CN108663525A - A kind of heart infarction heart failure magnetic particle microflow controlled biochip, detection method - Google Patents

A kind of heart infarction heart failure magnetic particle microflow controlled biochip, detection method Download PDF

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CN108663525A
CN108663525A CN201810645803.0A CN201810645803A CN108663525A CN 108663525 A CN108663525 A CN 108663525A CN 201810645803 A CN201810645803 A CN 201810645803A CN 108663525 A CN108663525 A CN 108663525A
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biochip
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heart failure
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张永勇
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Particle Cloud Technology (beijing) Co Ltd
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Particle Cloud Technology (beijing) Co Ltd
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Abstract

The present invention relates to a kind of heart infarction heart failure magnetic particle microflow controlled biochips, belong to POCT detection technique fields, including pcb board, there is microchannel on the pcb board, it is provided with biosensor in the microchannel, the biosensor is coated deposited antibodies on wafer platform and wafer platform, and the deposited antibodies can form immune complex with magnetic bead coupled antibody and marker protein, by measuring compound magnetoresistance signal power judgement symbol object albumen concentration on wafer platform.Magnetic bead has larger specific surface area in the technical solution, in conjunction with more antibody, improves the sensitivity of detection, specificity is higher than the chemoluminescence method of forth generation.The present invention also provides a kind of detection method using above-mentioned biochip, can simultaneous quantitative detection heart infarction, this kind of cardiac marker of heart failure, and improve detection Monitoring lower-cut, efficiently avoid missing inspection and false negative phenomenon.

Description

A kind of heart infarction heart failure magnetic particle microflow controlled biochip, detection method
Technical field
The present invention relates to a kind of magnetic particle microflow controlled biochips, mainly for detection of heart infarction heart failure mark in blood sample The detection of object；The present invention also provides a kind of detection methods using above-mentioned biochip, belong to POCT detection technique fields.
Background technology
Angiocardiopathy is to endanger human health and the serious disease of life, it has also become the No.1 of 21 century human health kills Hand.By the establishment of national cardiovascular disease center《Chinese cardiovascular disease report 2015》And《Chinese cardiovascular disease report 2016》Middle finger Go out, China's cardiovascular disease number of patients is still in rapid growth situation.According to statistics, cardiovascular disease number of patients in 2014 is 2.9 hundred million, By 2015, this number rose to 2.9 hundred million.Estimate that there are about 3,500,000 people to die of cardiovascular disease every year in China according to report, accounts for total The 43% of the cause of death.Wherein, acute myocardial infarction AMI is most common, most dangerous.The transformation of personal behavior mode and living habit, makes The risk that angiocardiopathy occurs for Chinese catches up with and surpasses rapidly the developed countries such as the U.S..
Angiocarpy inspection is " bottleneck " subject in entire cardiovascular field, because only that in the shortest possible time just It really diagnoses the illness, could early find, early treatment, utmostly prevent from disabling, lethal effect, improve patient's prognosis and life matter Amount.In recent years, increasingly deep for the research of cardiovascular biomarker, a large amount of clinical experiences and evidence are had accumulated, it is gradually bright Really their clinical indication and new research field, have pushed their clinical application.The detection of biomarker can be straight Connect the clinical diagnosis for influencing cardiovascular patient, risk stratification, therapeutic scheme selection and Index for diagnosis.
Cardiac marker full name is heart injury early sign object, refers to horizontal raising in blood in 6 hours after heart injury Marker, be the important Testing index of the heart diseases such as diagnosing myocardial infarction in clinic, myocardial ischemia, heart failure, include mainly Creatine kinase MB isoenzyme (CK-MB), cardiac troponin (cTn), b-type natriuretic peptide (BNP) etc. mark object.Cardiac marker POCT segments market the most fast business of middle speedup, and annual income scale alreadys exceed 900,000,000 dollars at present, average annual growth rate 14%.At a high speed The central factor of growth is the extensive support that the importance that these cardiac markers POCT is quickly detected has obtained circular for confirmation medicine, beautiful The tissues such as the clinical biochemical academy of sciences of state (NACB), U.S. clinical Chemical Society (AACC), European Society of Cardiology (ESC) are already The detection of these markers is included in heart disease prevention and diagnosis and treatment guide, and is gradually included in the meeting after 2000 POCT contents.
POCT becomes the highest product of growth rate in global medical instrument and medical diagnosis industry, wherein heart disease is real-time Examining becomes part with fastest developing speed during all POCT are examined.Statistical report shows that whole world IVD market scales about 44,000,000,000 are beautiful Member, wherein about 15,000,000,000 dollars of POCT market scales, it is contemplated that the markets POCT in 2018 will reach nearly 19,000,000,000 dollars, and its cardiac mark The POCT products of will object are estimated up to 1,200,000,000 dollars.Since instrument is portable, easy to operate, result is promptly and accurately etc. a series of excellent Point, POCT are quoted extensively.It is expected that cardiac marker in the markets POCT speedup up to 13.8%.
" heart mark when coronary artery disease and heart failure that China national health and the family planning committee formulated in 2015 Will analyte detection and clinical application " is pointed out：Should have preferable diagnosis, risk stratification and prognosis suitable for clinical cardiac marker Estimated value, analyzing detecting method should be special, sensitive, quick, cannot reach wanting for ＜ 60min in the emergency treatment detection turnaround time When asking, it is considered as using POCT modes.When being detected using POCT, quantitative analysis method should be used.American Academy of Clinical Biochemistry (NACB) to being pointed out in the suggestion of cardiac biomarkers detection：Laboratory should be preferably 30min or less turnovers in 1h Cardiac marker analyte detection is completed in time (turnaround time is defined as the time of the result from taking a blood sample to reporting)；POCT detections can be Obtained in 15-20 minutes the multinomial cardiac marker such as cTnI, BNP, D-Dime as a result, and the detection of conventional inspection section needs 1-2 Hour.Making a definite diagnosis to successful treatment for morbidity early stage key clinical index is extremely crucial, and POCT can save sample complexity Preprocessor and the time, shorten examine turnover period, can quickly obtain experimental result.
Currently, POCT clinical detection techniques include：
First generation detection technique competition law (radio immunoassay, RIA)：Impacted factor is more, RIA sample requirements It is more, due to usually requiring first to extract before detecting, operating procedure is not only increased, and usually only due to the rate of recovery of extraction 80%~90%, coefficient of variation (CV) is relatively large, reduces the precision of detection；
The non-competing method of second generation detection method (radio immunoassay, RIA)：Using the immunoassay of double-antibody sandwich, It need not extract, antibody specificity is high, and sensitivity, accuracy and specificity are got well than competition law, but are taken still longer, it is difficult to suitable For automating；
Third generation detection method enzyme immunoassay (EIA), stationary phase immunochromatographic method：Using double antibody plus heart method immunoassay Solid phase chromatography method includes colloidal gold, immunofluorescence etc., and antibody specificity is high, and sensitivity, accuracy and specificity are better than non-competing Method is striven, equipment may be implemented and detect semi-automatic detection, can only realize quantitative/half-quantitative detection, operation and sample and physical property It is affected to the characteristic ssensitivity of detection, batch internal difference between having larger batch, the coefficient of variation is generally 15%~30%；
Forth generation detection method chemiluminescence method, a kind of microdetermination technology of hypersensitivity, it has highly sensitive Degree, detection range be wide, it is easy to operate quickly, the advantages that marker stability is good, pollution-free, instrument simple economy.But the inspection Survey method still has the following deficiencies：Phenomena such as light background noise, sample photobleaching phenomenon, attenuated optical signal, is by the spirit to detection Sensitivity and specificity play interference effect, at the same equipment can not minimize with POCTization, mainly operated by professional, Wu Fashi Existing bedside detection, emergency tender detection and family's detection.
Invention content
The purpose of the present invention is to provide a kind of heart infarction heart failure magnetic particle microflow controlled biochips, have detection sensitivity Height, the short advantage of detection time.Specific technical solution is as follows：
A kind of heart infarction heart failure magnetic particle microflow controlled biochip, including pcb board have microchannel, institute on the pcb board It states and is provided with biosensor in microchannel, the biosensor is coated point sample on wafer platform and wafer platform Antibody, the deposited antibodies can form immune complex with magnetic bead coupled antibody and marker protein, flat by measuring wafer Compound magnetoresistance signal power judgement symbol object albumen concentration on platform.
As an improvement of the above technical solution, the deposited antibodies are heart infarction heart failure marker CTI, D-Dime, NT- ProBNP antibody.
As an improvement of the above technical solution, the biosensor is used as signal transmission carrier by gold thread.
As an improvement of the above technical solution, the microchannel includes liquid feeding end and outlet end, on the pcb board successively It is provided with first layer and the second layer, perforative sample holes and waste liquid hole are offered on the first layer, are had on the second layer Mixed zone and waste, the lateral microchannel of mixed zone side connection, far from mixed zone on the transverse direction microchannel One end has to be connected with sample holes, and the biosensor on the pcb board forms biosensor reaction zone with microchannel, raw Fluid one-way flow in the following order in object chip：Mixed zone → transverse direction microchannel → sample holes → liquid feeding end → miniflow is logical Road → biosensor reaction zone → outlet end → waste liquid hole → waste.
As an improvement of the above technical solution, microchannel confined layer and Whole Blood Filtration are additionally provided with above the second layer Layer.
