CN108828231A - A kind of cardio-pulmonary function marker magnetic particle microflow controlled biochip, detection method - Google Patents

Abstract

The present invention relates to a kind of cardio-pulmonary function marker magnetic particle microflow controlled biochips, belong to POCT detection technique field, including pcb board, there is microchannel on the pcb board, biosensor is provided in the microchannel, the biosensor is coated deposited antibodies on wafer platform and wafer platform, the deposited antibodies can form immune complex with magnetic bead coupled antibody and marker protein, pass through magnetoresistance signal power judgement symbol object protein concentration on compound on measurement wafer platform, the deposited antibodies are six marker CTI of cardio-pulmonary function, CK-MB, MYO, D-Dime, NT-proBNP, hs-CRP antibody.There is magnetic bead biggish specific surface area to improve the sensitivity of detection in conjunction with more albumen/antibody in the technical solution.The present invention also provides a kind of detection method using above-mentioned biochip, can simultaneous quantitative detect heart infarction, heart failure, the cardiac marker of pulmonary embolism, and improve detection Monitoring lower-cut, efficiently avoid missing inspection and false negative phenomenon.

Description

A kind of cardio-pulmonary function marker magnetic particle microflow controlled biochip, detection method

Technical field

The present invention relates to a kind of biochips, mainly for detection of six markers of cardio-pulmonary function, this hair in blood sample It is bright to additionally provide a kind of detection method using above-mentioned biochip, belong to POCT detection technique field.

Background technique

Cardiovascular disease is to endanger the serious disease of human health and 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 Out, cardiovascular disease number of patients in China's 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 cardiovascular disease 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 diagnosing myocardial infarction in clinic, myocardial ischemia, heart failure, the important Testing index of the heart diseases such as pulmonary embolism is main It to include Creatine kinase MB isoenzyme (CK-MB), cardiac troponin (cTnI), b-type natriuretic peptide (BNP)/N-terminal brain natriuretic peptide hormone Former (NT-proBNP), hs-CRP (hs-CRP), D dimer (D-Dime) etc..Cardiac marker POCT segments market The most fast business of middle speedup, annual income scale alreadys exceed 900,000,000 dollars at present, average annual growth rate 14%.The core of rapid growth because Element is the extensive support that the importance that these cardiac markers POCT is quickly detected has obtained circular for confirmation medicine, U.S. clinical biochemistry section The tissues such as institute (NACB), U.S. clinical Chemical Society (AACC), European Society of Cardiology (ESC) are already by these markers Detection be included in heart disease prevention and diagnosis and treatment guide, and POCT content is gradually included in the meeting after 2000.

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 in all POCT inspections.Statistical report shows about 44,000,000,000 beauty of whole world IVD market scale Member, wherein about 15,000,000,000 dollars of POCT market scale, it is contemplated that the market POCT in 2018 will reach nearly 19,000,000,000 dollars, and its cardiac mark The POCT product of will object is 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 market 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 mode.When detecting using POCT, quantitative analysis method should be used.American Academy of Clinical Biochemistry (NACB) it is pointed out in the suggestion detected to cardiac biomarkers：Laboratory should be preferably 30min or less turnover in 1h Cardiac marker analyte detection (time that the turnaround time is defined as the result from taking a blood sample to reporting) is completed in time；POCT detection can be Obtain in 15-20 minutes the multinomial cardiac marker such as BNP/NT-proBNP, CK-MB, cTnI as a result, and conventional inspection section examines Survey needs 1-2 hours.Making a definite diagnosis to successful treatment for morbidity early stage key clinical index is extremely crucial, and POCT can be saved The preprocessor of sample complexity and time shorten and examine turnover period, can quickly obtain experimental result.

Currently, POCT clinical detection technique includes：

First generation detection technique competition law (radio immunoassay, RIA)：Impacted factor is more, RIA sample requirement 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 time-consuming 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 biggish 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.

Summary of the invention

The purpose of the present invention is to provide a kind of cardio-pulmonary function marker magnetic particle microflow controlled biochips, have detection spirit Sensitivity is high, the short advantage of detection time.Specific technical solution is as follows：

A kind of cardio-pulmonary function marker magnetic particle microflow controlled biochip, including pcb board have miniflow on the pcb board Channel, biosensor is provided in the microchannel, and the biosensor is to wrap on wafer platform and wafer platform The deposited antibodies of quilt, the deposited antibodies can form compound with magnetic bead coupled antibody and marker protein, brilliant by measurement Compound magnetoresistance signal power judgement symbol object protein concentration on first platform, the deposited antibodies are six markers of cardio-pulmonary function CTI, CK-MB, MYO, D-Dime, NT-proBNP, hs-CRP antibody 1, the coupled antibody are six markers of cardio-pulmonary function CTI, CK-MB, MYO, D-Dime, NT-proBNP, hs-CRP antibody 2.

As an improvement of the above technical solution, when containing cardio-pulmonary function in blood sample (whole blood/plasma/serum/refers to blood) When six marker proteins, bead complexes aggregation will be formed by being coated on the wafer platform of deposited antibodies.

As an improvement of the above technical solution, the biosensor is by gold thread as signal transport vehicle.

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 mixed zone side connect lateral microchannel, separate mixed zone on the transverse direction microchannel One end has to be connected with sample holes, and the biosensor on the pcb board and microchannel form biosensor reaction zone, 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, there is 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 magnetizes magnetic bead, 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, quantifies cardio-pulmonary function marker protein concentration by standard curve algorithm.

Above-mentioned biochip magnetic bead has biggish 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 cardio-pulmonary function marker magnetic particle microflow controlled biochips, include the following steps：

Step 1, biochip pre-treatment are coated with six marker (CTI/CK-MB/ of cardio-pulmonary function on a biosensor MYO/D-Dime/NT-proBNP/hs-CRP) deposited antibodies；

Step 2, the preparation of 30nm+50nm magnetic bead and antibody coupling isolate and purify system：

I, the preparation of 30nm+50nm magnetic bead mixed liquor：Take Fe₃O₄Solution, which is added in ultrapure water, makes 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 sodium acetate final concentration of 0.2%~0.3%, reaction obtains the ferroso-ferric oxide superparamagnetic of 30nm under high temperature Magnetic bead black colloidal state suspension；

Take Fe₃O₄Solution, which is added in ultrapure 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%, reaction obtains the ferroso-ferric oxide super paramagnetic beads black colloidal state suspension of 50nm 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 stand-by；

30nm and 50nm ferroso-ferric oxide super paramagnetic beads black colloidal state mixing suspension is placed on magnetic patch, softly Supernatant is removed in absorption, and is cleaned with ethyl alcohol and ultrapure water, by 30nm the and 50nm ferroso-ferric oxide super paramagnetic beads washed point Dissipate in ethyl alcohol, ultrapure 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 The mixed liquor that silane, methanol, ultrapure water form obtains the 30nm+ containing epoxy-activated after heating, cleaning and drying The super quick magnetic bead of 50nm；

