CN108375559A - Cardiac troponin kit and preparation based on micro-fluidic chip and detection method - Google Patents
Cardiac troponin kit and preparation based on micro-fluidic chip and detection method Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0887—Laminated structure
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
Abstract
The cardiac troponin kit that the invention discloses a kind of based on micro-fluidic chip and preparation and detection method, the preparation method of the kit include the following steps：(1) micro-fluidic chip is coated with；(2) fluorescent microsphere marks；(3) micro-fluidic chip assembles；(4) preparation of product is calibrated；(5) the cardiac troponin cTnI kits based on micro-fluidic chip are obtained.The cTnI kits being prepared are measured using double antibody sandwich method, highly sensitive time-resolved fluorescence object is chosen as marker, the label of antibody is carried out on fluorescent microsphere, analysis detection, level of the prepared reagent performance up to same chemical illuminating reagent are carried out using the immune response of antibody pair.After immune response, after completely removing extra component using cleaning solution, reading is detected in conjunction with upper fluorescent microsphere to immune response, introducing developing solution is avoided and enters the problem of chip interior is reacted insufficient or read not in time after developing the color.
Description
Technical field
The invention belongs to field of immunodetection, are detected more particularly, to a kind of cardiac troponin based on micro-fluidic chip
Kit and its preparation and detection method.
Background technology
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 which to prepare include sample introduction, reaction, detaches, is 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 sample separation early period to be integrated with mating
Many difficulties are brought, while being fixed in sample, there is also many problems for elution etc..
Micro-fluidic chip detection mainly has following advantage：
1) device is small and not to 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 biology
The time required to experimental implementation shortens 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.
Acute myocardial infarction AMI (AMI) is irreversible Acute myocardial meronecrosis.4-6h levels of cTnI liter after the onset
Height, 18-24h reach peak, restore normal time average out to 99h, and small number of patients is down to normally for 1 week.CTnI is thromboembolism treatment simultaneously
Blood vessel whether again logical early stage sensitive indicator.CTnI has good correlation in terms of diagnosing myocardial infarction area, to independent
Skeletal muscle injury, wound, nephrosis will not cause cTnI to increase, and sensibility and specificity is superior, have to judging prognosis important
Meaning.Serum cTnI has clinical value in the judgement of myocardial inflammation and slight damage, in CABG peri-operation period cardiac muscles
It is also a sensitive index in terms of the judgement of damage.
A kind of micro-fluidic chip, including flexible polymer skin and base are disclosed in Chinese patent literature CN104614521A
Piece, flexible polymer skin and substrate are bonded together, and micro- channel system is equipped in the chip, and micro- channel system includes slow
Fliud flushing entrance, sample inlet, magnetic bead entrance, waste liquid outlet, micro-valve, Micropump area, magnetic bead plug area, race way and detection zone.
Above-mentioned technical proposal uses the lower turbidimetry of sensitivity, detects the immune response between immunomagnetic beads and determinand
The turbidity variation being formed by before and after complex compound progress reactant, to carry out the concentration of judgement determinand.In contrast, it is using
For this methodology of immunoturbidimetry, due to not needing additional pairing antibody and luminous marker, whole chip knot
Structure design is relatively simple.It is enriched with magnetic bead by the specific region that magnetic field is formed in chip raceway groove, sample continues through this part
It is combined with another survey magnetic bead after magnetic bead plug, is reunited to form more than two magnetic beads.
Structure using other prior arts or (1) chip is excessively complicated, can not make accurate regulation and control to reaction, clean
There are larger pollutions for step and liquid conductive；Or (2) have used multiple liquid chambers and solid phase carrier (magnetic bead and fluorescence
Microballoon uses simultaneously), reagent global design is excessively complicated；Or (3) immune response uses heterogeneous reaction, means solid phase carrier
It is reacted with the surface of solid phase carrier, reaction is insufficient, and sensitivity and repeatability are restricted.
Invention content
Highly sensitive time-resolved fluorescence object is chosen as label the technical problem to be solved in the present invention is to provide a kind of
Object carries out the label of antibody on fluorescent microsphere, carries out analysis detection using the immune response of antibody pair, prepared is agents
It can be up to the level of same chemical illuminating reagent, the more efficient more fully myocardium myo calcium based on micro-fluidic chip of immune response
The preparation method of albumen cTnI kits.