As an improvement of the above technical solution, the biosensor is connected by Wheatstone bridge mode, the biology Sensor includes an input terminal and two output ends, contains 0.1uM in the circuit of every group of output end2~100uM2Giant magnetoresistance material Material.
As an improvement of the above technical solution, be provided with magnetic excitation device on the outside of the biochip, deposited antibodies with After magnetic bead coupled antibody and marker protein form immune complex, clean unbonded magnetic bead extra on biosensor and Antibody protein, magnetic excitation device, which automatically turns on excitation uniform magnetic field, at this time makes magnetic bead magnetize, by magnetic on the biosensor The magnetic field that the magnetic bead of change generates causes the variation of giant magnetic resistance resistance, and then judges on biosensor the position of magnetic bead and dense Degree, heart infarction heart failure marker protein concentration is quantified by standard curve algorithm.
Above-mentioned biochip magnetic bead has larger specific surface area, can be in conjunction with more antibody, to improve detection Sensitivity；Without magnetism, unbonded magnetic bead on a sensor can not also excite magnetic for biomolecule and albumen simultaneously Field generates magnetoresistance signal, and specificity is higher than forth generation chemoluminescence method.
The present invention also provides a kind of detection methods using above-mentioned heart infarction heart failure magnetic particle microflow controlled biochip, including Following steps：
Step 1, biochip pre-treatment are coated with heart infarction heart failure marker (CTI/D-Dime/NT- on a biosensor ProBNP) deposited antibodies；
Step 2, prepared by 30nm+50nm magnetic beads and antibody coupling isolates and purifies system：
I, the preparation of 30nm+50nm magnetic beads mixed liquor：Take Fe3O4Solution is added in ultra-pure water so that ferroso-ferric oxide solution Final concentration 0.01%~0.03% is heated to 200 DEG C~300 DEG C in non magnetic concussion high-temperature heating system, and 10% vinegar is added Acid sodium solution so that the ferroso-ferric oxide superparamagnetic of 30nm is obtained by the reaction under high temperature for sodium acetate final concentration of 0.2%~0.3% Magnetic bead black colloidal state suspension；
Take Fe3O4Solution, which is added in ultra-pure water, makes ferroso-ferric oxide solution final concentration 0.01%~0.03%, non magnetic 200 DEG C~300 DEG C are heated in concussion high-temperature heating system, 10% sodium acetate solution is added so that sodium acetate is final concentration of 0.1%~0.2%, the ferroso-ferric oxide super paramagnetic beads black colloidal state suspension of 50nm is obtained by the reaction under high temperature；
The ferroso-ferric oxide of the ferroso-ferric oxide super paramagnetic beads black colloidal state suspension of 30nm and 50nm is super suitable Magnetic magnetic bead black colloidal state suspension 1：1 mixing is for use；
30nm and 50nm ferroso-ferric oxide super paramagnetic beads black colloidal state mixing suspensions are placed on magnetic patch, softly Supernatant is removed in absorption, and ethyl alcohol and ultra-pure water is used in combination to clean, by 30nm the and 50nm ferroso-ferric oxide super paramagnetic beads washed point Dissipate in ethyl alcohol, ultra-pure water, ammonium hydroxide mixed liquor in after be added tetraethyl orthosilicate, stirring form core-shell type silicon dioxide layer；
II, the Covalent bonding together of Epoxy functionalized group is added by γ-(2,3- the third oxygen of epoxy) propyl trimethoxy silicon The mixed liquor of alkane, methanol, ultra-pure water composition obtains the 30nm+ containing epoxy-activated after heating, cleaning and drying The super quick magnetic beads of 50nm；
III, it the super quick magnetic bead albumen coupling of superparamagnetic and isolates and purifies：
(1) Sulfo-SMCC (SS- sulfo groups are added in the super quick magnetic beads of 30nm+50nm by the activation of the super quick magnetic beads of 30nm+50nm Succinimido -4- (N- maleimidomehyls) cyclohexane -1- carboxylates), coupling buffer, ultrasonic disperse magnetic is added Pearl；
(2) the super quick magnetic beads of the 30nm+50nm being re-activated through sulfydryl are placed in magnetic separator and are detached, absorbed supernatant, add Enter coupling buffer cleaning, repeats and suspended again magnetic bead with coupling buffer after Magneto separate purification process, ultrasonic disperse waits for With；
(3) activation of coupling streptavidin/monoclonal antibody；
(4) the super quick magnetic bead of superparamagnetic is coupled streptavidin/monoclonal antibody：By activated streptavidin/Dan Ke Grand antibody is added in the magnetic bead activated, and coupling buffer is added, then test tube is placed on strong magnet and detaches by ultrasonic disperse Magnetic bead；
(5) super long-chain biological monoclonal antibody coupling：It is then mixed with monoclonal antibody solution with ultrapure water dissolution biotin It closes and is incubated a period of time, centrifuge removal supernatant, obtain overlength chain Bioconjugation monoclonal antibody；
Step 3, the detection of biotin-labeled pentylamine reaction system：Take blood sample (whole blood/plasma/serum/refers to blood) that life is added Object chip sample application zone dispenses biotin conjugated monoclonal antibodies and is shaken by automatic puncture into mixed zone in advance automatically into mixed zone Dynamic hybrid reaction 10-30 seconds is divided in magnetic bead coupled antibody in biochip vesica and is shaken by automatic puncture into mixed zone in advance Dynamic hybrid reaction enters reaction zone (biochip sensor) after 10-30 seconds, if containing heart infarction heart failure marker egg in sample In vain, the compound for forming magnetic bead is assembled into (Ab1-Ag-Ab2-bio-SA-MB) on biosensor, by measuring magnetoresistance signal Quantitative reaction goes out magnetic bead concentration, magnetic bead concentration is converted to albumen concentration by standard curve algorithm, and then judge heart infarction heart failure The concentration of marker protein.
As an improvement of the above technical solution, the step 1 specifically includes following three step：
I, biochip surface functionalization：
(1) preparation of activated polymerization agent is added in reactive compound and delays polymerizer, and pure water mixing ultrasound is added；
(2) protective coating solution is prepared, and isopropanol, activated polymerization agent, the second polymerization photosensitizer and crosslinking agent are uniformly mixed It closes；
(3) protective coating is evenly distributed to the biosensor surface of biochip by full-automatic spotting system, is led to Ultraviolet light excitation photoactive substance is crossed, the functional group in release catalysis is catalyzed monomer polymerization reactions, by ultraviolet irradiation so that protecting Shield coating forms stable solid-state function and protecting layer in biosensor surface；
(4) chip after solidification is respectively put into isopropanol and purified water and is cleaned by ultrasonic, goes by the cleaning of biosensor It is preserved except the residue of chip sensor surface, after being dried up by nitrogen for use；
II, biochip point sample and incubation：
(1) antibody protection liquid is prepared, and polyvinyl alcohol or polyvinylpyrrolidone are added under kaliumphosphate buffer system, adds Enter polysorbas20, Tween 80, tween 100 to be uniformly mixed；
(2) negative control substances are prepared, and BSA solution is configured with antibody protection liquid；
(3) deposited antibodies are prepared, and heart infarction heart failure marker (CTI/D-Dime/NT-proBNP) is prepared with antibody protection liquid Deposited antibodies, ultrasonic mixing；
(4) antibody and negative control are put on different biosensors respectively by full-automatic micro-sampling system, Liquid form and distribution situation on a biosensor are checked online by high-power microscope system；
(5) biochip after point sample is put into the closed container containing saturation potassium chloride and aluminium block and is incubated, make antibody The surface functional group stable bond of albumen and biosensor；
III, the closing of biochip：
(1) polysorbas20/80/100 is added in the preparation of chip confining liquid in trihydroxy methyl methylamino methane hydrochloride buffer solution Solution and ethanolamine solutions filter after mixing spare；
(2) closed protective liquid is prepared, and potassium chloride and sodium chloride are added under phosphate buffer, is filtered after mixing standby With；
(3) confining liquid is injected microchannel, closes extra site on biosensor by the closed system of biochip；
(4) cleaning and protection after biochip closing：Confining liquid protection liquid is injected into microchannel, cleans extra closing Liquid residual components；
(5) biochip after closing is put into drying box drying so that on biosensor by the drying of biochip Antibody or the albumen state that dehydration is stablized in low energy completely, it is closed after dry to vacuumize preservation.
As an improvement of the above technical solution, " step 3, the detection of biotin-labeled pentylamine reaction system " is replaced with：" exempt from Epidemiology double antibody sandwich method reaction system detects ", " detection of immunology double antibody sandwich method reaction system " specifically includes：It takes Biochip sample application zone is added automatically into mixed zone in blood sample (whole blood/plasma/serum/refers to blood), is divided in biology in advance In chip vesica magnetic bead coupled antibody by after automatic punctured into mixed zone mixing 10-30 second into reaction zone (biochip biography Sensor), if containing heart infarction heart failure marker protein in sample, the compound for forming magnetic bead is assembled on biosensor (Ab1-Ag-Ab2-MB), go out magnetic bead concentration by measuring magnetoresistance signal quantitative reaction, by standard curve algorithm by magnetic bead concentration It is converted to albumen concentration, and then judges the concentration of heart infarction heart failure marker protein.