III, it the super quick magnetic bead albumen coupling of superparamagnetic and isolates and purifies：

(1) Sulfo-SMCC (SS- sulfo group is added in the super quick magnetic bead of 30nm+50nm by the activation of the super quick magnetic bead of 30nm+50nm Succinimido -4- (N- maleimidomehyl) cyclohexane -1- carboxylate), coupling buffer, ultrasonic disperse magnetic is added Pearl；

(2) the super quick magnetic bead of the 30nm+50nm being re-activated through sulfydryl is placed in magnetic separator and is separated, absorbed supernatant, add Enter coupling buffer cleaning, repeats and suspended again magnetic bead after Magneto separate purification process with coupling buffer, ultrasonic disperse waits for With；

(3) coupling streptavidin/monoclonal antibody activation；

(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 separates by ultrasonic disperse Magnetic bead；

(5) super long-chain biological monoclonal antibody coupling：Biotin is dissolved with ultrapure water, it is then mixed with monoclonal antibody solution It closes and is incubated for a period of time, centrifuge separation removal supernatant 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 life is added Sample is dispensed biotin conjugated monoclonal antibodies vesica on chip and is pierced automatically in advance automatically into mixed zone in object chip sample application zone It is broken to enter mixed zone vibration mixing 10-30 seconds, magnetic bead coupling Avidin vesica is dispensed on chip in advance and is punctured automatically, with biotin The antibody samples compound object of coupling enters reaction zone (biochip sensor) after carrying out hybrid reaction 10-30 seconds, if sample Contain six marker proteins of cardio-pulmonary function in this, the compound aggregation (Ab1-Ag-Ab2- of magnetic bead will be formed on biosensor Bio-SA-MB), go out magnetic bead concentration by measuring magnetoresistance signal quantitative reaction, converted magnetic bead concentration by standard curve algorithm At protein concentration, and then judge the concentration of six marker proteins of cardio-pulmonary function.

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 is added and mixes ultrasound；

(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, to protect by ultraviolet irradiation 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 stand-by by being saved after being dried with nitrogen except the residue of chip sensor surface；

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 configure BSA solution with antibody protection liquid；

(3) deposited antibodies are prepared, and prepare six marker (CTI/CK-MB/MYO/D- of cardio-pulmonary function with antibody protection liquid Dime/NT-proBNP/hs-CRP) antibody, ultrasound mix；

(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 for, 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 and ethanolamine solutions filter spare after mixing；

(2) closed protective liquid is prepared, and potassium chloride and sodium chloride are added under phosphate buffer, filters 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 albumen are dehydrated the state stable in low energy completely, 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 detection ", " detection of immunology double antibody sandwich method reaction system " specifically includes：It takes Blood sample (whole blood/plasma/serum/refers to blood) is added to sample in biochip sample application zone and divides in advance automatically into mixed zone Magnetic bead coupled antibody vesica on biochip is punctured mixing automatically, if containing cardio-pulmonary function six marks in sample Object albumen will form the compound aggregation (Ab1-Ag-Ab2-MB) of magnetic bead on biosensor, quantitative by measurement magnetoresistance signal Magnetic bead concentration is reflected, magnetic bead concentration is converted to by protein concentration by standard curve algorithm, and then judge cardio-pulmonary function six The concentration of marker protein.

Above-mentioned detection method can be applicable in whole blood/plasma/serum/and refer to that blood sample detects, and be able to achieve micro (20-40ul) and add Sample trace detection is, it can be achieved that refer to blood examination brake；Detection time realized that highest 30~50 were immunized at 15 minutes~20 minutes Albumen/Molecular biomarkers detection, shortens Diagnostic Time, increases the rescue time of patient, which can simultaneous quantitative Heart infarction, heart failure, the cardiac marker of pulmonary embolism are detected, 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.

Detailed description of the invention

Fig. 1 is a kind of structural schematic diagram of cardio-pulmonary function marker magnetic particle microflow controlled biochip of the present invention.

Fig. 2 is the structural schematic diagram of another embodiment of the second layer in biochip of the invention.

Fig. 3 is that Troponin I (CTNI) quality-control product detects linear regression curve in example IV.

Fig. 4 is that myoglobins (MYO) quality-control product detects linear regression curve in example IV.

Fig. 5 is that creatine kinase isozyme (CK-MB) quality-control product detects linear regression curve in example IV.

Fig. 6 is that D dimer (D-Dime) quality-control product detects linear regression curve in example IV.

Fig. 7 is that N-terminal Natriuretic Peptide (NT-proBNP) quality-control product detects linear regression curve in example IV.

Fig. 8 is hs-CRP (hs-CRP) quality-control product linear regression curve in example IV.

Specific embodiment

As shown in Figure 1, the present invention provides a kind of cardio-pulmonary function marker magnetic particle microflow controlled biochip, feature exists In there is microchannel 11 on, including pcb board 10, pcb board 10, biosensor, bio-sensing are provided in microchannel 11 Device is coated deposited antibodies on wafer platform 20 and wafer platform 20, and deposited antibodies can be with magnetic bead coupled antibody and mark Object albumen forms immune complex, passes through magnetoresistance signal power rational judgment mark on compound on the different biosensors of measurement Object protein concentration, the deposited antibodies are cardio-pulmonary function six markers CTI, CK-MB, MYO, D-Dime, NT-proBNP, hs- CRP antibody 1, the coupled antibody are cardio-pulmonary function six markers CTI, CK-MB, MYO, D-Dime, NT-proBNP, hs- CRP antibody 2.When containing six marker proteins of cardio-pulmonary function in blood sample, it is coated with the biosensor of deposited antibodies On will be formed bead complexes aggregation.

The biochip includes pcb board 10, has microchannel 11, the biosensor setting on the pcb board 10 In in microchannel 11, microchannel 11 includes liquid feeding end 12 and outlet end 13, is disposed with first layer on the pcb board 10 30, the second layer 40, microchannel confined layer 50 and Whole Blood Filtration layer 60 offer perforative sample holes 31 and useless on first layer 30 Fluid apertures 32 has mixed zone 41 and waste 44 on the second layer 40, and 41 side of mixed zone connects lateral microchannel 42, laterally micro- One end on circulation road 42 far from mixed zone 41 has to be connected with sample holes 31, and fluid is unidirectional in the following order in biochip Flowing：Mixed zone 41 → 12 → microchannel of transverse direction microchannel 42 → sample holes, 31 → liquid feeding end, 11 (miniflow shown in Fig. 1 36 biosensors are provided in channel) → 13 → waste liquid of outlet end hole, 32 → waste 44, in order to realize in biochip The one-way flow of fluid can be arranged micro-valve and Micropump in the position of waste 44, generate vacuum cavitations by micro-valve and Micropump The flowing of fluid and flow direction, each region can be controlled by detector Minitype negative pressure air pump and its automatic program.

