bottle
Son is set 2-8 DEG C and is stirred to react no less than 20 hours, and EU marker is formed；
Step 1c, chromatographic column prepare: Sepharose 6B being filled chromatographic column, is balanced with Tris-HCl buffer；
Step 1d, fluorescent marker are collected: taking the EU marker formed in step 1b to cross the chromatographic column prepared in step 1c, then
It is eluted with Tris-HCl buffer, flow velocity 2ml/ pipe/5min, the first effluent of collection is fluorescent marker.
4. the detection method of microfluidic chip according to claim 1 based on magnetic bead technology, it is characterised in that: in step 2,
Magnetic bead is reacted with antibody-solutions method particularly includes: the Dynal Tosyl magnetic bead for taking 200 μ L, by 50mM pH9.0's
After borate buffer cleaning twice, the CRP antibody-solutions of 200 μ L and the borate buffer of 800 μ L, hybrid reaction, reaction is added
Concentration is 20mg/ml, is reacted 24 hours.
5. the detection method of microfluidic chip according to claim 1 based on magnetic bead technology, it is characterised in that: in step 2,
After reaction, magnetic bead is closed using the PB buffer containing 1%BSA and pH=7.4, after closing 24 hours, to magnetic bead
It is cleaned, and saves liquid using magnetic bead and redissolve, be configured to the solution of 0.4mg/ml；Wherein, it is containing 1%BSA's that magnetic bead, which saves liquid,
PBS buffer solution.
6. the detection method of microfluidic chip according to claim 1 based on magnetic bead technology, it is characterised in that: in step 9,
After cleaning, increased response liquid is injected to quantitative-reaction chamber of microcontroller chip by the enhancement solution inlet on microcontroller chip,
Magnetic attracting device is removed, and is aided with ultrasonic mixing processing.
7. the detection method of microfluidic chip according to claim 6 based on magnetic bead technology, it is characterised in that: increased response
Liquid is using glacial acetic acid and Potassium Hydrogen Phthalate as buffer ions pair, using β-NTA as the liquid of complexing agent.
8. the detection method of microfluidic chip according to claim 1 based on magnetic bead technology, it is characterised in that: in step 9,
After cleaning, air is injected to quantitative-reaction chamber by gas path device, excludes the cleaning solution in quantitative-reaction chamber.
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| CN108548931A (en) * | 2018-05-09 | 2018-09-18 | 南京岚煜生物科技有限公司 | Free thyroxine fT4 kits and preparation and detection method are detected based on micro-fluidic chip |
| CN108548932A (en) * | 2018-05-09 | 2018-09-18 | 南京岚煜生物科技有限公司 | Thyrotropic hormone TSH kits and preparation based on micro-fluidic chip and detection method |
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