Above-mentioned detection method can be applicable in whole blood/plasma/serum sample/refer to blood samples detection, can realize it is micro (20ul~ 40ul) sample-adding trace detection is, it can be achieved that refer to blood examination brake；Detection time at 15 minutes~20 minutes, and realize highest 30~ 50 immune proteins/Molecular biomarkers detection, shorten Diagnostic Time, increase the rescue time of patient, which can Simultaneous quantitative detects heart infarction, the cardiac marker of heart failure, and improves the sensitivity (Monitoring lower-cut) of detection, has widened detection The range of linearity efficiently avoids missing inspection and false negative phenomenon.And detection time is short, easy to operate, safety non-pollution is suitable for Emergency tender, emergency treatment, ICU and bedside detection, have wide range of applications.
Description of the drawings
Fig. 1 is a kind of structural schematic diagram of heart infarction heart failure magnetic particle microflow controlled biochip of the present invention.
Fig. 2 be the present invention biochip in the second layer another embodiment structural schematic diagram.
Fig. 3 is that Troponin I (CTNI) quality-control product detects linear regression curve in example IV.
Fig. 4 is that D dimer (D-Dime) quality-control product detects linear regression curve in example IV.
Fig. 5 is that N-terminal Natriuretic Peptide (NT-proBNP) quality-control product detects linear regression curve in example IV.
Specific implementation mode
As shown in Figure 1, the present invention provides a kind of heart infarction heart failure magnetic particle microflow controlled biochip, including pcb board 10, There is microchannel 11 on pcb board 10, be provided with biosensor in microchannel 11, biosensor be wafer platform 20 with And coated deposited antibodies on wafer platform 20, deposited antibodies can form immune multiple with magnetic bead coupled antibody and marker protein Object is closed, by measuring compound magnetoresistance signal power rational judgment marker protein concentration on wafer platform 20, wherein point sample is anti- Body is heart infarction heart failure marker CTI, D-Dime, NT-proBNP antibody 1, and coupled antibody is heart infarction heart failure marker CTI, D- Dime, NT-proBNP antibody 2.When containing heart infarction heart failure marker protein in blood sample, it is coated with the biology of deposited antibodies Bead complexes aggregation will be formed on sensor.
Further, microchannel 11 (36 biosensors are provided in microchannel shown in Fig. 1) include into Liquid end 12 and outlet end 13 are disposed with first layer 30, the second layer 40,50 and of microchannel confined layer on the pcb board 10 Whole Blood Filtration layer 60 offers perforative sample holes 31 and waste liquid hole 32 on first layer 30, has mixed zone 41 on the second layer 40 With waste 44, the lateral microchannel 42 of 41 side of mixed zone connection, one end far from mixed zone 41 on lateral microchannel 42 With being connected with sample holes 31, fluid one-way flow in the following order in biochip：Mixed zone 41 → transverse direction microchannel 42 → sample holes, 31 → liquid feeding end, 12 → microchannel, 11 → outlet end, 13 → waste liquid hole, 32 → waste 44, in order to realize biology Micro-valve and Micropump can be arranged in the position of waste 44 in the one-way flow of fluid in chip, generated by micro-valve and Micropump negative The flowing of voltage-controlled fluid processed and flow direction, each region can be controlled by detector Minitype negative pressure air pump and its automatic program System.
As shown in Fig. 2, can be with around mixed zone 41 on the second layer 40, microchannel confined layer 50 and Whole Blood Filtration layer 60 Sample application zone 43, the first vesica well 45, the second vesica well 46 and third vesica well 47 are offered, on the second layer 40 Sample application zone 43, the first vesica well 45, between the second vesica well 46 and third vesica well 47 and mixed zone 41 It is respectively arranged with the drainage channel 48 of connection；First vesica well 45, the second vesica well 46 and third vesica well The advance encapsulation of 47 difference is added Biotin-conjugated antibodies (bio-Ab2), the magnetic bead (SA-MB) that Streptavidin is coupled, cleans and delay Fliud flushing.Pre-packaged vesica well can be punctured automatically in detection process, make Biotin-conjugated antibodies and Streptavidin The magnetic bead of coupling enters mixed zone 41 and is mixed with the blood sample for entering mixed zone 41 from sample application zone 43.
The principle that biosensor is detected in said program is, using the principle of giant magnetoresistance (GMR), to be given birth to by measuring The magnetic field signal and resistance signal of magnetic bead on object sensor, calculating magnetic field signal and resistance signal reacting condition are incorporated in sensor The concentration of upper magnetic bead passes through standard curve algorithm quantitative measurment heart infarction, the cardiac marker of heart failure.This system can quantify the detection heart The whole blood/serum/plasma/of lung disease people at highest risk/patient refers to blood Applications of Cardiac Markers, judges patient's heart infarction process, myocardial damage Degree makes carly fruit drop for clinician, strives for the prime time of the rescue of patient by early discovery, early treatment.
Following two detection methods may be used in the biochip：
Method one, biotin-labeled pentylamine reaction system detection method
It takes 10ul~30ul (whole blood/serum/plasma/refers to blood) sample to be added in the sample application zone of biochip, passes through micro-valve Control sample flow with Micropump, have when sample reaches mixed zone biotin conjugated monoclonal antibodies vesica by it is automatic punctured into Enter mixed zone vibrations mixing 10 seconds~30 seconds, the vesica for having magnetic bead coupling streptavidin is punctured into mixed zone, with sample Product and Biotin-conjugated antibodies mixture shake hybrid reaction 10 seconds~30 seconds；
Enter in biosensor (coating heart infarction heart failure marker antibody 1 in advance) by micro-valve control channel switch, instead 100 seconds~120 seconds between seasonable；Detecting system punctures the unbonded magnetic bead and sample composite of cleaning buffer solution vesica cleaning automatically Object, scavenging period 1 minute~2 minutes；If containing higher concentration heart infarction marker protein in sample, will be formed on a sensor Bead complexes assemble (Ab1-Ag-Ab2-bio-SA- magnetic beads), and it is dense to go out higher magnetic bead by measurement magnetoresistance signal quantitative reaction Degree, if gone out by the magnetoresistance signal quantitative reaction of measurement weaker without containing/denier heart infarction heart failure marker protein in sample Magnetic bead concentration.Magnetic bead concentration is converted to albumen concentration by the standard curve algorithm software in detecting system, and automatically fixed Amount judges the concentration of heart infarction heart failure marker protein.The heart infarction heart failure marker protein of high concentration represents heart infarction heart failure process, Reference range is below to represent no heart infarction, heart failure；Reference range, which nearby represents, has heart infarction, heart failure risk.Whole inspection Surveying the time will complete in 15 minutes~20 minutes.
Method two, immunology double antibody sandwich method reaction system
It takes 10ul~30ul whole bloods/serum/plasma/to refer to the sample application zone that biochip is added in blood sample, passes through micro-valve Micropump Sample flow is controlled, when reaching sample mixed zone, has the vesica of magnetic bead coupled antibody and is punctured into mixed zone by automatic, with Sample mixture hybrid reaction 10 seconds~30 seconds；
Enter in biosensor (coating heart infarction heart failure marker antibody 1 in advance) by micro-valve control channel switch, instead 100 seconds~120 seconds between seasonable；Detecting system punctures the unbonded magnetic bead and antibody of cleaning buffer solution vesica cleaning, cleaning automatically 1 minute~2 minutes time；If containing higher concentration heart infarction heart failure marker protein in sample, magnetic will be formed on a sensor Pearl compound assembles (Ab1-Ag-Ab2-bio-SA- magnetic beads), goes out higher magnetic bead concentration by measuring magnetoresistance signal quantitative reaction, If it is dense without containing/denier Applications of Cardiac Markers albumen by the magnetoresistance signal quantitative reaction of measurement to go out weaker magnetic bead in sample Degree.The process and method one that converts is identical.
The quantitative detection system of magnetic particle micro-fluidic chip according to the present invention includes mainly hardware components and software portion Point.Hardware components include mainly：Device housings, display, battery and single chip microcomputer circuit board and field resistance signal inductor, it is micro- Type negative pressure mechanical motor, two-dimensional code scanning system etc. are divided into from module, and material is equipped with module, sensor sensing module, fluid path Control module, machine driving module, temperature control module, magnetoresistance signal detection module, circuit integrated control module, bluetooth module and GPS module；
Material is equipped with module：Vesica (the difference magnetic bead that packing Biotin-conjugated antibodies, Streptavidin are coupled in advance, Cleaning buffer solution etc.), sample application zone, mixed zone, detection zone (on biosensor pre-coated deposited antibodies), devil liquor recovery etc. is System；
Sensor sensing module：Biosensor contact contact (wafer platform 20) of biochip and signal pass transmission system System, Quick Response Code is read and its signal-obtaining, transmission, storage system and its auxiliary circuit；
Fluid path control module：Including the mixing vibrations after sample-adding, micro-valve, Micropump, the mixing vibrations of mixing pit, negative pressure gas Hole, Minitype negative pressure mechanical motor etc.；
Temperature control module：It is controlled including temperature, incubation reaction time control etc.；
Machine driving module：Into delivery device, micro machine, sensor, conveying track, mechanical arm etc.；
Magnetoresistance signal detection module：Uniform magnetic field generating means (helmholtz coil), the reading device of magnetoresistance signal, mould Quasi- digital switching device, storage device, Signal Analysis System and signal display system；
Circuit integrated module：Sensor signal sensor circuit, transmission circuit, amplifying circuit, filter circuit, squelch circuit, The circuit systems such as each Module Links control driving；
Bluetooth module：With the module and its circuit system that will detect signal and wire/wireless transmission；
GPS module：Have the function of GPS signal global location and its associated circuitry；
Print module：Minitype thermal printer and its auxiliary circuit connect system
Software section：The software programming of the quantitative detection system of magnetic particle microflow controlled biochip, main includes that machinery drives Dynamic program, display system program, magnetic excitation program, fluid path control program, detect program, parser, and standard curve is read, Conversion, the programs such as printing driving.