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, sample application zone 43.First vesica well 45, the second vesica well 46 and third capsule Bubble well 47 is in the magnetic bead for having dispensed Biotin-conjugated antibodies (bio-Ab2), Streptavidin coupling in advance using preceding difference (SA-MB), cleaning buffer solution is encapsulated in vesica, and detector punctures vesica automatically in the detection process, keeps biotin coupling anti- Body and the magnetic bead of Streptavidin coupling enter mixed zone 41 and are mixed with blood sample.

The principle that biosensor is detected in above scheme is, using the principle of giant magnetoresistance (GMR), to be given birth to by measurement 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, heart failure, the cardiac marker of pulmonary embolism.This system can determine Amount detection heart and lung diseases people at highest risk/patient whole blood/serum/plasma/refer to blood Applications of Cardiac Markers, judge patient's heart infarction process, Myocardial damage, pulmonary embolism degree make carly fruit drop for clinician, strive for the rescue of patient by early discovery, early treatment Prime time.

The biochip can use following two detection method：

Method one, biotin-labeled pentylamine reaction system detection method

Take 10ul~30ul whole blood/serum/plasma/to refer to blood sample, be added in the sample application zone of biochip, by micro-valve and Micropump control sample flow, after reaching sample mixed zone 41, have chain enzyme Biotin-conjugated antibodies vesica be punctured automatically into Enter mixed zone, automatic vibration mixes 10 seconds~30 seconds, and the vesica that detection device punctures magnetic bead coupling streptavidin automatically is pierced It is broken, with sample and Biotin-conjugated antibodies compound hybrid reaction 10 seconds~30 seconds；

Entered in biosensor (in advance coating Applications of Cardiac Markers antibody 1) by micro-valve control channel switch, when reaction Between 100 seconds~120 seconds；Detection system punctures the unbonded magnetic bead and sample composite object of cleaning buffer solution vesica cleaning automatically, clearly Wash the time 1 minute~2 minutes；If six marker proteins of cardio-pulmonary function in sample containing higher concentration, will be in sensor Upper formation bead complexes assemble (Ab1-Ag-Ab2-bio-SA- magnetic bead), go out higher magnetic by measurement magnetoresistance signal quantitative reaction Pearl concentration；If quantitative anti-by the magnetoresistance signal of measurement without containing/six marker proteins of denier cardio-pulmonary function in sample Weaker magnetic bead concentration should be gone out.Magnetic bead concentration is converted to protein concentration by the standard curve algorithm software in detection system, And automatic ration judges the concentration of six marker proteins of cardio-pulmonary function.Six marker proteins of cardio-pulmonary function of high concentration represent Heart infarction process, myocardial damage degree, the development degree such as heart failure pulmonary embolism represent no heart infarction, the heart reference range is below It declining, pulmonary embolism occurs, and cardiac muscle is without impaired, and nearby six marker protein concentration of cardio-pulmonary function represent heart infarction to reference range, Heart failure, myocardial damage, the High risk groups such as pulmonary embolism.The whole detection time will complete in 15-20 minutes.

Method two, immunology double antibody sandwich method reaction system

It takes 10ul~30ul whole blood/serum/plasma/to refer to that chip sample application zone 43 is added in blood sample, is controlled by micro-valve Micropump Sample flow, when reaching sample mixing pit, the vesica for having magnetic bead coupled antibody is punctured, with sample composites hybrid reaction 10 seconds~30 seconds；

Entered in biosensor (in advance coating Applications of Cardiac Markers antibody 1) by micro-valve control channel switch, when reaction Between 100 seconds~120 seconds；Detection system punctures the unbonded magnetic bead and albumen of cleaning buffer solution vesica cleaning, scavenging period 1 automatically Minute~2 minutes；If six marker proteins of cardio-pulmonary function in sample containing higher concentration will form multiple on a sensor Object aggregation (Ab1-Ag-Ab2- magnetic bead) is closed, higher magnetic bead concentration is gone out by measurement magnetoresistance signal quantitative reaction；If in sample not Containing/six marker proteins of denier cardio-pulmonary function, weaker magnetic bead concentration is gone out by the magnetoresistance signal quantitative reaction of measurement. The process and method one that converts is identical.

The quantitative detection system of magnetic particle micro-fluidic chip according to the present invention mainly includes hardware components and software portion Point.Hardware components mainly include：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 integration 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, two dimensional code is read and its signal-obtaining, transmission, storage system and its auxiliary circuit；

Fluid path control module：Including the mixing vibration after sample-adding, micro-valve, Micropump, the mixing vibration 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：Pass in and out bin device, micro machine, sensor, conveying track, mechanical arm etc.；

Magnetoresistance signal detection module：Uniform magnetic field generating device (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 test 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 mechanical drive 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 with one or more biosensors (illustrations 1 and figure in each microchannel Example is carried out using 36 biosensors in 2), biosensor uses the structure of wafer platform 20, each wafer platform 20 At convex shape, each biosensor has individual gold thread transmitting device and address code for surface, can individually record its generation Signal, the signal of the generation of quantization sensing device in a manner of Wheatstone bridge.Biosensor is in a manner of Wheatstone bridge Connection, including an input, two export, and contain 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 device 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 the variation of induction 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 Wheatstone bridge by multilayer film giant magnetic resistance structure It out, will be electric by biosensor interface in magnetic signal instrument and its auxiliary circuit (amplifying circuit and squelch circuit) Pressure signal is transmitted to analog-digital commutator and is converted into digital signal.

Bio-sensor signal analysis：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 output：Bio-sensor signal analyzes result can be by display chip or other outputs Equipment (LED, VGA display) shown, can also directly be printed by controlling print circuit or wired be set by external Standby/Bluetooth system externally transmits testing result.

A specific embodiment of the invention is described with reference to the accompanying drawings and examples, to better understand this hair It is bright.

Embodiment one

A kind of cardio-pulmonary function marker magnetic particle microflow controlled biochip of the present invention, biosensor have been coated with cardiopulmonary in advance Six marker (CTI/CK-MB/MYO/D-Dime/NT-proBNP/hs-CRP) point sample monoclonal antibodies of function, then by One layer 30, the second layer 40, microchannel confined layer 50 and Whole Blood Filtration layer 60 paste the microchannel in pcb board 10 in sequence 11 tops carry out reaction under conditions of closed, achieve the effect that micro- full laboratory to close microchannel 11.First Layer 30 is biosensor confined layer, contains sample holes 31 and waste liquid hole 32；The second layer 40 is microchannel layer, connects sample holes 31, the access of biosensor and waste liquid hole 32, while guiding sample to be tested (biological from mixed zone 41 to detection zone by Micropump Position where sensor), 44 one-way flow of waste；Microchannel confined layer 50 is third layer, guarantees that sample is close at one It is flowed in the pipeline closed, while Whole Blood Filtration layer 60 contains whole blood filtration system close to the position of mixed zone 41, it is red to play prevention Blood cell enters microchannel 11, avoids blocking channel, improves the accuracy of testing result.

