CN108226536A - The micro-fluidic immune detection chip and method of a kind of cardiac muscle troponin I - Google Patents
The micro-fluidic immune detection chip and method of a kind of cardiac muscle troponin I Download PDFInfo
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- CN108226536A CN108226536A CN201810138655.3A CN201810138655A CN108226536A CN 108226536 A CN108226536 A CN 108226536A CN 201810138655 A CN201810138655 A CN 201810138655A CN 108226536 A CN108226536 A CN 108226536A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention discloses the micro-fluidic immune detection chips and method of a kind of cardiac muscle troponin I.In detection chip, sample reservoir, reaction detection pond and waste liquid pool are successively set on the main road of microchannel, one end of microchannel main road is equipped with injection port, the other end of microchannel main road is connected with the first pump, one end of microchannel branch is connected by the second pump with savings pond, the other end of microchannel branch is connected on the microchannel main road between reaction detection pond and waste liquid pool, microchannel branch road is equipped with reagent B filling ponds and high level calibration pool successively, low value calibration pool is in parallel with high level calibration pool, the microchannel branch that reagent B is filled between pond and high level calibration pool also connects branch by a microchannel and is connected with the microchannel main road between sample reservoir and reaction detection pond.Sample processing time can not only be greatly shortened by the present invention, reduce cost, moreover it is possible to improve the precision of detection, realize the functions such as the self-service detection of mobile detection and family.
Description
Technical field
The present invention relates to technical field of biological, more specifically, more particularly to a kind of miniflow of cardiac muscle troponin I
Control immune detection chip and method.
Background technology
Cardiac troponin (cTn) is the regulatory protein of mycardial contractility, it is made of the subunit of three kinds of different genes：
Serum cardiac troponin T (cTnT), cardiac muscle troponin I (cTnI) and cTnC (cTnC).The detection of cTnI is three
Medium sensitivity is highest, therefore testing requirements precision is also highest, and conventional means are difficult to do greatly very in the detection of the project
High accuracy and precision.But cTnI has very important clinical meaning, is mainly manifested in：(1) cTnI is not by bone
The influence that flesh is damaged, and occur relatively early after morbidity, the duration is long, has its unique Cardiac-specific and diagnosis
The longer advantage of window phase is that current diagnosis myocardial damage preferably determines marker.(2) release of cTnI and myocardial infarction face
Product is linear related, and 9h correlations are best after AMI, and the concentration of 9h cTnI can estimate the area of myocardial infarction after AMI.Cardiac muscle stalk
Dead area and the decrease of ventricular function and ventricular arythmia have close relationship, estimate the area pair of myocardial infarction
Follow-up condition assessment prognosis has very great help.(3) cTnI has higher sensibility and specificity to detection minor myocardial damage,
Available for the monitoring of heart peri-operation period, some researches show that cTnI is to the predictive value highest of postoperative minor myocardial damage.
(4) whether cTnI has unique Cardiac-specific, Skeletal muscle injury or Chronic Renal Failure Patients can be differentiated with myocardial damage.
The detection of cardiac muscle troponin I at present mainly by extremely sensitive enzyme immunoassay (ELISA) do quantitative detection or
Person makees qualitative detection with quick solid-phase immunity chromatography.Major defect is：Sample consumption is big, operation sequence is complicated, cost
It is high, time-consuming, and the personnel for generally requiring profession are operated, seriously hinder application that ACS quickly detects immediately and
It promotes.
Microfluidic chip technology, also known as chip lab (Lab on a Chip) are a kind of biology, chemistry, medicine
The basic operation units such as sample preparation, reaction, separation, the detection of analytic process are integrated on one piece several square centimeters of chip,
It is automatically performed the technology of analysis overall process.Chip is mainly made of microchannel, Micropump, micro-valve, reaction microchamber room etc., has height
It is integrated, automation and Biochemical Lab various functions feature.Biochemical manipulation is carried out on micro-fluidic chip has sample
Processing easily and fast, it is at low cost the advantages that, and under micro-scale, the specific surface area of sample is much larger than macroscopical sample, very short
The interior of time can more fully complete the lower biochemical reaction that time-consuming of many macroscopic views, and possess than traditional detection means
More accurately testing result.Therefore, the zymetology carried out for the purpose of drug screening or biochemistry detection analysis is reacted, is immune
Reaction, PCR reactions, ELISA (ELISA) etc. realize on micro-fluidic chip, can not only be by sample processing time substantially
Degree shortens, and reagent and instrument cost are greatly lowered, moreover it is possible to improve the precision of detection and realize that mobile detection and family are self-service
The functions such as formula detection.