The testing principle of magnetic signal instrument：Microflow controlled biochip contains multiple parallel to each other, section inverted trapezoidal The snakelike microchannel 11 of structure is provided in one or more in each microchannel and is provided with biosensor (the application 36 biosensors are used to carry out examples in attached drawing 1 and Fig. 2), biosensor uses the structure of wafer platform 20, Mei Gejing At convex shape right over first platform 20, each biosensor has individual gold thread transmitting device and address code, can individually remember Record the signal of its generation, the signal of the generation of quantization sensing device in a manner of Wheatstone bridge.Biosensor is with favour stone electricity The mode of bridge connects, including an input, two outputs, contains 0.1uM in every group of output circuit2~100uM2Giant magnetic resistance.
Biochip sensor signal reaction：On cleaning biosensor after extra unbonded magnetic bead and albumen, Detector excites uniform magnetic field generating means to generate uniform high-intensity magnetic field magnetization magnetic bead automatically, when antibody/long-chain of magnetic bead coupling When biosensor combines and assembles, fixed magnetic bead on a biosensor is magnetized Avidin.By magnetic on biosensor The magnetic field that the magnetic bead of change generates causes the variation of giant magnetic resistance resistance, is surveyed by incuding the variation of biosensor self-resistance Measure the quantity of magnetic bead and position on biosensor.
The transmission and processing of bio-sensor signal：Contain a series of input and output pin on microflow controlled biochip, DC power-supply circuit, circuit for regulating and controlling, field circuit, signal amplification circuit etc.；There is output giant magnetoresistance on each biosensor One Wheatstone bridge of effect signal, when there is externally-applied magnetic field to be added on the fixed magnetic bead of biosensor, biosensor The resistance variations of induction are turned resistance signal becoming voltage signal defeated by multilayer film giant magnetic resistance structure by Wheatstone bridge Go out, it will be electric by biosensor interface in magnetic signal instrument and its auxiliary circuit (amplifying circuit and squelch circuit) Pressure signal transmission to analog-digital commutator is converted into digital signal.
Bio-sensor signal is analyzed：The digital signal of analog-digital converter output is acted in auxiliary circuits such as address decoders Under, input micro-chip processor, using in memory analysis software and standard curve read analysis software for digital signal into Row processing.
Bio-sensor signal exports：Bio-sensor signal analysis result can pass through display chip or other outputs Equipment (LED, VGA display) shown, wired can also be set by controlling print circuit directly printing or by external Standby/Bluetooth system externally transmits testing result.
The specific implementation mode of the present invention is described with reference to the accompanying drawings and examples, to be better understood from this hair It is bright.
Embodiment one
A kind of heart infarction heart failure magnetic particle microflow controlled biochip of the present invention, biosensor have been coated with heart infarction heart failure three in advance Item marker (CTI/D-Dime/NT-proBNP) point sample monoclonal antibody, then by first layer 30, the second layer 40, microchannel Confined layer 50 and Whole Blood Filtration layer 60 are pasted in sequence above the microchannel 11 of pcb board 10, to close microchannel 11, so that reaction is carried out under conditions of closed, achievees the effect that micro- full laboratory.First layer 30 is biosensor confined layer, Contain sample holes 31 and waste liquid hole 32；The second layer 40 is microchannel layer, connection sample holes 31, biosensor and waste liquid hole 32 Access, while guiding sample to be tested from mixed zone 41 to detection zone (position where biosensor), waste liquid by Micropump 44 one-way flow of area；Microchannel confined layer 50 is third layer, ensures that sample flows in a closed pipeline, while whole blood Filter layer 60 contains whole blood filtration system close to the position of mixed zone 41, plays and red blood cell is prevented to enter microchannel 11, avoid Blocking channel improves the accuracy of testing result.
Three marker (CTI/D-Dime/NT-proBNP) magnetic particle microflow controlled biochips of heart infarction heart failure of the present invention, 30nm+50nm magnetic beads are coupled streptavidin dosage：0.1ug~0.5ug, 1 dosage of antibody：0.1ug~0.5ug, biotinylation 2 dosage of antibody：0.1ug~0.5ug, BSA dosage：0.1ug~0.25ug.Wherein there are two types of marker monoclonal antibodies, antibody 1 For point sample, antibody 2 is for being coupled.
Embodiment two：
Three marker (CTI/D-Dime/NT-proBNP) magnetic particle microflow controlled biochip systems of heart infarction heart failure of the present invention Preparation Method：
The method being coupled using superparamagnetic nanometer magnetic particle, superparamagnetic nano magnetic fine-grained particles include a diameter of 30nm and straight Diameter is the mixing magnetic bead of 50nm, and the reaction mechanism of nanometer magnetic particle is Covalent bonding together, vinegar of the ferroso-ferric oxide in various concentration The effect of sour sodium is reduced into the super quick magnetic particle mixing suspension of 30nm and 50nm black colloidal superparamagnetics, and anti-with amino Body/Avidin coupling.Quantitative detecting reagent preparation process is as follows,
1, the preparation of the super quick magnetic particle of 30nm+50nm superparamagnetics：
(1) prepared by 30nm magnetic beads：Take 0.75ml~2.25ml 4%Fe3O4Solution is added in 300ml ultra-pure waters so that four oxygen Change three ferrous solution final concentrations 0.01%~0.03%, 200 DEG C~300 DEG C be heated in non magnetic concussion high-temperature heating system, 10% sodium acetate (CH of 6ml~10ml are added3COONa) solution makes sodium acetate (CH3) final concentration of 0.2% COONa~ 0.3%, the ferroso-ferric oxide super paramagnetic beads black colloidal state suspension for obtaining 30nm in 12 hours is reacted under high temperature.
(2) prepared by 50nm magnetic beads：Take 0.75ml~2.25ml 4%Fe3O4Solution is added in 300ml ultra-pure waters so that four oxygen Change three ferrous solution final concentrations 0.01%~0.03%, 200 DEG C~300 DEG C be heated in non magnetic concussion high-temperature heating system, 10% sodium acetate (CH of 3ml~6ml are added3COONa) solution makes sodium acetate (CH3COONa) final concentration of 0.1%~0.2%, The ferroso-ferric oxide super paramagnetic beads black colloidal state suspension for obtaining 50nm in 12 hours is reacted under high temperature.
(3) 30nm and 50nm magnetic beads mix：By the ferroso-ferric oxide super paramagnetic beads black colloidal state suspension of 30nm With the ferroso-ferric oxide super paramagnetic beads black colloidal state suspension 1 of 50nm：1 mixing is for use.