Six marker (CTI/CK-MB/MYO/D-Dime/NT-proBNP/hs-CRP) detections of cardio-pulmonary function of the present invention Biochip, 30nm+50nm magnetic bead are coupled streptavidin dosage：0.1ug~0.5ug, 1 dosage of antibody：0.1ug~0.5ug, 2 dosage of biotinylated antibody：0.1ug~0.5ug, BSA dosage：0.1ug~0.25ug.Wherein marker monoclonal antibody has two Kind, antibody 1 is used for point sample, and antibody 2 is for being coupled.

Embodiment two：

Six marker (CTI/CK-MB/MYO/D-Dime/NT-proBNP/hs-CRP) detections of cardio-pulmonary function of the present invention Biochip preparation method：

The method being coupled using superparamagnetic nanometer magnetic particle, it is 30nm and straight that superparamagnetic nano magnetic fine-grained particles, which include diameter, 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 superparamagnetic, and anti-with amino Body/Avidin coupling.Quantitative detecting reagent preparation step is as follows,

1, the preparation of the super quick magnetic particle of 30nm+50nm superparamagnetic：

(1) prepared by 30nm magnetic bead：Take 0.75ml~2.25ml 4%Fe₃O₄Solution, which is added in 300ml ultrapure water, makes four oxygen Change three ferrous solution final concentrations 0.01%~0.03%, be heated to 200 DEG C~300 DEG C in non magnetic concussion high-temperature heating system, 10% sodium acetate (CH of 6ml~10ml is added₃COONa) solution makes sodium acetate (CH₃) final concentration of 0.2% COONa~ 0.3%, reaction obtains the ferroso-ferric oxide super paramagnetic beads black colloidal state suspension of 30nm for 12 hours under high temperature.

(2) prepared by 50nm magnetic bead：Take 0.75ml~2.25ml 4%Fe₃O₄Solution, which is added in 300ml ultrapure water, makes four oxygen Change three ferrous solution final concentrations 0.01%~0.03%, be heated to 200 DEG C~300 DEG C in non magnetic concussion high-temperature heating system, 10% sodium acetate (CH of 3ml~6ml is added₃COONa) solution makes sodium acetate (CH₃COONa) final concentration of 0.1%~0.2%, Reaction obtains the ferroso-ferric oxide super paramagnetic beads black colloidal state suspension of 50nm for 12 hours under high temperature.

(3) 30nm and 50nm magnetic bead mixes：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 stand-by.

(4) purifying of 30nm and 50nm magnetic bead complex colloid 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 oxide is 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 superparamagnetic 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 with ultrapure water to neutrality, reaches 7.0 with pH value test paper detection PH.It washs 30nm+50nm ferroso-ferric oxide super paramagnetic beads be scattered in the mixed liquor of ethyl alcohol, ultrapure 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 oxide superparamagnetic, Ferroso-ferric oxide (Fe₃O₄) it with the additional proportion of TEOS (tetraethyl orthosilicate) is 10：1~10：3 room temperatures are without magnetic mechanical stirring 12 Hour~18 hours formation core-shell type silicon dioxide layer (SiO₂)；

2, the Covalent bonding together of the super quick Epoxy functionalized group of magnetic particle of 30nm+50nm superparamagnetic：

(1) 30nm+50nm magnetic bead activation act：It is respectively that the super quick magnetic of 30nm+50nm superparamagnetic is micro- with ethyl alcohol and ultrapure 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 Water (10%) volume ratio composition mixed liquor, with HCL adjust pH value to 4,50 DEG C water-bath 12 hours~18 hours, use toluene It is respectively washed 3 times~5 times with acetone, obtains the 30nm containing 100umol/g~300umol/g epoxy-activated after 60 DEG C of drying The super quick magnetic bead of+50nm；

(2) 30nm+50nm Beads enrichment purifies：The super quick magnetic bead of the 30nm+50nm being re-activated through sulfydryl is placed in Magneto separate It is separated 90 minutes~120 minutes for 4 DEG C in device, slowly absorbs supernatant as far 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 stand-by；

3, coupling monoclonal antibody/streptavidin activation：

(1) liquid dosage：

Reaction buffer：1*PBS, pH8.0,5mM EDTA

Save buffer：1*PBS, pH7.2,5mM EDTA；

Traut's reagent (TR-2- imido grpup sulfane hydrochloride)：Weighed in the 1.6mL test tube stored at 4 DEG C in advance= (TR, Mw=137.63)

(2) streptavidin/monoclonal antibody activation：

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 are rotated 3 minutes~5 minutes；

5. measuring Thiolation streptavidin/monoclonal antibody concentration 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 (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)

4) by magnetic bead test tube as separating magnetic bead on strong magnet, 4 DEG C of isolating environment, disengaging time 1 hour~2 hours；

5) slowly supernatant is exhausted, does not touch the magnetic bead of aggregation；

6) washing of magnetic bead is separated：Be added 400ul~800ul PBST buffer (1xPBS, pH7.2,5mM EDTA, 0.01%~0.05% polysorbas20) after vortex mixing, by magnetic bead test tube as separating 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, concentration is adjusted to 2mg/ml~5mg/ml；

5, overlength chain biotin/monoclonal antibody coupling：

(1) overlength chain biotin solution is prepared：Biotin is dissolved to 4ug/ul~5ug/ul with ultrapure water, crosses 0.22um filter Film is stand-by；

(2) cardio-pulmonary function marker monoclonal antibody solution is prepared：Six marker antibody of cardio-pulmonary function are diluted respectively To 1ug/ul~2ug/ul, it is stand-by to cross 0.22um filter membrane；

(3) overlength chain biotin monoclonal antibody is coupled：Overlength chain biotin is slowly added to coupled monoclonal to resist It mixes in liquid solution and is incubated for 30 minutes~60 minutes on oscillator；

(4) overlength chain biotin/monoclonal antibody coupling isolates and purifies：It is added in centrifugal column and is centrifuged 1 minute for 1500 turns, it is small Heart Aspirate supernatant, be added 300ul~500ul PBS, 1500 turns, again be centrifuged 1 minute, in triplicate；Supernatant is removed, is used PBS dilutes overlength chain Bioconjugation monoclonal antibody to 500-1000ug/ml, and 4 DEG C save 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, 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 line 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 transport vehicle, ensure that the accuracy of signal transmission, reduce the loss and 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 according to structure with laser The structural member (3~4 layers of structure) of different layers is respectively cut in cutting mill；

(2) biochip structural member is processed：Appearance, the size, 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 compound, addition 50%~100% (delays Polymerizer), purified water is added and mixes ultrasound；

Protective coating solution is prepared：Protective coating solution includes the isopropanol of volume ratio 1%~5%, and 10%~30% is living 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 surface covering protective agent, 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 solidification 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, by being dried with 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 chip sensor, distribution are observed under electron microscope, and UV solidification effect etc. is detected；

9, the point sample of six marker antibody of cardio-pulmonary function：

(1) antibody protection liquid is prepared：1%~5%PVA (polyvinyl alcohol) or PVP are 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 filtration after mixing；

(2) negative control substances are prepared：0.5mg/ml~2mg/ml BSA solution is configured with antibody protection liquid, is mixed 0.22um membrane filtration.