In order to solve the above technical problems, the technical solution adopted by the present invention is, it should the myocardium myo calcium based on micro-fluidic chip
The preparation method of albumen cTnI kits, includes the following steps：
(1) micro-fluidic chip is coated with：CTnI antibody 440 is taken, is added into the CB buffer solutions of pH9.6, according to one single chip
25 μ L are added in the reaction chamber of micro-fluidic chip, are cleaned to chip after warm bath；
(2) fluorescent microsphere marks：CTnI antibody 129 is taken to be added into fluorescent microsphere solution, and the mixing that is vortexed, wherein glimmering
Light microballoon is by activation process after reaction, terminate liquid to be added and is terminated to reaction, envelope is added in rear eccentric cleaning
It closes liquid to be closed, the fluorescent microsphere after closing carries out eccentric cleaning, is preserved using microballoon storing liquid；
(3) micro-fluidic chip assembles：Seal treatment is carried out in advance to the reaction chamber of micro-fluidic chip, after confining liquid addition
Drying and processing is carried out under baking oven, is added in the step (2) in middle level chip back reaction chamber lower part after closure
Fluorescent microsphere solution, is dried in vacuo in vacuum drying oven, and droplet form is presented in fluorescent microsphere in the chips, does not spread；
After chip is ready by the way of ultrasonic bond, according to chip structure successively by hemofiltration pad, it is loaded gasket,
Chip interior is added in conductive rubber, blotting paper and sealing ring, and lower layer chip is completed to merge according to ultrasonic bond line；
(4) preparation of product is calibrated：Calibration product are made of component A and B component；
According to the estimated concentration of antigen concentrate, calibration product dilution is used to dilute antigen concentrate as calibration product antigen
Mother liquor is spare；
Calibration product dilution is calibration product component A, a concentration of 0ng/mL, 2-8 DEG C of preservation；
Calibration product B component：CTnI antigen mother liquors are diluted to 25ng/mL, 2-8 DEG C of preservation with calibration product dilution；
(5) the cardiac troponin cTnI kits based on micro-fluidic chip are obtained.
In the present invention, calibration product dilution be mainly Tris-HCl buffer system, a concentration of 50mM, pH8.5, including
Cow's serum, BSA, surfactant R PE1740, RPE2520 and Sodium azide；
In the step (3), dilution label cTnI microballoons, in advance by microballoon water bath sonicator mixing, take 20 microlitres of microballoons+
80 microlitres are drained preservation liquid, and mixing water bath sonicator adds 5 microlitres of microballoons, vacuum in the chip lower layer reactive tank middle closed
Dry 30min, the chip handled well is assembled, ultrasonic bond, last aluminium foil bag preserves and drier is added.
In the above-mentioned technical solutions, reagent be based on micro-fluidic chip as carrier, the fluid channel inside micro-fluidic chip
Immune detection reaction is carried out in reactor, one single chip is tested for single part and used；The kit being prepared is detected,
Manually after sample-adding, instrument realizes liquid flowing control of the determinand in micro-fluidic chip, the regulation and control and reaction of immune response
After cleaning step, instrument fluorescence signal and calculates specific response value by collecting；The reagent can be used for assisting examining
Disconnected AMI morbidities patient, and have good monitoring and diagnostic effect to the patient for suffering from secondary cardiovascular complication；Skill of the present invention
The cTnI kits that art scheme is prepared are measured using double antibody sandwich method, and an anti-cTnI antibody is coated on miniflow
It controls in chip reaction chamber, as stationary phase, another anti-cTnI antibody is coated on fluorescent microsphere, and immune microsphere is drained in advance
It is fixed in micro-fluidic chip, when micro-fluidic chip adding mouth is added in sample, is flowed into inside fluid channel by air pump sample, sample
CTnI antigens priority to be measured in this is captured by fluorescent microsphere coated antibody and chip coated antibody respectively.Surpass in reaction chamber
Extra unbonded antigen and fluorescent microsphere are removed after phonoresponse, after cleaning, by swashing under 360nm wavelength to micro-fluidic chip
Hair, detects fluorescence of the fluorescent microsphere of capture at 615nm, the fluorescence signal of its generation is measured, with relative light units (RLU)
It is indicated.The RLU of cTnI antigen concentrations and Instrument measuring in sample is at certain functional relation, by calculating acquisition automatically
Concentration value.