Invention content
The purpose of the present invention is to provide the micro-fluidic immune detection chip and method of a kind of cardiac muscle troponin I, with gram
Clothes are the problems of in the prior art.
In order to achieve the above object, the technical solution adopted by the present invention is as follows：
A kind of micro-fluidic immune detection chip of cardiac muscle troponin I, the chip including chip upper cover and band structure are described
The chip of band structure be equipped with injection port, sample reservoir, microchannel, reagent B filling pond, high level calibration pool, low value calibration pool,
Reaction detection pond, waste liquid pool, savings pond, the second pump and the first pump, the microchannel include microchannel main road, microchannel branch
Road, the sample reservoir, reaction detection pond and waste liquid pool are successively set on the main road of microchannel, and the one of the microchannel main road
End is equipped with injection port, and the other end of the microchannel main road is connected with the first pump, and one end of the microchannel branch passes through second
Pump is connected with savings pond, and the other end of the microchannel branch is connected to the microchannel master between reaction detection pond and waste liquid pool
On the road, the microchannel branch road is equipped with reagent B filling ponds and high level calibration pool, the low value calibration pool and high value calibration successively
Pond is in parallel, the microchannel branch between reagent B filling pond and high level calibration pool also by a microchannel connect branch with
Microchannel main road connection between sample reservoir and reaction detection pond；Hemofiltration film is equipped in the sample reservoir.
Further, one end of the microchannel branch is equipped with the second pump interface, and first is equipped in second pump interface
Medical rubber plug, the output terminal of second pump are connected by syringe needle with the second pump interface.
Further, the other end of the microchannel main road is equipped with the first pump interface, and the is equipped in first pump interface
Two Medical rubber plugs, the output terminal of first pump are connected by syringe needle with the first pump interface.
The present invention also provides a kind of detection sides of the micro-fluidic immune detection chip according to above-mentioned cardiac muscle troponin I
Method, this method include the following steps,
S1, quantitative whole blood or blood serum sample are injected into test chip by injection port；
S2, the first pump startup vacuumize, and the fluid both ends in the microchannel main road of test chip is made to generate pressure differential, sample
Sample reservoir is flowed under the action of surface tension, and serum enters reaction detection after hemofiltration membrane filtration in sample reservoir
Pond；
S3, sample and pre-filled reagent A association reaction in reaction detection pond are flowed into, after reaction fully, the first pump will
Extra sample takes inflow waste liquid pool away, is then shut off the first pump；
S4, the second pump startup by save pond in water be drawn into reagent B filling pond, and be transported to reaction detection pond,
In high level calibration pool and low value calibration pool, the second pump is closed, so that reagent is fully reacted；
S5, the first pump is opened, reagent extra in reaction detection pond, high level calibration pool and low value calibration pool is siphoned away, flowed
Enter in waste liquid pool；
S6, external equipment equipment are to reaction detection pond, three ponds of high level calibration pool and low value calibration pool are detected, locate
Reason and display.
Compared with prior art, the advantage of the invention is that：Sample processing time can not only be greatly shortened by the present invention,
Reagent cost is greatly lowered, moreover it is possible to and the precision of detection is improved, realizes the functions such as the self-service detection of mobile detection and family,
Bring foreseeable great economic benefit.
Description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention, for those of ordinary skill in the art, without creative efforts, can be with
Other attached drawings are obtained according to these attached drawings.
Fig. 1 is the structure diagram of the micro-fluidic immune detection chip of cardiac muscle troponin I of the present invention.
Fig. 2 is the schematic internal view of the micro-fluidic immune detection chip of cardiac muscle troponin I of the present invention.
Fig. 3 is the stereogram of the micro-fluidic immune detection chip of cardiac muscle troponin I of the present invention.
Fig. 4 is the stereogram of the internal structure of the micro-fluidic immune detection chip of cardiac muscle troponin I of the present invention.
Reference sign：1st, injection port, 2, sample reservoir, 3, microchannel, 4, test chip, the 5, second pump interface,
6th, reagent B fills pond, and 7, high level calibration pool, 8, low value calibration pool, 9, reaction detection pond, 10, waste liquid pool, the 11, first pump interface,
12nd, pond is saved, the 13, second pump, the 14, first Medical rubber plug, the 15, second Medical rubber plug, the 16, syringe needle, 17, first pump,
21st, chip upper cover, 22, the chip of band structure.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with the accompanying drawings, so that advantages and features of the invention energy
It is easier to be readily appreciated by one skilled in the art, so as to make a clearer definition of the protection scope of the present invention.