(3) deposited antibodies are prepared：Protect liquid compound concentration at cardio-pulmonary function six of 0.5mg/ml~2mg/ml with antibody Marker deposited antibodies, ultrasound mix 0.22um membrane filtration.

(4) compartment system of six marker antibody of cardio-pulmonary function and negative control on chip sensor：By it is complete from Dynamic micro-sampling system puts antibody and negative control in different sensors respectively, each sensor point sample 5nl~10nl, entirely Automatic spotting system can be simultaneously by high-power microscope system to liquid form, droplet position, point of drop on a sensor Cloth situation carries out online quality control；

(5) after six marker antibody spot samples of cardio-pulmonary function sensor hatching combination：It will be enriched in cardio-pulmonary function six marks Will object antibody and negative control biochip are put into the closed container containing saturation potassium chloride and aluminium block and are incubated overnight for 4 DEG C, egg White combination abundant with the function group of chip sensor, stable；

Six marker point sample distribution maps of cardio-pulmonary function (according to 6 × 6 biosensor distributed architectures)

A

B

C

D

E

F

1 (liquid outlet)

hs-CRP

D-Dime

CK-MB

BSA

2

hs-CRP

D-Dime

CK-MB

3

hs-CRP

D-Dime

CK-MB

BSA

4

NT-proBNP

MYO

CTNI

5

NT-proBNP

MYO

CTNI

6

NT-proBNP

MYO

CTNI

BSA (liquid-inlet)

10, after point sample biochip closing：

(1) preparation of chip confining liquid：In the slow addition 0.1%~0.5% of Tris (trihydroxy methyl methylamino methane) hydrochloric acid 1%~5% ethanolamine solutions, 0.22um membrane filtration after mixing is added in polysorbas20/80/100 solution；

(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 filtration 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, microchannel, sensor response area, micro- full experiment reaction system of waste System, monitors the chip after each assembling on-line by Electronic Speculum system, including appearance, the integrality of passage of particles, leads to The passability in road, 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, closing non-specific sites, off-period 10 minutes~30 minutes；

(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, the microenvironment of deposited antibodies and the stability of micro-structure are protected, protection liquid is 10 by the time Minute~30 minutes；

(6) drying system of microflow controlled biochip：It is 1 hour dry that chip after closing is put into 37 DEG C of thermostatic drying chambers ~2 hours, so that the antibody or albumen on chip sensor are dehydrated the state stable in low energy completely.Closed pumping after drying Vacuum saves stand-by；

11, six marker magnetic particle microflow controlled biochip quantitative detecting reagent intermediate detections of cardio-pulmonary function：

(1) intermediate includes：It is affine to have dispensed magnetic bead coupling for the biochip of point sample, the sample diluting liquid dispensed Plain vesica, the Biotin-conjugated antibodies vesica dispensed, the cleaning solution buffer vesica dispensed, by the above intermediate and chip Intermediate detection is carried out after assembling；

(2) intermediate detects：Take enterprise's internal control product (5~7 different antigen concentration points) 25ul-50ul that core is added respectively Microflow controlled biochip detector is inserted into piece sample application zone, selects corresponding detection menu to be detected, detector punctures idol automatically The antibody vesica of connection biotin mixes 30 seconds automatically with sample and mixed zone；Detector punctures magnetic bead coupling Avidin automatically and enters Mixed zone react 100-120 second, detector puncture automatically cleaning solution vesica clean automatically (1 minute~2 minutes) clean be not associated with Magnetic bead, 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；Performance detection will meet the technical requirements mark of setting It is quasi-；

12, the packing of the microflow controlled biochip quantitative detecting reagent vesica of six marker detections of cardio-pulmonary function：It is even Join the magnetic bead of Avidin, the antibody of couple biotin, the packing of cleaning solution buffer passes through full-automatic micro sub-packaging system, capsule Bubble closure system is packaged liquid；

The 30nm+50nm magnetic bead 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/ vesica；

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, to encapsulation amount, seals airtightness etc. and is detected；

13, the production 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, the biochip quantitative detection biochip semi-finished product detection of six marker detections of cardio-pulmonary function：

Detection is sampled according to the semi-finished product after the assembling of the standard of technical requirements；

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 application zone, are inserted into equipment Chip entrance starts corresponding detection menu and detects respectively to six mark quality control products of cardio-pulmonary function, and detector is 15 Minute~20 minutes automatic reading results；

Detect acceptable standard：Detection project meets parameter request (Monitoring lower-cut, the range of linearity, the correlation of technical requirements Property, precision, accuracy, specificity etc.), whole blood ' negative ' specimens test value is shown as negative, and detected value is not interfered with.

Embodiment three

The specific steps of six marker detections of cardio-pulmonary function of the present invention：

(1) enterprise's quality-control product and biochip immue quantitative detection reagent box are taken out from refrigerator, balance to room temperature；

(2) chip detector is opened, carries out System self-test and preheating 5 minutes；

(3) taking-up biochip is torn equipped with chip aluminium foil bag by required, marks detection Quality Control kind in id information position Class, the information such as concentration.

(4) it is loaded：Phase is added with the internal control product 25ul that 100ul micropipettor pipettes six markers of cardio-pulmonary function respectively It answers in chip sample application zone, chip is inserted into detector；

(5) it detects：The corresponding Applications of Cardiac Markers detection module starting detection of detector is selected, equipment will be at 15 minutes~20 points Clock reads result automatically；

(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

Six marker (CTI/CK-MB/MYO/D-Dime/NT-proBNP/hs-CRP) detections of cardio-pulmonary function of the present invention The preparation and testing result explanation of biochip immue quantitative detection reagent box 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)：It will not be diluted to respectively various concentration point with frozen-dried protective liquid by synantigen, vibrate Device mixing is stand-by.