In addition, the micro-fluidic chip used in above-mentioned preparation method is announces in Chinese patent literature CN107219360A
Technical solution, applicant introduced herein；The highly sensitive time-resolved fluorescence object of selection is micro- in fluorescence as marker
The label that antibody is carried out on ball carries out analysis detection using the immune response of antibody pair, and prepared reagent performance is up to same
The level of chemical illuminating reagent.Reaction avoids single line using homogeneous reaction by ultrasonic mixing in micro-fluidic chip chamber
The chromatographic flow of journey is reacted so that reaction efficiency higher is more abundant.After immune response, extra component is gone completely using cleaning solution
After removing, reading is detected in conjunction with upper fluorescent microsphere to immune response, introducing developing solution is avoided and enters chip interior reaction
The problem of being read not in time after insufficient or colour developing.
Fluorescent marker is fixed in the middle level chip stage casing of micro-fluidic chip, in lower layer chip coated antibody, micro-fluidic core
Piece is coated and is dried using isolation buffer liquid in advance, and fluorescence is added after the surface layer of plastic chip forms layer protecting film
Microballoon marker, is both fixed on chip interior by way of drying, after sample and dilution is added, can answer again
It is molten and reacted with determinand, and component (i.e. confining liquid) is isolated and is not involved in reaction, and fluorescent marker and immune can be placed
Magnetic bead keeps bioactivity during the drying process, and avoids that non-specific binding occurs with chip.
Further improve is that in the step (2), the method for the fluorescent microsphere activation process is：Weigh EDC
It is configured as the solution of 1mg/mL using Mes buffer solutions, measures 1mg carboxyl fluorescent microspheres, microballoon solid content is 10mg/mL, body
Product is 50 μ L, is placed in EP pipes, and the dilution of 0.45mL Mes buffer solutions is added, 5 μ L are taken from the EDC activator solutions of 1mg/mL
It is rapidly added in microspheres solution, vortex mixing, the ultrasound 10min in ultrasonic cleaning machine, is placed on roller bearing vortex mixer and shakes instead
Answer 20min, total reaction time 30min.
Further improve is, in the step (3), the confining liquid also have 2%BSA, 0.5%Tween-20,
10% trehalose and 0.2% mannitol.
Further improve is that in the step (3), the method for Seal treatment is：Reaction to micro-fluidic chip
Chamber carries out Seal treatment in advance, wherein needed in the back side reaction chamber of second layer chip add 6 μ L+6 μ L confining liquid into
Row is closed, and in the positive reaction chamber of third layer, the confining liquid for being respectively necessary for 5 μ L+5 μ L+5 μ L of addition is closed.
Further improve is, in the step (3), the micro-fluidic chip is three-chip type structure, including by upper
Upper layer chip, middle level chip and the lower layer chip stacked gradually under；Upper layer chip, middle level chip and lower layer chip are two-by-two
Between positioning column, location hole be connected by way of realize mutual stacking.
The invention solves another problem be to provide a kind of cardiac troponin cTnI examination based on micro-fluidic chip
Agent box includes micro-fluidic chip, further includes having calibration product, the calibration product are made of component A and B component, wherein calibration product
Dilution is calibration product component A, a concentration of 0ng/mL；Calibration product B component is with the calibration product dilution by cTnI antigens
Mother liquor is diluted to 25ng/mL；Storage is dried in the antibody reagent of fluorescent microsphere label in the micro-fluidic chip；It is another
CTnI antibody is coated in micro-fluidic chip reaction chamber.
Kit in above-mentioned technical proposal is the heart based on micro-fluidic chip being prepared using the method for the present invention
Flesh troponin cTnI kits, cTnI kits are measured using double antibody sandwich method, and an anti-cTnI antibody is coated on
In micro-fluidic chip reaction chamber, as stationary phase, another anti-cTnI antibody is coated on fluorescent microsphere, can be used for assisting examining
Disconnected AMI morbidities patient, and have good monitoring and diagnostic effect to the patient for suffering from secondary cardiovascular complication.