Refering to shown in Fig. 1-Fig. 4, the present invention provides a kind of micro-fluidic immune detection chip 4 of cardiac muscle troponin I, including
The chip 22 of chip upper cover 21 and band structure, the chip 22 of the band structure are equipped with injection port 1, sample reservoir 2, microchannel
3rd, reagent B fills pond 6, high level calibration pool 7, low value calibration pool 8, reaction detection pond 9, waste liquid pool 10, savings pond 12, second pump
13 and first pump 17.
The microchannel 3 includes microchannel main road, microchannel branch, the sample reservoir 2, reaction detection pond 9 and useless
Liquid pool 10 is successively set on the main road of microchannel, and one end of the microchannel main road is equipped with injection port 1, the microchannel main road
The other end is connected with the first pump 17, and one end of the microchannel branch is connected by the second pump 13 with savings pond 12, described micro-
The other end of passage branches is connected on the microchannel main road between reaction detection pond 9 and waste liquid pool 10, the microchannel branch
On successively be equipped with reagent B filling pond 6 and high level calibration pool 7, the low value calibration pool 8 be in parallel with high level calibration pool 7, the examination
Agent B fill microchannel branch between pond 6 and high level calibration pool 7 also by a microchannel connect branch and sample reservoir 2 and
Microchannel main road connection between reaction detection pond 9；Hemofiltration film is equipped in the sample reservoir 2.
One end of the microchannel branch is equipped with the second pump interface 5, and the first medical grade rubber is equipped in second pump interface 5
Plug 14, the output terminal of second pump 13 are connected by syringe needle 16 with the second pump interface 5.
The other end of the microchannel main road is equipped with the first pump interface 11, and it is medical to be equipped with second in first pump interface 11
Rubber stopper 15, the output terminal of first pump 17 are connected by syringe needle 16 with the first pump interface 11.
It is pre-buried that test chip 4 in the present invention does reagent A (capture antibody) in reaction detection pond 9 in advance, is filled out in reagent B
It fills and reagent B pre-buried (labelled antibody of coupling fluorescein) is in pond 6.
The basic principle that the present invention realizes is：Fluid has the function of surface tension in microchannel, using test chip 4
External pump has pressure differential by vacuumizing fluid in microchannel, and the effect of pressure differential can be such that fluid is flowed in microchannel
Dynamic, the distance of flowing is related with the size of microchannel surface roughness, the coefficient of viscosity of fluid and pressure differential etc..Changed by surface
Microchannel surface can be modified as water-wetted surface by property technology, and the resistance of fluid in the channel can become smaller, and it is residual to also avoid liquid
It stays in runner and does not flow away.When stopping driving, fluid can stop no longer to be flowed in situ pump.
The present invention specific implementation the step of be：
The first step prepares chip：It can pass through injection, photoetching, burn into revolving die, coining for the micro-fluidic chip of detection
The methods of be fabricated.Chip can be process by materials such as glass, PDMS, PC plastic, PMMA, and chip material can be more
Kind or one kind therein.Chemical modification is carried out to chip surface structure according to demand after chip manufacture, can be hydrophilic
Modified or hydrophobically modified.
Second step, reagent are pre-filled：By reagent by physical absorption, chemical crosslinking, sol-gel embed, microballon fix,
The modes such as film is fixed, crystallization is handled and is freezed are embedded into reaction detection pond, reagent filling pond and height value calibration pond.
Third step, chip bonding and encapsulation：The bonding of chip prepares the difference of material according to chip and selects different keys
Conjunction mode.Including Direct Bonding encapsulation, ultrasonic bond encapsulation, laser bonding encapsulation, solution auxiliary bonding packaging, thermal bonding envelope
Dress introduces Intermediate Layer Bonding encapsulation (glue, film etc.) etc..