CTI linear criterion product (5 concentration)：0.02ng/ml~45ng/ml (0.02ng/ml, 0.1ng/ml；1ng/ml； 5ng/ml；15ng/ml；45ng/ml)；

CK-MB linear criterion product：1ng/ml~400ng/ml (1ng/ml, 5ng/ml；10ng/ml；50ng/ml；100ng/ ml；500ng/ml)；

MYO linear criterion product：5ng/ml~900ng/ml (5ng/ml, 10ng/ml；100ng/ml；200ng/ml； 500ng/ml；1000ng/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)

Hs-CRP (hs-CRP) linear criterion product：0.05mg/L~30mg/L (50ng/ml~30000ng/ Ml), standard items include 50ng/ml, 100ng/ml, 1000ng/ml, 5000ng/ml, 10000ng/ml, and 30000ng/ml six Concentration point.

The preparation of buffer is lyophilized：Under 100mM Tris-HCL buffer solution system, 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 stand-by to cross 0.22um filter membrane by Proclin-300.

The preparation of Troponin I (CTNI)/myoglobins (MYO) standard items and freeze-drying (1000ul/ branch)：

1. Troponin I (CTNI) sterling antigen freeze-dried powder is diluted to 1000ng/ml, taking 45ul concentration is 1000ng/ Ml Troponin I (CTNI) is added in freeze-drying pipe, and it is the highly concentrated speed cone of 45ng/ml that the freeze-drying buffer for pipetting 945ul, which mixes, Quasi- product；Myoglobins I (MYO) sterling antigen freeze-dried powder is diluted to 10ug/ml (10000ng/ml), takes the 100ul concentration to be 10000ng/ml myoglobins I (MYO) is added in freeze-drying pipe, and it is 1000ng/ml high that the freeze-drying buffer for pipetting 900ul, which mixes, Dense velocity standard product；

2. being separately added into the standard items and corresponding freeze-drying buffer of high concentration 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 save stand-by, validity period is 2 years.

Troponin I (CTNI)/myoglobins (MYO) standard items are with tabulation

CTNI	Sterling antigen (ul)	It is lyophilized buffer (ul)	MYO	Sterling antigen (ul)	It is lyophilized buffer (ul)
						0.02ng/ml	4(5.0ng/ml)	996	5ng/ml	5 (taking 1000ng/ml)	995
0.1ng/ml	2.2 (taking 45ng/ml)	997.8	10ng/ml	10 (taking 1000ng/ml)	990
						1.0ng/ml	1 (taking 1000ng/ml)	999	100ng/ml	10 (taking 10000ng/ml)	990
5.0ng/ml	5 (taking 1000ng/ml)	995	200ng/ml	20 (taking 10000ng/ml)	980
						15ng/ml	15 (taking 1000ng/ml)	985	500ng/ml	50 (taking 10000ng/ml)	950
45ng/ml	45 (taking 1000ng/ml)	945	1000ng/ml	100 (taking 10000ng/ml)	900

The preparation of creatine kinase isozyme (CK-MB)/D dimer (D-Dime) standard items and freeze-drying (1000ul/ branch)：

1. creatine kinase isozyme (CK-MB) sterling antigen freeze-dried powder is diluted to 10000ng/ml；By D dimer (D- Dime) sterling antigen freeze-dried powder is diluted to 100ug/ml (100000ng/ml)；

2. being separately added into the standard items and corresponding freeze-drying buffer of high concentration according to following table, mix well；

Freeze-drying：Be distributed into 100ul/ be placed on be lyophilized on freeze dryer after -80 DEG C save stand-by, validity period is 2 years

Creatine kinase isozyme (CK-MB)/D dimer (D-Dime) standard items are with tabulation

CK-MB	Sterling antigen (ul)	It is lyophilized buffer (ul)	D-Dime	Sterling antigen (ul)	It is lyophilized buffer (ul)
						1ng/ml	2 (taking 500ng/ml)	998	25ng/ml	5 (taking 5000ng/ml)	995
5ng/ml	10 (taking 500ng/ml)	990	50ng/ml	10 (taking 5000ng/ml)	990
						10ng/ml	1 (taking 1000ng/ml)	999	500ng/ml	5 (taking 10000ng/ml)	995
50ng/ml	5 (taking 10000ng/ml)	995	1000ng/ml	10 (taking 10000ng/ml)	990
						100ng/ml	10 (taking 10000ng/ml)	990	2500ng/ml	25 (taking 10000ng/ml)	975
500ng/ml	20 (taking 10000ng/ml)	980	5000ng/ml	50 (taking 100000ng/ml)	950

N-terminal Natriuretic Peptide (NT-proBNP)/hs-CRP (hs-CRP) standard items preparation and freeze-drying (1000ul/ branch)

1. N-terminal Natriuretic Peptide (NT-proBNP) sterling antigen freeze-dried powder, which is diluted to 100ug/ml, pipettes 10ul Managing the interior 990 freeze-drying buffers that are added to freeze-drying is 1ug/ml (1000000pg/ml)；Hs-CRP (hs-CRP) is frozen Dry powder is diluted to 100ug/ml (100000ng/ml).

2. being separately added into the standard items and corresponding freeze-drying buffer of high concentration 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 save stand-by, validity period is 2 years.

N-terminal Natriuretic Peptide (NT-proBNP)/hs-CRP (hs-CRP) standard items are with tabulation

Remarks：The following standard of standard items/quality-control product tracing basis carries out standard items/quality-control product and traces to the source, assignment, transmitting Deng：

《GB 21415-2008-T calibration object 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》

《External diagnosis reagent analyzes Performance Evaluation series guideline》

(2) preparation of quality-control product

Quality-control product buffer：It is by processing 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 various concentration, 4 DEG C are saved for use, and validity period 1 month.

Specifically it see the table below：

CTNI	MYO	CK-MB	D-Dime	NT-proBNP	Hs-CRP
						0.02ng/ml	5ng/ml	1ng/ml	25ng/ml	15pg/ml	50ng/ml(0.05mg/L)
0.1ng/ml	10ng/ml	10ng/ml	50ng/ml	150pg/ml	100ng/ml(0.1mg/L)
						5ng/ml	200ng/ml	100ng/ml	500ng/ml	1500pg/ml	1000ng/ml(1mg/L)
45ng/ml	1000ng/ml	400ng/ml	2500ng/ml	10000pg/ml	10000ng/ml(10mg/L)
						90ng/ml	2000ng/ml	800ng/ml	5000ng/ml	20000pg/ml	30000ng/ml(30mg/L)

Specific quality-control product：

Intersect quality-control product：By projects sterling antigen diluent to 1000ng/ml, 4 DEG C are saved for use, and validity period 1 month.

By processing mixed human serum/blood plasma or calf serum feminine gender quality-control product：To 100ul/ branch, -80 DEG C are protected for packing Deposit validity period 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, is imitated the phase 1 month.

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 save for use, and validity period 2 weeks.

2, the biochip immue quantitative detection reagent box test data of six marker detections of cardio-pulmonary function, test result is such as Under：

1, 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 value	Y- calculated value	Y- residual error
				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.

The positive determines：It is positive greater than 0.02ng/ml or there is heart infarction risk；

Feminine gender determines：It is negative findings either normal population less than 0.02ng/ml.