(4) purifying of 30nm and 50nm magnetic beads colloid mixture suspension：By 30nm+50nm ferroso-ferric oxide super paramagnetic beads Black colloid mixed liquor is placed on magnetic patch, and gentle aspiration removes supernatant, and sediment is that 30nm+50nm ferroso-ferric oxides are super suitable The super quick magnetic bead of magnetic, is placed in closed container and is put into insulating box, and 40 DEG C~70 DEG C dry 8 hours~12 hours.By dry 30nm+ The super quick magnetic bead of 50nm ferroso-ferric oxide superparamagnetics redissolves ultrasonic disperse 50 minutes, tetra- oxygen of 30nm+50nm of dispersion in HCl solution Change the super quick magnetic bead of three-iron superparamagnetic to be washed repeatedly to neutrality with ultra-pure water, reaches 7.0 with pH value test paper detection PH.It washs Tetra- chlorination three-iron super paramagnetic beads of 30nm+50nm be scattered in the mixed liquor of ethyl alcohol, ultra-pure water, ammonium hydroxide (volume ratio 75： 25：1) tetraethyl orthosilicate (TEOS), is added into the super quick magnetic bead mixed liquor of above-mentioned 30nm+50nm ferroso-ferric oxides superparamagnetic, Ferroso-ferric oxide (Fe3O4) with the additional proportion of TEOS (tetraethyl orthosilicate) it is 10：1~10：3 room temperatures are without magnetic mechanical stirring 12 Hour~form core-shell type silicon dioxide layer (SiO within 18 hours2)；
2, the Covalent bonding together of the super quick Epoxy functionalized group of magnetic particle of 30nm+50nm superparamagnetics：
(1) 30nm+50nm magnetic beads activation act：It is respectively that the super quick magnetic of 30nm+50nm superparamagnetics is micro- with ethyl alcohol and ultra-pure water Grain cleaning 3 times~5 times is added by γ-(2,3- the third oxygen of epoxy) propyl trimethoxy silicane (15%), methanol (75%), ultrapure The mixed liquor of water (10%) volume ratio composition adjusts pH value to 4,50 DEG C of water-baths 12 hours~18 hours with HCL, uses toluene It is respectively washed 3 times~5 times with acetone, the 30nm containing 100umol/g~300umol/g epoxy-activateds is obtained after 60 DEG C of drying The super quick magnetic beads of+50nm；
(2) 30nm+50nm Beads enrichments purify：The super quick magnetic beads of the 30nm+50nm being re-activated through sulfydryl are placed in Magneto separate It is detached 90 minutes~120 minutes for 4 DEG C in device, slowly absorbs supernatant as possible, not touch the magnetic bead of aggregation.Addition 400ul~ The coupling buffer of 800ul cleans 3 times~5 times repeatedly, repeats the coupling buffer that Magneto separate purification process uses 100ul later Again suspend magnetic bead, and ultrasonic disperse is for use；
3, coupling monoclonal antibody/streptavidin activation：
(1) liquid dosage：
Reaction buffer：1*PBS, pH8.0,5mM EDTA
Preserve buffer solution：1*PBS, pH7.2,5mM EDTA；
Traut's reagents (TR-2- imido grpup sulfanes hydrochloride)：Weigh in advance in the 1.6mL test tubes stored at 4 DEG C= (TR, Mw=137.63)
(2) activation of streptavidin/monoclonal antibody：
1. streptavidin/monoclonal antibody to be activated is diluted to 1mg/ml~3mg/ml with reaction buffer；
2. according to TR moles：Streptavidin/monoclonal antibody=40：1 ratio calculate TR solution (0.5mg/ml~ Additive amount 1mg/ml)；
3. 0.5mg/ml~1mg/ml TR solution is added in streptavidin/monoclonal antibody solution, incubation at room temperature 1 Hour~2 hours；
4. albumen is added in desalination centrifugal column, 2000 rotate 3 minutes~5 minutes；
5. measuring the concentration of Thiolation streptavidin/monoclonal antibody with ultraviolet specrophotometer；
4, magnetic bead is coupled streptavidin/monoclonal antibody：
1) activated streptavidin/monoclonal antibody is added to the activation 30nm+50nm of the 100ul of 5mg/ml In magnetic bead, the coupling buffer of 100ul, vortex ultrasound 30 seconds to 1 minute is added；
2) it reacts 3 hours~5 hours at room temperature, it is primary every 10 minutes~30 minutes ultrasounds；
3) magnetic is added in 10ul NEM (n-ethylmaleimide) buffer solution (50mg/ml, the NEM dissolved in DMSO) In pearl solution, react at room temperature 30 minutes to 1 hour；(NEM：N-ethylmaleimide；DMSO：Dimethyl sulfoxide (DMSO))
4) by magnetic bead test tube as detaching magnetic bead, 4 DEG C of isolating environment, disengaging time 1 hour~2 hours on strong magnet；
5) slowly supernatant is exhausted, does not touch the magnetic bead of aggregation；
6) washing of magnetic bead is detached：Be added 400ul~800ul PBST buffer solutions (1xPBS, pH7.2,5mM EDTA, 0.01%~0.05% polysorbas20) after vortex mixing, by magnetic bead test tube as detaching magnetic bead on strong magnet, 4 DEG C of isolating environment, point 1 hour from the time~2 hours；It washs 5 times~8 times repeatedly, removes unbonded magnetic bead and streptavidin/monoclonal antibody；
7) magnetic bead is resuspended in 400ul~600ul PBST buffer solutions, concentration is adjusted to 2mg/ml~5mg/ml；
5, overlength chain biotin/monoclonal antibody coupling：
(1) overlength chain biotin solution is prepared：With ultrapure water dissolution biotin to 4ug/ul~5ug/ul, 0.22um filters are crossed Film is for use；
(2) three marker monoclonal antibody solutions of heart infarction heart failure are prepared：By three marker antibody difference of heart infarction heart failure It is diluted to 1ug/ul~2ug/ul, it is for use to cross 0.22um filter membranes；
(3) overlength chain biotin monoclonal antibody is coupled：Overlength chain biotin is slowly added to coupled monoclonal to resist It is incubated 30 minutes~60 minutes on mixing oscillator in liquid solution；
(4) overlength chain biotin/monoclonal antibody coupling isolates and purifies：1500 leave the heart 1 minute in addition centrifugal column, small Heart Aspirate supernatant, is added 300ul~500ul PBS, 1500 turns, centrifuges 1 minute again, in triplicate；Supernatant is removed, is used PBS dilutes overlength chain Bioconjugation monoclonal antibody to 500-1000ug/ml, and 4 DEG C preserve for use；
6, the processing technology of biochip：
(1) production and processing of biochip：The process of manufacture of biochip carries out under the conditions of 100,000 grades of cleanliness factors, By wafer platform 20, pcb board 10 (printed circuit board), metal transmission line is by full-automatic ball bonding system (binding), entirely certainly Dynamic die bond system, full automatic point colloid system, full-automatic UV fixed lines system, the full-automatic chip manufacture such as full-automatic chip cleaning system Module produces the processing of automatic on-line monitoring biochip automatically.The metal transmission line of biochip and sensor of the present invention uses Be gold thread as signal transmission carrier, ensure that the accuracy of signal transmission, reduce loss and the loss of signal transmission, improve The sensitivity and specificity of detection.
(2) detection of biochip：
On-line monitoring：Under high magnification microscope, the chip of flow line production is inspected by random samples, carries out appearance detection, it is micro- See detection, physical detection (detection chip signal transmission pathway detects average variation)；
(3) intermediate detects：Quality inspection, including appearance detection, microstructure detection, physical detection are sampled according to sampling principle (signal transduction access, signal value variation of different point difference chips etc.)；
7, biochip structural member is processed：
(1) biochip structural member is processed：According to Chip scale design structure part structure chart, cut with laser according to structure The structural member (3~4 layers of structure) of different layers is respectively cut in cutting mill；
(2) biochip structural member is processed：Appearance, size, the specification of the different structure part layer processing of on-line monitoring；
(3) assembling of biochip structural member：The structure that different layers are cleaned with isopropanol, according to assembling figure respectively by each layer It pastes and fits together, the positioning of each structural member is detected；
(4) cleaning of the structural member after assembling：Structural member after assembling is cleaned with isopropanol；
8, biochip is surface-functionalized：
(1) surface-functionalized liquid dosage：
The preparation of activated polymerization agent：The aqueous isopropanol of 5mg~20mg reactive compounds, addition 50%~100% (delays Polymerizer), purified water mixing ultrasound is added；
Protective coating solution is prepared：Protective coating solution includes the isopropanol of volume ratio 1%~5%, and 10%~30% lives Change polymerizer, the 5%~20% the second polymerization photosensitizers；5%~15% crosslinking agent；
(2) point sample of the coating for surface protection agent on chip sensor：It will by the advanced full-automatic spotting system of Germany Protective coating is uniformly distributed in chip, and each sensor point sample needs 5nl~10nl face coat protective agents, by equipment to liquid Form is dripped, droplet position, drop distribution consistency degree is monitored on-line on sensor；By ultraviolet light (200nm~300nm, 50Mw~100Mw) UV cure 1 minute~3 minutes；
(3) cleaning of chip sensor：Chip after solidification is respectively put into isopropanol and purified water and is cleaned by ultrasonic, is gone Except the residue of chip sensor surface, dried up by nitrogen；
(4) detection of the biochip after surface-functionalized：Sampling observation appearance, microcosmic knot are carried out to surface-functionalized chip Structure, the form of the surface-functionalized layer of observation chip sensor, distribution, UV solidification effects etc. are detected under electron microscope；
9, the point sample of heart infarction heart failure marker antibody：
(1) antibody protection liquid is prepared：1%~5%PVA (polyvinyl alcohol) or PVP is added under phosphate-buffered liquid system 0.1%~0.5% polysorbas20, Tween 80, (20/80/100 sorb of polyvinyl chloride of tween 100 is added in (polyvinylpyrrolidone) Acid anhydrides monolaurate), 0.22um membrane filtrations after mixing；
(2) negative control substances are prepared：0.5mg/ml~2mg/ml BSA solution, mixing are configured with antibody protection liquid 0.22um membrane filtrations
(3) deposited antibodies are prepared：Protect liquid compound concentration in the heart infarction heart failure three of 0.5mg/ml~2mg/ml with antibody Marker (CTI/D-Dime/NT-proBNP) deposited antibodies, ultrasonic mixing 0.22um membrane filtrations.