The invention solves another problem be to provide it is a kind of based on micro-fluidic chip detection cardiac troponin cTnI
Method, the reagent obtained using the preparation method of the cardiac troponin cTnI kits above-mentioned based on micro-fluidic chip
Box includes the following steps：
(1) pipettor is loaded, and the sample for drawing 100 μ L is added in well, and micro-fluidic chip is entered in detecting instrument,
Gas path device is slowly inflated, and pushes sample to be moved along, gas is flowed out by ventilation mouth, and liquid is using PS as the micro-fluidic core of base material
It is flowed into reaction chamber in piece, sample contact reacts the conductive rubber of cabin end, and capacitance variations can touch, and closes runner valve
Door simultaneously closes off air circuit breaker, stops pressurization；
(2) instrument opens Vltrasonic device, and ultrasonic mixing 3-10 minutes, ultrasonic probe was placed in three layers of chip bottom, is located at anti-
The fore-end for answering chamber avoids the influence of directly ultrasonic chip coated antibody, after reaction gas push sample carry out to
Preceding movement dries up well and runner sample, and cleaning fluid path device starts, and into reaction chamber, mixing 1-3 minutes carries out
Cleaning；Gas push cleaning solution at adding mouth continues to move, and dries up reaction chamber and runner liquid；
(3) excitation light source is opened, immune response is measured in conjunction with fixed fluorescent microsphere, instrument reads chip reaction
Part fluorescence intensity below chamber is reported after obtaining data by calculating and providing accordingly result.
Sample or Sample dilution are entered by adding mouth (middle level of micro-fluidic chip) inside micro-fluidic chip, to sample-adding
The additional certain pressure of mouth, after liquid flows into reaction chamber, contacts the Fluorescent microsphere marker of oven-dried condition, passes through conduction in advance
Rubber controls the indoor total amount of liquid of reaction chamber, stops the pressure of adding mouth, is ultrasonically treated, makes to the reaction cabin of chip
It obtains coated antibody on chip and carries out hybrid reaction with Fluorescent microsphere marker test substance, the reaction time is i.e. reachable at 5-10 minutes
To the plateau of reaction；After reaction, cleaning solution is injected to chip interior by the side port of micro-fluidic chip, by extra sample
This and fluorescent marker are cleaned to devil liquor recovery area, after cleaning, then by chip side port one section of air of injection, it is indoor to exclude chamber
Cleaning solution；Cleaning step in triplicate, after to be cleaned, directly carry out laser excitation detection to chip, passes through Computation immunity
Fluorescent microsphere content in reaction bonded extrapolates the content of determinand in sample, therefore can analyze to be measured in calculating sample
The concentration of object.
Specific implementation mode
It is further described with reference to embodiments of the present invention：
The cardiac troponin cTnI kits based on micro-fluidic chip of the present invention, include micro-fluidic chip, also wrap
Calibration product are included, the calibration product are made of component A and B component, wherein calibration product dilution is calibration product component A, concentration
For 0ng/mL；Calibration product B component is that cTnI antigen mother liquors are diluted to 25ng/mL with the calibration product dilution；Fluorescent microsphere
Storage is dried in the antibody reagent of label in the micro-fluidic chip；Another cTnI antibody is coated on micro-fluidic chip reaction
Intracavitary.