2, myoglobins (MYO) test data

Myoglobins (MYO) quality-control product detection linear regression curve is shown in Fig. 4.

It is as follows that myoglobins (MYO) quality-control product detects linear regression equation data statistic：

Equation：Y=a+b*x, a=9.16639, b=0.90445, r²=0.99928.

X	Y- response value	Y- calculated value	Y- residual error
				5.0000	5.2000	13.6886	8.4886
10.0000	9.5000	18.2109	8.7109
				100.0000	98.5000	99.6116	1.1116
1000.0000	950.0000	913.6184	-36.3816
				2000.0000	1800.0000	1818.0704	18.0704

Data amount check 5

Residual sum of squares (RSS) 1799.33331

Data analyze conclusion and yin and yang attribute determination method：

Myoglobins (MYO) quality-control product linearly dependent coefficient of the present invention：r²=0.99928, precision (cv%)：2.6%, The range of linearity：5ng/ml~900ng/ml, positive coincidence rate：100% negative match-rate：100%, no cross reaction.

The positive determines：Greater than 5ng/ml be positive findings or have heart infarction risk；

Feminine gender determines：It is negative findings either normal population less than 5ng/ml

3, creatine kinase isozyme (CK-MB) test data

Creatine kinase isozyme (CK-MB) quality-control product detection linear regression curve is shown in Fig. 5.

It is as follows that creatine kinase isozyme (CK-MB) quality-control product detects linear regression equation data statistic

Equation：Y=a+b*x, a=9.46229, b=0.88329, r²=0.99606.

X	Y- response value	Y- calculated value	Y- residual error
				1.0000	1.3000	10.3456	9.0456
10.0000	10.5000	18.2952	7.7952
				100.0000	98.5000	97.7909	-0.7091
400.0000	395.0000	362.7769	-32.2231
				800.0000	700.0000	716.0915	16.0915

Data amount check 5

Residual sum of squares (RSS) 1440.35453

Data analyze conclusion and yin and yang attribute determination method：

Creatine kinase isozyme (CK-MB) quality-control product linearly dependent coefficient of the present invention：r²=0.99606, precision (cv%)：1.2%, the range of linearity：1ng/ml~400ng/ml, positive coincidence rate：100% negative match-rate：100%, no friendship Fork reaction.

The positive determines：Greater than 1ng/ml be positive findings or have heart infarction risk；

Feminine gender determines：It is negative findings either normal population less than 1ng/ml.

If the present invention tests Troponin I (CTNI), myoglobins (MYO), creatine kinase isozyme (CK-MB) is Positive findings, indication need to carry out emergent management in conjunction with other physical inspection results myocardial infarction high-risk stage of attack.

4, D dimer (D-Dime) test data

D dimer (D-Dime) quality-control product detection linear regression curve is shown in Fig. 6.

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 value	Y- calculated value	Y- residual error
				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 analyze 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.

The positive determines：It is positive greater than 25ng/ml or there is pulmonary embolism risk；

Feminine gender determines：It is negative either normal population less than 25ng/ml.

5, 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. 7.

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 value	Y- calculated value	Y- residual error
				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 analyze 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.

The positive determines：It is positive greater than 15pg/ml or there is heart failure risk；

Feminine gender determines：It is feminine gender less than 15pg/ml or just produces crowd.

6, hs-CRP (hs-CRP) quality-control product test data

Hs-CRP (hs-CRP) quality-control product detection linear regression curve is shown in Fig. 8.

Hs-CRP (hs-CRP) quality-control product detects linear regression equation data statistic

Equation：Y=a+b*x, a=0.23091, b=0.83556, r²=0.99773.

X	Y- response value	Y- calculated value	Y- residual error
				0.0500	0.0480	0.2727	0.2247
0.1000	0.0900	0.3145	0.2245
				1.0000	0.9000	1.0665	0.1665
10.0000	9.5000	8.5865	-0.9135
				30.0000	25.0000	25.2978	0.2978

Data amount check 5

Residual sum of squares (RSS)：1.05168

Data analyze conclusion and yin and yang attribute determination method：

The linearly dependent coefficient of hs-CRP (hs-CRP) quality-control product detection of the present invention：R2=0.99773 is accurate It spends (cv%)：2.7%, the range of linearity：0.05mg/L~30mg/L, positive coincidence rate：100% negative match-rate：100%, nothing Cross reaction.

The positive determines：It is positive greater than 0.05mg/L or there is cardiovascular and cerebrovascular disease risk；

Feminine gender determines：It is negative either normal population less than 0.05mg/L.

Hs-CRP is not only that " risk factor (the rist fastor) " of cardiovascular and cerebrovascular disease is even more to treat inspection The marker " monnitor " of survey is one of dangerous the most powerful predictive factor of cardiovascular event.In whole blood hs-CRP level with The generation of atherosclerosis and acute cerebral infarction (ACI), severity and prognosis are closely related.Hs-CRP content and stalk simultaneously Unleavened dough product, neurological functional deficit are related, and are one of the indexs of Patients with Cerebral Infarction lesion degree.

Normal value judgement：

Adult and children：0.068mg/L~8.2mg/L, intermediate value 0.58mg/L；

Newborn or Cord blood：≤0.6mg/L；

After birth the 4th day to 1 month baby：≤1.6mg/L；

Women when childbirth：≤47mg/L.

It is distributed by positive skewness, credibility interval, the analysis such as difference analysis and credibility interval is the results show that most of super quick The range of normal value of c reactive protein exists：0.05mg/L~1.15mg/L, but different methodologies, not agnate, Chinese and western has Certain difference.

In above embodiments, the mixing of multiple groups part solution 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 are fallen within the scope of the claimed invention.

Claims (10)

1. a kind of cardio-pulmonary function marker magnetic particle microflow controlled biochip, which is characterized in that including pcb board, on the pcb board With microchannel, biosensor is provided in the microchannel, the biosensor is wafer platform and wafer Coated deposited antibodies on platform, the deposited antibodies can with magnetic bead be coupled antibody and marker protein be coupled to be formed it is immune Compound passes through compound magnetoresistance signal power judgement symbol object protein concentration on measurement wafer platform.

2. a kind of cardio-pulmonary function marker magnetic particle microflow controlled biochip as described in claim 1, which is characterized in that described Deposited antibodies are six marker CTI, CK-MB, MYO, D-Dime, NT-proBNP, Hs-CRP antibody 1 of cardio-pulmonary function, the idol Len antibody is myocardium five marker CTI, CK-MB, MYO, D-Dime, NT-proBNP, Hs-CRP antibody 2, works as blood sample When containing six marker proteins of cardio-pulmonary function in (whole blood/plasma/serum/refers to blood), it is coated with the wafer platform of deposited antibodies On will be formed bead complexes aggregation.