(4) three marker (CTI/D-Dime/NT-proBNP) antibody of heart infarction heart failure and negative control are in chip sensor On compartment system：Antibody and negative control are put in different sensors respectively by full-automatic micro-sampling system, each Sensor point sample 5nl~10nl, full-automatic spotting system can be simultaneously by high-power microscope system to liquid form, drop positions It sets, the distribution situation of drop on a sensor carries out online quality control；
(5) after three marker (CTI/D-Dime/NT-proBNP) antibody spot samples of heart infarction heart failure sensor incubation knot It closes：It will be enriched in three marker (CTI/D-Dime/NT-proBNP) antibody of heart infarction heart failure and negative control chip sensor be put into It is incubated overnight for 4 DEG C in closed container containing saturation potassium chloride and aluminium block, the function group of albumen and chip sensor is abundant, surely Fixed combination；
10, after point sample biochip closing：
(1) preparation of chip confining liquid：Delay in Tris (trihydroxy methyl methylamino methane) hydrochloric acid and is added 0.1%~0.5% The solution of polysorbas20/80/100, is added 1%~5% ethanolamine solutions, 0.22um membrane filtrations after mixing；
(2) closed protective liquid is prepared：0.1%~0.5% potassium chloride of addition under phosphate buffer, addition 5%~ 10% sodium chloride, 0.22um membrane filtrations after mixing；
(3) assembling of biochip structural member：Structural member is aligned with the location hole on chip sensor, with special glue It assembles closed.Form complete closed mixed zone, mixed zone, microchannel, sensor response area, micro- full experiment of waste Reaction system monitors the chip after each assembling on-line by Electronic Speculum system, including appearance, passage of particles it is complete Property, the passability in channel, the position etc. of each department；
(4) closing of microflow controlled biochip：Fully automatic system, setting are closed using the chip of full-automatic micro high throughput 1ul/ minutes~5ul/ minutes flow velocitys of flow velocity close microchannel and different sensors position using the confining liquid of 50ul~100ul Point closes non-specific sites, 10 minutes~30 minutes off-period；
(5) cleaning and protection after microflow controlled biochip closing：Confining liquid is emptied, closing pipe line is cleaned, is added 1ul/ minutes~5ul/ minutes flow velocitys are arranged in the closed protective liquid of 100ul, flow velocity, and the confining liquid of 50ul~80ul protects liquid, clearly Extra confining liquid residual components are washed, it is 10 by the time to protect the microenvironment of deposited antibodies and the stability of micro-structure, protection liquid Minute~30 minutes；
(6) drying system of microflow controlled biochip：Chip after closing is put into 37 DEG C of thermostatic drying chambers to dry 1 hour ~2 hours so that antibody or the albumen state that dehydration is stablized in low energy completely on chip sensor.Closed pumping after drying Vacuum preserves for use；
11, three marker (CTI/D-Dime/NT-proBNP) magnetic particle microflow controlled biochips of heart infarction heart failure are quantitatively examined Test agent intermediate detects：
(1) intermediate includes：The biochip of point sample, the sample diluting liquid dispensed have dispensed coupling Avidin The magnetic bead vesica of 30nm+50nm, the antibody vesica of the couple biotin dispensed, the cleaning solution buffer solution vesica dispensed will Intermediate detection is carried out after the above intermediate and chip assembling；
(2) intermediate detects：Enterprise's internal control product (5~7 different antigen concentration points) 25ul~50ul is taken to be added respectively Microflow controlled biochip detector is inserted into chip sample application zone, selects corresponding detection menu to be detected, detector punctures automatically The antibody vesica of couple biotin enters mixed zone and mixes automatically 10-30 seconds；Detector punctures coupling Avidin vesica (work automatically Make a concentration of 1mg/ml~2mg/ml) and 30nm+50nm magnetic beads vesica (working concentration be 1mg/ml~2mg/ml) reaction 3 minutes ~5 minutes, detector punctured cleaning solution vesica and cleans (1 minute~3 minutes) the unbonded magnetic beads of cleaning automatically automatically, antigen, Biotin-conjugated antibodies.Detector carries out Data Detection and assay readings automatically；
(3) the detection data analysis of intermediate：The minimum detectability of each detectable substance, line are calculated by standard curve fit Property range, correlation, precision, accuracy, specificity carry out data analysis；The technology that performance detection will meet setting requires mark It is accurate；
12, the microflow controlled biochip of three markers (CTI/D-Dime/NT-proBNP) of heart infarction heart failure detection is quantitatively examined Test agent has been coupled the magnetic bead of Avidin, the antibody of couple biotin, the packing of cleaning solution buffer solution：By full-automatic micro Sub-packaging system, vesica closure system are packaged liquid；
The 30nm+50nm magnetic beads diluted of Avidin will be coupled at working concentration (1mg/ml~2mg/ml), Each chip vesica dispensed loading amount is 25ul~30ul；It is every that the antibody of biotin coupling is diluted to working concentration 1mg/ml~2mg/ml A chip vesica dispensed loading amount is 25ul~30ul, washing lotion 1*PBS, dispensed loading amount 30-50ul/ vesicas；
The parameter of full automatically subpackaging closure system is set, the packing liquid subpackage sealing of chip vesica, equipment on-line prison are carried out Examining system monitors liquid subpackage amount, and vesica leak detection etc. is monitored；
Quality inspects chip vesica by random samples online, and to encapsulation amount, sealing seal etc. is detected.
13, the making of standard curve：The company standard that will trace to the source product make fit standard curve on corresponding software, will Standard curve reading and writing data to chip reads contact corresponding position；
14, three marker (CTI/D-Dime/NT-proBNP) magnetic particle microflow controlled biochips of heart infarction heart failure are quantitatively examined Survey the detection of biochip semi-finished product：
Semi-finished product after being assembled according to technology required standard are sampled detection；
It is detected including appearance, physical detection and performance detection
Performance detection：Enterprise's internal control product are balanced to the internal control product for taking various concentration respectively to room temperature, intersect internal control product, essence Density internal control product, specific internal control product, the 25ul such as whole blood internal control product are added in corresponding semi-finished product chip sample holes, are inserted into equipment Chip entrance starts corresponding detection menu respectively to three marker (CTI/D-Dime/NT-proBNP) Quality Controls of heart infarction heart failure Product are detected, and detector is automatic at 15 minutes~20 minutes to read result；
Detect acceptable standard：Detection project meets parameter request (Monitoring lower-cut, the range of linearity, the correlation of technology requirement Property, precision, accuracy, specificity etc.), whole blood ' negative ' specimens test value is shown as negative, and detected value is not interfered with.
Embodiment three
Three marker (CTI/D-Dime/NT-proBNP) magnetic particle microflow controlled biochip inspections of heart infarction heart failure of the present invention The specific steps of survey：
(1) enterprise's quality-control product and three marker (CTI/D-Dime/NT-proBNP) magnetic particles of heart infarction heart failure is micro-fluidic Biochip immue quantitative detection reagent box takes out from refrigerator, balance to room temperature；
(2) chip detector is opened, carry out System self-test and preheated 5 minutes；
(3) taking-up biochip is torn equipped with chip aluminium foil bag by required, detection Quality Control kind is marked in id information position Class, the information such as concentration.
(4) it is loaded：Phase is added in the internal control product 25ul for pipetting three markers of heart infarction heart failure respectively with 100ul micropipettors It answers in the sample application zone of biochip, chip is inserted into detector；
(5) it detects：Select the corresponding heart infarction heart failure marker detection module of detector to start detection, equipment will 15 minutes~ 20 minutes automatic reading results；
(6) linear analysis：The positive data of reading is analyzed into data, including Monitoring lower-cut, linear model on standard curve It encloses, related coefficient, accuracy etc. is analyzed；
(7) basis：The result of detection meets the requirement of enterprise product technical standard.
Example IV
Three marker (CTI/D-Dime/NT-proBNP) magnetic particle microflow controlled biochips of heart infarction heart failure of the present invention are fixed Measure preparation and the testing result explanation of detection kit quality-control product：
1, the preparation method of linear criterion product and quality-control product of the present invention：
(1) linear criterion product (5~7)：Various concentration point will not be diluted to respectively with frozen-dried protective liquid by synantigen, vibrated Device mixing is for use.
CTI linear criterions product (5 concentration)：0.02ng/ml~45ng/ml (0.02ng/ml, 0.1ng/ml；1ng/ml； 5ng/ml；15ng/ml；45ng/ml)
D-Dime (D dimer) linear criterion product：25ng/ml~5000ng/ml (25ng/ml, 50ng/ml；500ng/ ml；1000ng/ml；2500ng/ml；5000ng/ml)
NT-proBNP linear criterion product：15pg/ml~30000pg/ml (15pg/ml, 150pg/ml；1500pg/ml； 10000pg/ml；20000pg/ml；30000pg/ml)
The preparation of buffer solution is lyophilized：Under 100mM Tris-HCL buffer solution systems, be added 1%~3%BSA, 1%~5% Glycine, 5%~10% trehalose, 0.1%~0.5% neomycinsulphate, 5%~10% horse serum, 0.1%~0.3% It is for use to cross 0.22um filter membranes by Proclin-300.
The preparation and freeze-drying (1000ul/ branch) of Troponin I (CTNI) standard items：
1. Troponin I (CTNI) sterling antigen freeze-dried powder is diluted to 1000ng/ml, a concentration of 1000ng/ of 45ul are taken Ml Troponin Is (CTNI) are added in freeze-drying pipe, and the freeze-drying buffer solution mixing for pipetting 945ul is the highly concentrated speed cones of 45ng/ml Quasi- product；
2. being separately added into the standard items of high concentration and corresponding freeze-drying buffer solution according to following table, mix well；
3. being lyophilized：Be distributed into 100ul/ be placed on be lyophilized on freeze dryer after -80 DEG C preserve for use, the term of validity is 2 years.
Troponin I (CTNI)/D-Dime standard items are with tabulation
CTNI Sterling antigen (ul) Buffer solution (ul) is lyophilized D-Dime Sterling antigen (ul) Buffer solution (ul) is lyophilized
0.02ng/ml 4(5.0ng/ml) 996 25ng/ml 5 (taking 5000ng/ml) 995
0.1ng/ml 2.2 (taking 45ng/ml) 997.8 50ng/ml 10 (taking 5000ng/ml) 990
1.0ng/ml 1 (taking 1000ng/ml) 999 500ng/ml 5 (taking 10000ng/ml) 995
5.0ng/ml 5 (taking 1000ng/ml) 995 1000ng/ml 10 (taking 10000ng/ml) 990
15ng/ml 15 (taking 1000ng/ml) 985 2500ng/ml 25 (taking 10000ng/ml) 975
45ng/ml 45 (taking 1000ng/ml) 945 5000ng/ml 50 (taking 100000ng/ml) 950
The preparation and freeze-drying (1000ul/ branch) of N-terminal Natriuretic Peptide (NT-proBNP) standard items
1. N-terminal Natriuretic Peptide (NT-proBNP) sterling antigen freeze-dried powder, which is diluted to 100ug/ml, pipettes 10ul It is 1ug/ml (1000000pg/ml) to manage the interior 990 freeze-drying buffer solutions that are added to freeze-drying；
2. being separately added into the standard items of high concentration and corresponding freeze-drying buffer solution according to following table, mix well；
Freeze-drying：Be distributed into 100ul/ branch be placed on be lyophilized on freeze dryer after -80 DEG C preserve for use, the term of validity is 2 years.