4th step, with quantitative capillary tube, either other equipment injects quantitative whole blood or blood serum sample into chip, and two
A external first pump and the second pump are inserted into chip, and insert port is embedded with Medical rubber plug, and guarantee is not in gas leakage and liquid
The phenomenon that body is flow backwards, specific testing process is as follows：
S1, quantitative whole blood or blood serum sample are injected into test chip 4 by injection port 1；
S2, the first pump 17, which start, to be vacuumized, and the fluid both ends in the microchannel main road of test chip 4 is made to generate pressure differential,
Sample flows into sample reservoir 2 under the action of surface tension, and serum enters instead after hemofiltration membrane filtration in sample reservoir 2
Answer detection cell 9；
Sample and pre-filled reagent A association reaction in S3, inflow reaction detection pond 9, after reaction fully, the first pump 17
Extra sample is taken away and flows into waste liquid pool 10, is then shut off the first pump 17；
S4, the second pump 13 start is drawn into reagent B fillings pond 6, and be transported to reaction inspection by the water saved in pond 12
It surveys in pond 9, high level calibration pool 7 and low value calibration pool 8, the second pump 13 is closed, so that reagent is fully reacted；
S5, the first pump 17 is opened, reagent extra in reaction detection pond 9, high level calibration pool 7 and low value calibration pool 8 is inhaled
It walks, flows into waste liquid pool 10；
S6, external equipment equipment are to reaction detection pond, three ponds of high level calibration pool and low value calibration pool are detected, locate
Reason and display.
The principle of reaction detection of the present invention is double-antibody method principle, in test chip 4, pre-buried reagent A (capture in advance
Antibody) and reagent B (coupling fluorescein labelled antibody), the serum after filtering comes into full contact with reaction after flowing to reaction detection pond 9, will
Antigen capture in serum completes the combination of antigen and antibody, after the completion of reaction, extra serum is taken away, then the second pump
13 start, and water is pumped into chip solubilising reagent B and is transported in reaction detection pond 9, high level calibration pool 7 and low value calibration pool 8, react
The antibody with fluorescent marker is recombined with the antigen in reaction tank combined with capture antibody in detection cell 9, is formed dual anti-
Body presss from both sides the structure of antigen；It is then that antigen is combined with fluorescein labelled antibody in high level calibration pool 7 and low value calibration pool 8.It is combined
Antigen on all with fluorescent marker, only there is absorption to some wavelength or certain section of wavelength.Pass through optical detection side
Formula can rapidly detect the antigen in serum, and can accurately judge and correct detection according to the calibration of high low value
As a result.
The detection chip of the present invention has included calibration system, it is ensured that testing result is accurate.
The calibrating principle of calibration system is：The quantitative of various concentration known to filling resists respectively in high level and low value calibration pool
Original, the antigen of the cardiac muscle troponin I of filling higher concentration in high level calibration pool, filling low concentration is anti-in low value calibration pool
Original, when calibration, the antigen in two calibration pools is combined with reagent B (labelled antibody of coupling fluorescein), detection when
It waits with regard to that can detect 2 different values：Low value and high level, and can all there is one to be corresponded to often by each concentration in every detection device
The standard calibration curve of a detected value, in practical detection process, when we do not have calibration system, the result of detection
It will rely on and can be calculated with the standard calibration curve in instrument, system according to the comparison in the data and calibration curve of detection
The concentration of tester's Blood Center flesh Troponin I, however be not so simple in practical detection, between sample, chip
Between it is all variant, simple dependence standard calibration curve is susceptible to testing result and deviation and inaccuracy occurs, is there is height
In the case that low value is calibrated, 2 high low values of measure can be compared with standard curve, form a reduction formula, each chip
All there is otherness, therefore the high low value that each chip measures may be different, reduction formula is also different, each chip
The result of pattern detection also can make corresponding adjustment according to the calibration reduction formula of this chip, so as to more accurately determine
The testing result of the sample.
The whole process for detecting cardiac muscle troponin I is integrated into micro-fluidic core by the present invention using micro-fluidic chip
On piece, using the driving of additional pump, the entrance to sample, reaction is controlled, right using fluorescent marker and optical detection apparatus
Reaction result is detected.
The present invention has the following advantages：1. greatly reduce the dosage of sample and reagent, it is only necessary to the whole blood of 1-100 μ L
Or serum can just complete detection process, corresponding reagent dosage is also significantly reduced；2. sample processing time is reduced,
The hemofiltration film added in chip, can quickly detach serum, overcome tradition using centrifuging serum isolation technics time-consuming
Feature improves the efficiency of sample pretreatment；3. detecting instrument microminiaturization, portability can realize clinical and mobile inspection
The detection mode that survey or family popularize, brings great convenience to medical worker and patient；4. shorten detection time and
Improve accuracy of detection, under minute yardstick, the specific surface area of reagent and sample much larger than under macro-scale, reagent and sample
More fully and rapid with reference to meeting, therefore detection time also greatly reduces, while accuracy of detection is also more accurate.