In the present embodiment, the preparation method of the above-mentioned cardiac troponin cTnI kits based on micro-fluidic chip, packet
Include following steps：
(1) micro-fluidic chip is coated with：
CTnI antibody 440 (Biospacific) 1mg is taken, adds the CB buffer solutions of 50mM pH9.6 to 1ml, according to single core
25 μ L of piece are added in micro-fluidic chip reaction chamber, and warm bath cleaned chip after 2 hours；
(2) fluorescent microsphere marks：
The solution that EDC is configured as 10mg/mL using Mes buffer solutions is weighed, is further diluted to using Mes buffer solutions afterwards
For the solution of 1mg/mL.1mg carboxyl fluorescent microspheres are measured using 200 μ L pipettors, microballoon solid content is 10mg/mL, and volume is
For 50 μ L；It is placed in EP pipes, the dilution of 0.45ml Mes buffer solutions is added；Take 5 μ L rapid from the EDC activator solutions of 1mg/mL
It is added in microspheres solution, vortex mixing is placed on concussion reaction on roller bearing vortex mixer with ultrasound 10min in ultrasonic cleaning machine
20min, total reaction time 30min；The precipitation that this step generates belongs to normal phenomenon, usually disappears after being coupled with target antibody
It loses, activation ratio (microballoon is than activator) is 1:0.01；Activation terminates, and centrifugal treating does not take cTnI antibody 129 directly
(Biospacific) (according to antibody concentration, quality is 0.2 with microballoon ratio:1) fluorescent microsphere solution is added, and it is mixed to be vortexed at once
It is even；After reacting 5min, microballoon generates more apparent deposited phenomenon, and using Probe Ultrasonic Searching, power 5%, ultrasonic 3S is spaced 3S；
It after ultrasonic 5min, is placed in roller bearing mixing and reacts 2 hours, the reaction was continued has no that precipitation occurs again；Reaction terminates, and 500 μ L are added
Terminate liquid terminates reaction, and roller bearing reacts eccentric cleaning after 20min, and the confining liquid that 0.5mL is added is closed, closing 1
Hour；Microballoon after closing carries out eccentric cleaning, and rotating speed 15000rpm, centrifugation time 45min, cleaning is primary, makes 0.5ml's
Microballoon storing liquid is preserved；Microballoon is carrying out it before probe type ultrasonic resuspension dispersion, ultrasonic power 5%, ultrasound
Time 10min, ultrasound works 3S suspend 3S；
(3) micro-fluidic chip assembles：
Carrying out Seal treatment in advance to the reaction chamber of micro-fluidic chip, (confining liquid contains 2%BSA, 0.5%Tween-
20,10% trehalose and 0.2% mannitol), the envelope for adding 6 μ L+6 μ L is wherein needed in the back side reaction chamber of middle level chip
It closes liquid to be closed, in the positive reaction chamber of lower layer chip, the confining liquid for being respectively necessary for 5 μ L+5 μ L+5 μ L of addition is sealed
It closes, drying and processing 5min is carried out under 45 DEG C of baking ovens after confining liquid addition, under middle level chip back reaction chamber after closure
The fluorescent solutions of 15 μ L are added in portion, 30min are dried in vacuo in 45 DEG C of vacuum drying ovens, small liquid is presented in fluorescent microsphere in the chips
Form is dripped, is not spread；
After chip is ready by the way of ultrasonic bond, according to chip structure successively by hemofiltration pad, it is loaded gasket,
Chip interior is added in conductive rubber, blotting paper and sealing ring, and three layers of chip are completed to merge according to ultrasonic bond line.
(4) preparation of product is calibrated
According to the estimated concentration of antigen concentrate, antigen concentrate is diluted to a certain concentration (such as with calibration product dilution
500ng/mL), spare as calibration product antigen mother liquor, -20 DEG C of antigen mother liquor or less preserves, the term of validity 12 months.
Calibration product antigen mother liquor is taken, it is diluted with calibration product dilution with certain dilution ratio, dilution ratio is at least
For 2 or more, (such as dilution ratio is 1:10,1:100).Above-mentioned prepared two cTnI are calibrated into product antigen mother liquor dilution
The curve determination calibration product antigen mother liquor that definite value or the foundation of last batch cTnI calibration objects are carried out with the reagent of reference system is each dilute
The cTnI concentration for degree of releasing measures at least multiple holes, and result is multiplied by after respective extension rate and show that respective calibration product antigen is female
All calibration product antigen mother liquid concentrations are obtained the estimated concentration of calibration product antigen mother liquor after results are averaged by liquid concentration.
The preparation of calibration product A：Calibration product dilution is calibration product A, a concentration of 0ng/mL, 2-8 DEG C of preservation；
The preparation of calibration product B：CTnI antigen mother liquors are diluted to 25ng/mL, 2-8 DEG C of preservation with calibration product dilution.