N-terminal Natriuretic Peptide (NT-proBNP) standard items are with tabulation
NT-proBNP Sterling antigen (ul) Buffer solution (ul) is lyophilized
15pg/ml 10(1500pg/ml) 990
150pg/ml 4.3(35000pg/ml) 995.7
1500pg/ml 42.86(35000pg/ml) 957.14
10000pg/ml 10(1000000pg/ml) 990
20000pg/ml 20(1000000pg/ml) 980
30000pg/ml 30(1000000pg/ml) 970
Remarks：The following standard of tracing basis of standard items/quality-control product carries out standard items/quality-control product and traces to the source, and assignment is transmitted Deng：
《GB 21415-2008-T calibration objects and control physical measurement are traced to the source》；
《GB/T 19702-2005/ISO 15193：The measurement measured in 2002 in-vitro diagnosis medical instrument biogenic samples The explanation of reference measure program》
《GB/T 19703-2005/ISO 15194：The measurement measured in 2002 in-vitro diagnosis medical instrument biogenic samples The explanation of reference material》；
《External diagnosis reagent calibration object, quality-control product investigative technique guideline》
《The serial guideline of external diagnosis reagent analytical performance assessment》
(2) preparation of quality-control product
Quality-control product buffer solution：It is by handling mixed human serum/blood plasma (50 people~100 people mixing) or calf serum mould Quasi- clinical detection whole blood sample environment
The preparation of positive quality control product：4 parts of each items selection senior middle school low value and minimum detectability company standard dried frozen aquatic products, essence 10 parts of density quality-control product, with the standard items dry powder of quality-control product buffer solution various concentration, 4 DEG C preserve for use, the term of validity 1 month.
Specifically it see the table below：
CTNI D-Dime NT-proBNP
0.02ng/ml 25ng/ml 15pg/ml
0.1ng/ml 50ng/ml 150pg/ml
5ng/ml 500ng/ml 1500pg/ml
45ng/ml 2500ng/ml 10000pg/ml
90ng/ml (huck quality-control products) 5000ng/ml (huck quality-control products) 20000pg/ml (huck quality-control products)
Specific quality-control product：
Intersect quality-control product：By projects sterling antigen diluent to 1000ng/ml, 4 DEG C preserve for use, the term of validity 1 month.
By handling mixed human serum/blood plasma or calf serum feminine gender quality-control product：To 100ul/ branch, -80 DEG C are protected for packing Deposit the term of validity 1 year.
Interfering substance quality-control product：By triglycerides, hemoglobin, bilirubin is diluted according to following table method, dispenses 100ul/ Branch, 4 DEG C of preservations, imitates 1 month phase.
Title Triglyceride Hemoglobin Bilirubin Remarks
Concentration 10mg/ml 10mg/ml 0.6mg/ml
Sterling amount 10mg 10mg 0.6mg
PBS To 1000ul 50ng/ml 1000ng/ml
Whole blood control：5 parts of fresh whole blood sample, 4 DEG C preserve for use, the term of validity 2 weeks.
2, three marker (CTI/D-Dime/NT-proBNP) magnetic particle micro-fluidic biologicals of heart infarction heart failure of the present invention are tested Chip immue quantitative detection reagent box test data, test result is as follows：
Troponin I (CTNI) quality-control product test data
Troponin I (CTNI) quality-control product data analysis of the present invention
Troponin I (CTNI) quality-control product detection linear regression curve is shown in Fig. 3.
Troponin I (CTNI) quality-control product detects linear regression equation data statistic
Equation：Y=a+b*x, a=0.84507, b=0.89826, r2=0.99704.
X Y- response values Y- calculated values Y- residual errors
0.0200 0.0180 0.8630 0.8450
0.1000 0.0900 0.9349 0.8449
5.0000 4.9000 5.3364 0.4364
15.0000 14.9000 14.3189 -0.5811
45.0000 44.5000 41.2666 -3.2334
90.0000 80.0000 81.6882 1.6882
Data amount check 6
Residual sum of squares (RSS) 15.26066
Data analysis conclusion of the present invention and yin and yang attribute determination method：
Troponin I (CTNI) quality-control product linearly dependent coefficient of the present invention：r2=0.99704, precision (cv%)：4%, The range of linearity：0.02ng/ml~45ng/ml, positive coincidence rate：100%, negative match-rate：100%, no cross reaction.
Positive judgement：It is positive more than 0.02ng/ml or there is heart infarction increased risk；
Feminine gender judgement：It is normal population less than 0.02ng/ml.
2, D dimer (D-Dime) test data
D dimer (D-Dime) quality-control product detection linear regression curve is shown in Fig. 4.
It is as follows that D dimer (D-Dime) quality-control product detects linear regression equation data statistic：
Equation：Y=a+b*x, a=-0.94343, b=0.99730, r2=0.99999.
X Y- response values Y- calculated values Y- residual errors
25.0000 26.0000 23.9891 -2.0109
50.0000 47.5000 48.9217 1.4217
500.0000 490.0000 497.7078 7.7078
2500.0000 2505.0000 2492.3127 -12.6873
5000.0000 4980.0000 4985.5687 5.5687
Data amount check 5
Residual sum of squares (RSS) 257.45437
Data analysis conclusion and yin and yang attribute determination method：
The linearly dependent coefficient of D dimer (D-Dime) quality-control product detection of the present invention：r2=0.99999, precision (cv)： 1.1%, the range of linearity：25ng/ml~5000ng/ml, positive coincidence rate：100% negative match-rate：100%, it is no to intersect instead It answers.
Positive judgement：It is positive more than 25ng/ml or there is pulmonary embolism risk；
Feminine gender judgement：It is normal population less than 25ng/ml.
3, N-terminal Natriuretic Peptide (NT-proBNP) quality-control product test data
N-terminal Natriuretic Peptide (NT-proBNP) quality-control product detection linear regression curve is shown in Fig. 5.
N-terminal Natriuretic Peptide (NT-proBNP) quality-control product detects linear regression equation data statistic
Equation：Y=a+b*x, a=-35.10050, b=0.99432, r2=0.99999.
X Y- response values Y- calculated values Y- residual errors
15.0000 13.0000 -20.1856 -33.1856
150.0000 140.0000 114.0481 -25.9519
1500.0000 1400.0000 1456.3853 56.3853
10000.0000 9900.0000 9908.1378 8.1378
30000.0000 29800.0000 29794.6145 -5.3855
Data amount check 5
Residual sum of squares (RSS) 5049.31364
Data analysis conclusion and yin and yang attribute determination method：
The linearly dependent coefficient of N-terminal Natriuretic Peptide (NT-proBNP) quality-control product detection of the present invention：r2= 0.99999, precision (cv%)：2.1%, the range of linearity：15pg/ml~30000pg/ml, positive coincidence rate：100%, it is negative Coincidence rate：100%, no cross reaction.
Positive judgement：There is heart failure risk more than 15pg/ml；
Feminine gender judgement：It is positive production crowd less than 15pg/ml.
In above example, multigroup part of solution mixing is carried out according to percent by volume in case of no particular description Mixing.
Although above having used general explanation and specific embodiment, the present invention is described in detail, at this On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore, These modifications or improvements without departing from theon the basis of the spirit of the present invention belong to the scope of protection of present invention.

Claims (10)

1. a kind of heart infarction heart failure magnetic particle microflow controlled biochip, which is characterized in that have including pcb board, on the pcb board micro- Circulation road, biosensor is provided in the microchannel, and the biosensor is on wafer platform and wafer platform Coated deposited antibodies, the deposited antibodies can form immune complex with magnetic bead coupled antibody and marker protein, pass through Measure compound magnetoresistance signal power judgement symbol object albumen concentration on wafer platform.
2. a kind of heart infarction heart failure magnetic particle microflow controlled biochip as described in claim 1, which is characterized in that the point sample is anti- Body is heart infarction heart failure marker CTI, D-Dime, NT-proBNP antibody, when in blood sample (whole blood/plasma/serum/refers to blood) When containing heart infarction heart failure marker protein, bead complexes aggregation will be formed on biosensor.
3. a kind of heart infarction heart failure magnetic particle microflow controlled biochip as described in claim 1, which is characterized in that the biology passes Sensor is used as signal transmission carrier by gold thread.
4. a kind of heart infarction heart failure magnetic particle microflow controlled biochip as described in claim 1, which is characterized in that the miniflow is logical Road includes liquid feeding end and outlet end, and first layer and the second layer are disposed on the pcb board, offers and passes through on the first layer The sample holes worn and waste liquid hole have mixed zone and waste, the lateral miniflow of mixed zone side connection on the second layer Channel, it is described transverse direction microchannel on far from mixed zone one end have be connected with sample holes, the bio-sensing on the pcb board Device and microchannel form biosensor reaction zone, fluid one-way flow in the following order in biochip：Mixed zone → Lateral microchannel → sample holes → liquid feeding end → microchannel → biosensor reaction zone → outlet end → waste liquid hole → useless Liquid zone.