Although being described in conjunction with the accompanying embodiments of the present invention, patent owner can be in appended claims
Within the scope of make various deformations or amendments, as long as no more than the present invention the described protection domain of claim, all should
Within protection scope of the present invention.
Claims (4)
1. a kind of micro-fluidic immune detection chip of cardiac muscle troponin I, it is characterised in that：Including chip upper cover and band structure
Chip, the chip of the band structure are equipped with injection port, sample reservoir, microchannel, reagent B fillings pond, high level calibration pool, low
Value calibration pond, reaction detection pond, waste liquid pool, savings pond, second pump and first pump, the microchannel include microchannel main road,
Microchannel branch, the sample reservoir, reaction detection pond and waste liquid pool are successively set on the main road of microchannel, the microchannel
One end of main road is equipped with injection port, and the other end of the microchannel main road is connected with the first pump, one end of the microchannel branch
It is connected by the second pump with savings pond, the other end of the microchannel branch is connected between reaction detection pond and waste liquid pool
On the main road of microchannel, microchannel branch road is equipped with reagent B filling ponds and high level calibration pool successively, the low value calibration pool with
High level calibration pool is in parallel, and the microchannel branch between the reagent B filling ponds and high level calibration pool is also connected by a microchannel
The microchannel main road connect between branch and sample reservoir and reaction detection pond connects；Hemofiltration is equipped in the sample reservoir
Film.
2. the micro-fluidic immune detection chip of cardiac muscle troponin I according to claim 1, it is characterised in that：It is described micro-
One end of passage branches is equipped with the second pump interface, and the first Medical rubber plug is equipped in second pump interface, second pump
Output terminal is connected by syringe needle with the second pump interface.
3. the micro-fluidic immune detection chip of cardiac muscle troponin I according to claim 1, it is characterised in that：It is described micro-
The other end of channel main road is equipped with the first pump interface, and the second Medical rubber plug, first pump are equipped in first pump interface
Output terminal connected by syringe needle with the first pump interface.
4. a kind of inspection of the micro-fluidic immune detection chip of cardiac muscle troponin I according to claim 1-3 any one
Survey method, it is characterised in that：This method includes the following steps,
S1, quantitative whole blood or blood serum sample are injected into test chip by injection port；
S2, the first pump startup vacuumize, and the fluid both ends in the microchannel main road of test chip are made to generate pressure differential, sample is in table
Sample reservoir is flowed under the action of the tension of face, and serum enters reaction detection pond after hemofiltration membrane filtration in sample reservoir；
Sample and pre-filled reagent A association reaction in S3, inflow reaction detection pond, after reaction fully, the first pump will be extra
Sample take inflow waste liquid pool away, be then shut off the first pump；
The water saved in pond is drawn into reagent B fillings pond, and is transported to reaction detection pond, high level by S4, the second pump startup
In calibration pool and low value calibration pool, the second pump is closed, so that reagent is fully reacted；
S5, the first pump is opened, reagent extra in reaction detection pond, high level calibration pool and low value calibration pool is siphoned away, flowed into useless
In liquid pool；
S6, external equipment equipment are to reaction detection pond, three ponds of high level calibration pool and low value calibration pool are detected, handle and
Display.
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| CN201810138655.3A CN108226536B (en) | 2018-02-10 | 2018-02-10 | Micro-fluidic immunodetection chip for cardiac troponin I |
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| CN201810138655.3A CN108226536B (en) | 2018-02-10 | 2018-02-10 | Micro-fluidic immunodetection chip for cardiac troponin I |
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Cited By (5)
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| CN109211870A (en) * | 2018-11-17 | 2019-01-15 | 郑州亲和力科技有限公司 | A kind of micro-fluidic fluorescence immunoassay chip of rapid quantitative detection cTnI |
| CN109765358A (en) * | 2019-01-18 | 2019-05-17 | 江苏医联生物科技有限公司 | The chemical treatment method of micro-fluidic chip application protein detection |
| CN110026256A (en) * | 2019-04-19 | 2019-07-19 | 深圳市亚辉龙生物科技股份有限公司 | Micro-fluidic chip |
| CN116539701A (en) * | 2023-07-07 | 2023-08-04 | 北京几何科技有限公司 | Reagent card for urine detection and detection method |
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