In the present embodiment, calibration product dilution is mainly the buffer system of Tris-HCl, a concentration of 50mM, pH8.5, packet
Containing cow's serum, BSA, surfactant R PE1740, RPE2520 and Sodium azide；
In the step (3), dilution label cTnI microballoons, in advance by microballoon water bath sonicator mixing, take 20 microlitres of microballoons+
80 microlitres are drained preservation liquid, and mixing water bath sonicator adds 5 microlitres of microballoons, vacuum in the chip lower layer reactive tank middle closed
Dry 30min, the chip handled well is assembled, ultrasonic bond, last aluminium foil bag preserves and drier is added.
Based on the method for micro-fluidic chip detection cardiac troponin cTnI, the reagent being prepared using the above method
Box specifically has：
Pipettor is loaded, and the sample for drawing 100 μ L is added in well, micro-fluidic chip is entered in detecting instrument, gas
Road device is slowly inflated, and pushes sample to be moved along, gas is flowed out by ventilation mouth, and liquid flows into the chip using PS as base material
In reaction chamber.Sample contact reacts the conductive rubber of cabin end, and capacitance variations can touch, and closes runner valve, closes simultaneously
It holds one's breath way switch, stops pressurization；
Instrument opens Vltrasonic device, and ultrasonic mixing 3-10 minutes, ultrasonic probe was placed in three layers of chip bottom, is located at reaction chamber
The fore-end of room avoids the influence of directly ultrasonic chip coated antibody.Gas push sample is carried out to Forward after reaction
It is dynamic, well and runner sample are dried up, cleaning fluid path device starts, and into reaction chamber, mixing 1-3 minutes is cleaned；
Gas push cleaning solution at adding mouth continues to move, and dries up reaction chamber and runner liquid；Excitation light source is finally opened,
Immune response is measured in conjunction with fixed fluorescent microsphere；Instrument reads part fluorescence intensity below chip reaction chamber,
It is reported after obtaining data by calculating and providing accordingly result.
In the present embodiment, coated antibody optimum amount determines in the following ways：
To determine the optimal use concentration of CTNI coated antibodies, we respectively grope the dosage of TNI440, will
TNI440 carries out incubation reaction with the concentration of 15 holes μ L/, 25 μ L/mL, 35 μ L/mL respectively, detection company's calibration object (S1-S6),
Two various concentration samples and three quality-control products, by comparing S2/S1 ratios (sensitivity), the RLU values of S6, company's calibration object
The accuracy of correlation coefficient r (linear) and sample and quality-control product selects best coated antibody dosage.It the results are shown in Table 1.
From in table 1 it can be found that be crosslinked under concentration at 3, linear dependence result indifference, the first and it is last a kind of
It is inaccurate to be coated with Quality Control measured value under volume, and S2/S1 is also lower than second, thus it is final it is selected be suitably crosslinked a concentration of two kinds it is anti-
Body equivalent is added, and is 2 μ g/mg.
Table 1
For the coating processes of fluorescent microsphere, oven drying method is to be dried in the middle part of the chip lower layer that fluorescent microsphere is added dropwise after closure
Chip is assembled after two hours dry；It is in the middle part of the chip lower layer of fluorescent microsphere dropwise addition after closure, in drying box to drain method
Middle carry out vacuum drying, vacuum degree 0.1MPa are 1 hour dry.Compare the detection company calibration object under two kinds of painting methods
Measured value situation, the measured value of S1/S2, the RLU values of S6.
It is analyzed according to the correction data of labeling method, detection background result difference is little under two methods, still
One-step method detection high level is higher than two-step method, and coincidence rate when considering the simplification of microballoon marking process and measuring sample is drafted
The label of fluorescent microsphere is carried out using one-step method.For the painting method of fluorescent microsphere when using oven drying method, the background of detection is higher,
And the discrimination that calibration object measures can not draw away, and use when draining mode and being coated, background is promoted, but overall performance is examined
It surveys and is better than oven drying method.In conclusion fluorescent microsphere is marked using one-step method, the fluorescent microsphere after label is applied using method of draining
It covers in the chip.