5. a kind of heart infarction heart failure magnetic particle microflow controlled biochip as claimed in claim 4, which is characterized in that the second layer Top is additionally provided with microchannel confined layer and Whole Blood Filtration layer.
6. a kind of heart infarction heart failure magnetic particle microflow controlled biochip as described in claim 1, which is characterized in that the biology passes Sensor is connected by Wheatstone bridge mode, and the biosensor includes an input terminal and two output ends, every group of output Contain 0.1uM in the circuit at end2~100uM2Giant magnetic resistance.
7. a kind of heart infarction heart failure magnetic particle microflow controlled biochip as claimed in claim 6, which is characterized in that the biochip Outside is provided with magnetic excitation device, after deposited antibodies and magnetic bead coupled antibody and marker protein form immune complex, Unbonded magnetic bead and antibody protein extra on biosensor is cleaned, it is uniform to automatically turn on excitation for magnetic excitation device at this time Magnetic field makes magnetic bead magnetize, and the magnetic field that the magnetic bead being magnetized on the biosensor generates causes the change of giant magnetic resistance resistance Change, and then judge the position of magnetic bead and concentration on biosensor, heart infarction heart failure marker egg is quantified by standard curve algorithm White concentration.
8. applying a kind of detection method of heart infarction heart failure magnetic particle microflow controlled biochip described in claim 1, feature exists In including the following steps：
Step 1, biochip pre-treatment are coated with heart infarction heart failure marker (CTI/D-Dime/NT- on a biosensor ProBNP) deposited antibodies；
Step 2, prepared by 30nm+50nm magnetic beads and antibody coupling isolates and purifies system：
I, the preparation of 30nm+50nm magnetic beads mixed liquor：Take Fe3O4Solution is added in ultra-pure water so that ferroso-ferric oxide solution is dense eventually Degree 0.01%~0.03% is heated to 200 DEG C~300 DEG C in non magnetic concussion high-temperature heating system, and 10% sodium acetate is added Solution so that the ferroso-ferric oxide super paramagnetic beads of 30nm are obtained by the reaction under high temperature for sodium acetate final concentration of 0.2%~0.3% Black colloidal state suspension；
Take Fe3O4Solution, which is added in ultra-pure water, makes ferroso-ferric oxide solution final concentration 0.01%~0.03%, in non magnetic concussion It is heated to 200 DEG C~300 DEG C in high-temperature heating system, 10% sodium acetate solution is added so that sodium acetate final concentration of 0.1%~ 0.2%, the ferroso-ferric oxide super paramagnetic beads black colloidal state suspension of 50nm is obtained by the reaction under high temperature；
By the ferroso-ferric oxide superparamagnetic magnetic of the ferroso-ferric oxide super paramagnetic beads black colloidal state suspension of 30nm and 50nm Pearl black colloidal state suspension 1：1 mixing is for use；
30nm and 50nm ferroso-ferric oxide super paramagnetic beads black colloidal state mixing suspensions are placed on magnetic patch, gentle aspiration Supernatant is removed, is used in combination ethyl alcohol and ultra-pure water to clean, 30nm the and 50nm ferroso-ferric oxide super paramagnetic beads washed is scattered in Ethyl alcohol, ultra-pure water, ammonium hydroxide mixed liquor in after be added tetraethyl orthosilicate, stirring form core-shell type silicon dioxide layer；
II, the Covalent bonding together of Epoxy functionalized group, be added by γ-(2,3- the third oxygen of epoxy) propyl trimethoxy silicane, It is super to obtain the 30nm+50nm containing epoxy-activated after heating, cleaning and drying for the mixed liquor of methanol, ultra-pure water composition Quick magnetic bead；
III, the super quick magnetic bead coupling protein of superparamagnetic is coupled and isolates and purifies：
(1) Sulfo-SMCC (SS- sulfosuccinics are added in the super quick magnetic beads of 30nm+50nm by the activation of the super quick magnetic beads of 30nm+50nm Imide -4- (N- maleimidomehyls) cyclohexane -1- carboxylates), coupling buffer, ultrasonic disperse magnetic bead is added；
(2) the super quick magnetic beads of the 30nm+50nm being re-activated through sulfydryl are placed in magnetic separator and are detached, absorb supernatant, be added even Join buffer solution for cleaning, repeats and suspended again magnetic bead with coupling buffer after Magneto separate purification process, ultrasonic disperse is for use；
(3) activation of coupling streptavidin/monoclonal antibody；
(4) the super quick magnetic bead of superparamagnetic is coupled streptavidin/monoclonal antibody：Activated streptavidin/monoclonal is resisted Body is added in the magnetic bead activated, and coupling buffer is added, then test tube is placed on strong magnet and detaches magnetic by ultrasonic disperse Pearl；
(5) super long-chain biological monoclonal antibody coupling：With ultrapure water dissolution biotin, then mixes and incubate with monoclonal antibody solution A period of time is educated, removal supernatant is centrifuged, obtains overlength chain Bioconjugation monoclonal antibody；
Step 3, the detection of biotin-labeled pentylamine reaction system：Take blood sample (whole blood/plasma/serum/refers to blood) that biological core is added Piece sample application zone sample dispenses biotin conjugated monoclonal antibodies vesica automatically into mixed zone, on chip and is punctured entrance automatically in advance Mixed zone vibrations mixing 10-30 seconds dispenses magnetic bead coupling Avidin vesica and punctures vibrations hybrid reaction 10- by automatic in advance on chip Enter reaction zone (biochip sensor) after 30 seconds, if containing heart infarction heart failure marker protein, biosensor in sample On will be formed magnetic bead compound aggregation (Ab1-Ag-Ab2-bio-SA-MB), by measure magnetoresistance signal quantitative reaction go out magnetic bead Magnetic bead concentration is converted to albumen concentration by standard curve algorithm, and then judges the dense of heart infarction heart failure marker protein by concentration Degree.
9. detection method as claimed in claim 8, which is characterized in that the step 1 specifically includes following three step：
I, biochip surface functionalization：
(1) preparation of activated polymerization agent is added in reactive compound and delays polymerizer, and pure water mixing ultrasound is added；
(2) protective coating solution is prepared, and isopropanol, activated polymerization agent, the second polymerization photosensitizer and crosslinking agent are uniformly mixed；
(3) protective coating is evenly distributed to the biosensor surface of biochip by full-automatic spotting system, passes through purple Outside line excites photoactive substance, the functional group in release catalysis to be catalyzed monomer polymerization reactions, by ultraviolet irradiation so that protection applies Layer forms stable solid-state function and protecting layer in biosensor surface；
(4) chip after solidification is respectively put into isopropanol and purified water and is cleaned by ultrasonic by the cleaning of biosensor, goes to decore The residue of piece sensor surface preserves for use after being dried up by nitrogen；
II, biochip point sample and incubation：
(1) antibody protection liquid is prepared, and polyvinyl alcohol is added under kaliumphosphate buffer system or polyvinylpyrrolidone, addition are spat Temperature 20, Tween 80, tween 100 are uniformly mixed；
(2) negative control substances are prepared, and BSA solution is configured with antibody protection liquid；
(3) deposited antibodies are prepared, and heart infarction heart failure marker (CTI/D-Dime/NT-proBNP) point sample is prepared with antibody protection liquid Antibody, ultrasonic mixing；
(4) antibody and negative control are put on different biosensors respectively by full-automatic micro-sampling system, is passed through High-power microscope system checks liquid form and distribution situation on a biosensor online；
(5) biochip after point sample is put into the closed container containing saturation potassium chloride and aluminium block and is incubated, make antibody protein With the surface functional group stable bond of biosensor；
III, the closing of biochip：
(1) solution of polysorbas20/80/100 is added in the preparation of chip confining liquid in trihydroxy methyl methylamino methane hydrochloride buffer solution And ethanolamine solutions, it filters after mixing spare；
(2) closed protective liquid is prepared, and potassium chloride and sodium chloride are added under phosphate buffer, is filtered after mixing spare；
(3) confining liquid is injected microchannel, closes extra site on biosensor by the closed system of biochip；
(4) cleaning and protection after biochip closing：Confining liquid protection liquid is injected into microchannel, it is residual to clean extra confining liquid Retained part；
(5) biochip after closing is put into drying box drying so that the antibody on biosensor by the drying of biochip Or the albumen state that dehydration is stablized in low energy completely, it is closed after dry to vacuumize preservation.
10. detection method as claimed in claim 8 or 9, which is characterized in that by " step 3, biotin-labeled pentylamine reaction system Detection " replaces with：" detection of immunology double antibody sandwich method reaction system ", " the immunology double antibody sandwich method reaction system Detection " specifically includes：Blood sample (whole blood/plasma/serum/refers to blood) is taken, blood sample is added to biochip sample application zone Middle blood sample is divided in magnetic bead coupled antibody in biochip vesica and is punctured into mixed by automatic in advance automatically into mixed zone Enter reaction zone (biochip sensor) after closing area 10-30 seconds, if containing heart infarction heart failure marker protein in sample, biology The compound for forming magnetic bead is assembled into (Ab1-Ag-Ab2-MB) on sensor, goes out magnetic bead by measuring magnetoresistance signal quantitative reaction Magnetic bead concentration is converted to albumen concentration by standard curve algorithm, and then judges the dense of heart infarction heart failure marker protein by concentration Degree.
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