3. a kind of cardio-pulmonary function marker magnetic particle microflow controlled biochip as described in claim 1, which is characterized in that described Biosensor is by gold thread as signal transport vehicle.

4. a kind of cardio-pulmonary function marker magnetic particle microflow controlled biochip as described in claim 1, which is characterized in that described Microchannel includes liquid feeding end and outlet end, and first layer and the second layer are disposed on the pcb board, is opened on the first layer Equipped with perforative sample holes and waste liquid hole, there is mixed zone and waste on the second layer, the mixed zone side connection is horizontal To microchannel, on the transverse direction microchannel, one end far from mixed zone has is connected with sample holes, the life on the pcb board Object sensor and microchannel form biosensor reaction zone, fluid one-way flow in the following order in biochip：It is mixed Close area → transverse direction microchannel → sample holes → liquid feeding end → microchannel → biosensor reaction zone → outlet end → waste liquid Hole → waste.

5. a kind of cardio-pulmonary function marker magnetic particle microflow controlled biochip as claimed in claim 4, which is characterized in that described Microchannel confined layer and Whole Blood Filtration layer are additionally provided with above the second layer.

6. a kind of cardio-pulmonary function marker magnetic particle microflow controlled biochip as described in claim 1, which is characterized in that described Biosensor is connected by Wheatstone bridge mode, and the biosensor includes an input terminal and two output ends, often Contain 0.1uM in the circuit of group output end²~100uM²Giant magnetic resistance.

7. a kind of cardio-pulmonary function marker magnetic particle microflow controlled biochip as claimed in claim 6, which is characterized in that described There is on the outside of biochip magnetic excitation device, in deposited antibodies and carry magnetic bead coupled antibody and marker protein forms and exempts from After epidemic disease compound, unbonded magnetic bead and antibody protein extra on biosensor is cleaned, magnetic excitation device is automatic at this time Opening excitation uniform magnetic field magnetizes magnetic bead, and the magnetic field that the magnetic bead being magnetized on the biosensor generates causes giant magnetoresistance material Expect the variation of resistance, and then judge the position of magnetic bead and concentration on biosensor, cardiopulmonary function is quantified by standard curve algorithm It can marker protein concentration.

8. applying a kind of cardio-pulmonary function marker magnetic particle microflow controlled biochip described in claim 1, which is characterized in that packet Include following steps：

Step 1, biochip pre-treatment are coated with six marker (CTI/CK-MB/MYO/ of cardio-pulmonary function on a biosensor D-Dime/NT-proBNP/hs-CRP) deposited antibodies；

Step 2, the preparation of 30nm+50nm magnetic bead and antibody coupling isolate and purify system：

I, the preparation of 30nm+50nm magnetic bead mixed liquor：Take Fe₃O₄Solution, which is added in ultrapure water, makes ferroso-ferric oxide solution 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 sodium acetate final concentration of 0.2%~0.3%, reaction obtains the ferroso-ferric oxide super paramagnetic beads of 30nm under high temperature Black colloidal state suspension；

Take Fe₃O₄Solution, which is added in ultrapure 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%, reaction obtains the ferroso-ferric oxide super paramagnetic beads black colloidal state suspension of 50nm 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 stand-by；

30nm and 50nm ferroso-ferric oxide super paramagnetic beads black colloidal state mixing suspension is placed on magnetic patch, gentle aspiration Supernatant is removed, and is cleaned with ethyl alcohol and ultrapure water, disperses 30nm the and 50nm ferroso-ferric oxide super paramagnetic beads washed in Ethyl alcohol, ultrapure 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, ultrapure 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- sulfosuccinic is added in the super quick magnetic bead of 30nm+50nm by the activation of the super quick magnetic bead of 30nm+50nm Imide -4- (N- maleimidomehyl) cyclohexane -1- carboxylate), coupling buffer, ultrasonic disperse magnetic bead is added；

(2) the super quick magnetic bead of the 30nm+50nm being re-activated through sulfydryl is placed in magnetic separator and is separated, absorb supernatant, be added even Join buffer solution for cleaning, repeats and suspended again magnetic bead after Magneto separate purification process with coupling buffer, ultrasonic disperse is stand-by；

(3) coupling streptavidin/monoclonal antibody activation；

(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 separates magnetic by ultrasonic disperse Pearl；

(5) super long-chain biological monoclonal antibody coupling：Biotin is dissolved with ultrapure water, then mixes and incubates with monoclonal antibody solution It educates a period of time, centrifuge separation removal supernatant 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 Sample is automatically into mixed zone in piece sample application zone, dispensed in advance on chip biotin conjugated monoclonal antibodies vesica punctured automatically into Enter mixed zone vibration mixing 10-30 seconds, dispenses magnetic bead coupling Avidin vesica on chip in advance and punctured automatically, is coupled with biotin Antibody samples compound object carry out hybrid reaction 10-30 second after into reaction zone (biochip sensor), if in sample Containing six marker proteins of cardio-pulmonary function, the compound for forming magnetic bead is assembled into (Ab1-Ag-Ab2-bio- on biosensor SA-MB), go out magnetic bead concentration by measuring magnetoresistance signal quantitative reaction, magnetic bead concentration is converted to by egg by standard curve algorithm White concentration, and then judge the concentration of six marker proteins of cardio-pulmonary function.

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 is added and mixes ultrasound；

(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, and the functional group in release catalysis is catalyzed monomer polymerization reactions, by ultraviolet irradiation protection is applied 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, it is stand-by by being saved after being dried with 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 configure BSA solution with antibody protection liquid；

(3) deposited antibodies are prepared, and prepare six marker (CTI/CK-MB/MYO/D-Dime/ of cardio-pulmonary function with antibody protection liquid NT-proBNP/hs-CRP) antibody, ultrasound mix；

(4) antibody and negative control are put on different biosensors by full-automatic micro-sampling system respectively, 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 for, make antibody protein With the surface functional group stable bond of biosensor；

III, the closing of biochip：

(1) polysorbas20/80/100 solution is added in the preparation of chip confining liquid in trihydroxy methyl methylamino methane hydrochloride buffer And ethanolamine solutions, it is filtered after mixing spare；

(2) closed protective liquid is prepared, and potassium chloride and sodium chloride are added under phosphate buffer, filters 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 albumen is dehydrated the state stable 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：Take blood sample (whole blood/plasma/serum/refers to blood) be added in biochip sample application zone sample automatically into Enter mixed zone, the magnetic bead coupled antibody vesica being divided on biochip in advance is punctured mixing automatically, if contained in sample Six marker proteins of cardio-pulmonary function will form the compound aggregation (Ab1-Ag-Ab2-MB) of magnetic bead on biosensor, pass through Measurement magnetoresistance signal quantitative reaction goes out magnetic bead concentration, magnetic bead concentration is converted to protein concentration by standard curve algorithm, in turn Judge the concentration of six marker proteins of cardio-pulmonary function.