In the present embodiment, using micro-fluidic chip as detection platform, the detection of cTnI projects has been carried out, with immune layer
The cTnI projects comparison of platform is analysed, the sensitivity of detection reagent has great raising, and due to being used in immune response
The reaction system of phase avoids chromatography release uneven and the linear single problem of reaction, has in accuracy in detection greatly
Improvement；Storage is dried in fluorescent microsphere labelled antibody reagent in the chip, uses exclusive isolation buffer liquid component,
Liquid reagent can effectively be separated, so that it is not susceptible to random flowing, while can be fast when sample and dilution is added
Instant solution simultaneously keeps original bioactivity.Isolation buffer liquid also acts as simultaneously prevents the absorption of fluorescent marker in the chip
Effect, fluorescent marker are contacted due to not direct with chip, and nonspecific absorption is reduced.
Mixing method of the Probe Ultrasonic Searching as micro-fluidic chip is used, ultrasonication is in the left side side of chip, in not shadow
In the optical property premise for ringing the finish of chip, ultrasonic energy transmission will mark fluorescent microsphere in reaction chamber again to be resisted
Body is redissolved and is reacted, in single chamber the reaction of single liquid ensure that no cross contamination.The effect of supersound process can
The efficiency of immune response is greatly improved, was reduced to reach the flat of reaction within 5-10 minutes by 30 minutes or more of popular response
The platform phase.
Dypass has been used to clean and the sample-adding pattern of addition enhancement solution on micro-fluidic chip, using seal washer,
Under the premise of ensureing that leakage does not occur for side port, cleaning solution or enhancement solution are injected to the side of reaction chip chamber interior by external pressure
On the one hand method simplifies the structure of chip, avoid occurring multiple chambers in chip, on the other hand to the reaction in micro-fluidic chip
Chamber plays the role of fully cleaning, and avoids the reflux of liquid and the pollution of sample；Using time-resolved fluorescence as glimmering
Light marker, detection signal compare traditional immunologic function test reagent and are greatly improved, and the design of chip reaction chamber uses
Transparent optical device can effectively detect optical signal, and fluorescent molecular is stabilized, not by the shadow of time and excitation number
It rings.
The basic principles and main features and advantage of the present invention have been shown and described above.The technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, and the above embodiments and description only describe the originals of the present invention
Reason, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes and improvements
It all fall within the protetion scope of the claimed invention.The claimed scope of the invention is by appended claims and its equivalent circle
It is fixed.
Claims (7)
1. a kind of preparation method of the cardiac troponin cTnI kits based on micro-fluidic chip, which is characterized in that including with
Lower step：
(1) micro-fluidic chip is coated with：CTnI antibody 440 is taken, is added into the CB buffer solutions of pH9.6, according to 25 μ L of one single chip
It is added in the reaction chamber of micro-fluidic chip, chip is cleaned after warm bath；
(2) fluorescent microsphere marks：CTnI antibody 129 is taken to be added into fluorescent microsphere solution, and the mixing that is vortexed, wherein fluorescence is micro-
Ball is by activation process after reaction, terminate liquid to be added and is terminated to reaction, confining liquid is added in rear eccentric cleaning
It is closed, the fluorescent microsphere after closing carries out eccentric cleaning, is preserved using microballoon storing liquid；
(3) micro-fluidic chip assembles：Seal treatment is carried out in advance to the reaction chamber of micro-fluidic chip, is being dried after confining liquid addition
Drying and processing is carried out under case, and the fluorescence in the step (2) is added in middle level chip back reaction chamber lower part after closure
Microspheres solution is dried in vacuo in vacuum drying oven, and droplet form is presented in fluorescent microsphere in the chips, does not spread；
After chip is ready by the way of ultrasonic bond, according to chip structure successively by hemofiltration pad, it is loaded gasket, it is conductive
Chip interior is added in rubber, blotting paper and sealing ring, and three layers of chip are completed to merge according to ultrasonic bond line；
(4) preparation of product is calibrated：Calibration product are made of component A and B component；
According to the estimated concentration of antigen concentrate, calibration product dilution is used to dilute antigen concentrate as calibration product antigen mother liquor
It is spare；
Calibration product dilution is calibration product component A, a concentration of 0ng/mL, 2-8 DEG C of preservation；
Calibration product B component：CTnI antigen mother liquors are diluted to 25ng/mL, 2-8 DEG C of preservation with calibration product dilution；
(5) the cardiac troponin cTnI kits based on micro-fluidic chip are obtained.
2. the preparation method of the cardiac troponin cTnI kits according to claim 1 based on micro-fluidic chip,
It is characterized in that, in the step (2), the method for the fluorescent microsphere activation process is：EDC is weighed using Mes buffer solutions to match
It is set to the solution for 1mg/mL, measures 1mg carboxyl fluorescent microspheres, microballoon solid content is 10mg/mL, and volume is 50 μ L, is placed in
In EP pipes, the dilution of 0.45mL Mes buffer solutions is added, takes 5uL to be rapidly added microballoon from the EDC activator solutions of 1mg/mL molten
In liquid, vortex mixing, the ultrasound 10min in ultrasonic cleaning machine is placed on concussion reaction 20min, overall reaction on roller bearing vortex mixer
Time is 30min.
3. the preparation method of the cardiac troponin cTnI kits according to claim 1 based on micro-fluidic chip,
Be characterized in that, in the step (3), the confining liquid also have 2%BSA, 0.5%Tween-20,10% trehalose and
0.2% mannitol.
4. the preparation method of the cardiac troponin cTnI kits according to claim 3 based on micro-fluidic chip,
It is characterized in that, in the step (3), the method for Seal treatment is：It is closed in advance to the reaction chamber of micro-fluidic chip
Processing wherein needs the confining liquid for adding 6 μ L+6 μ L to be closed in the back side reaction chamber of second layer chip, third layer front
Reaction chamber in, be respectively necessary for addition 5 μ L+5 μ L+5 μ L confining liquid closed.
5. the preparation method of the cardiac troponin cTnI kits according to claim 1 based on micro-fluidic chip,
It is characterized in that, in the step (3), the micro-fluidic chip is three-chip type structure, including is stacked gradually from top to bottom upper
Layer chip, middle level chip and lower layer chip；Upper layer chip, middle level chip and lower layer chip pass through positioning between any two
The mode that column, location hole are connected realizes mutual stacking.
6. a kind of cardiac troponin cTnI kits based on micro-fluidic chip, include micro-fluidic chip, which is characterized in that
Further include having calibration product, the calibration product are made of component A and B component, wherein calibration product dilution is calibration product component A,
A concentration of 0ng/mL；Calibration product B component is that cTnI antigen mother liquors are diluted to 25ng/mL with the calibration product dilution；Fluorescence
Storage is dried in the antibody reagent of microballoon label in the micro-fluidic chip；Another cTnI antibody is coated on micro-fluidic chip
In reaction chamber.
7. a kind of method based on micro-fluidic chip detection cardiac troponin cTnI, which is characterized in that use claim 1-5
The kit that the preparation method of cardiac troponin cTnI kit of any one of them based on micro-fluidic chip obtains, including
Following steps：
(1) pipettor is loaded, and the sample for drawing 100 μ L is added in well, micro-fluidic chip is entered in detecting instrument, gas circuit
Device is slowly inflated, and pushes sample to be moved along, gas is flowed out by ventilation mouth, and liquid is in the micro-fluidic chip using PS as base material
It flows into reaction chamber, sample contact reacts the conductive rubber of cabin end, and capacitance variations can touch, and closes runner valve, together
When close air circuit breaker, stop pressurization；
(2) instrument opens Vltrasonic device, and ultrasonic mixing 3-10 minutes, ultrasonic probe was placed in three layers of chip bottom, is located at reaction chamber
The fore-end of room avoids the influence of directly ultrasonic chip coated antibody, and gas push sample is carried out to Forward after reaction
It is dynamic, well and runner sample are dried up, cleaning fluid path device starts, and into reaction chamber, mixing 1-3 minutes is cleaned；
Gas push cleaning solution at adding mouth continues to move, and dries up reaction chamber and runner liquid；
(3) excitation light source is opened, immune response is measured in conjunction with fixed fluorescent microsphere, instrument reads chip reaction chamber
Below part fluorescence intensity, obtain data after by calculate and provide accordingly result report.
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