CN105785054A - Chemical-luminescent microfluidic disk for quantitative detection of procalcitonin and using method thereof - Google Patents
Chemical-luminescent microfluidic disk for quantitative detection of procalcitonin and using method thereof Download PDFInfo
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Abstract
The invention discloses a chemical-luminescent microfluidic disk for quantitative detection of procalcitonin and a using method thereof. The chemical-luminescent microfluidic disk comprises a microfluidic disk body, and a microfluidic detecting unit is arranged on the microfluidic disk body; the microfluidic detecting unit comprises a whole-blood injecting tank, a whole-blood separating channel, a blood cell storing tank, a plasma conveying runner, a mixing/detecting tank, a procalcitonin monoclonal antibody coated magnetic particle solution injecting tank, a cleaning solution injecting tank, an external magnet area containing a magnet internally, a waste liquid tank, an alkaline phosphatase labeled procalcitonin monoclonal antibody solution injecting tank and an enhanced chemical-luminescent solution injecting tank. The chemical-luminescent microfluidic disk can achieve functions of whole-blood separation, plasma quantification, cultivating mixture and cleaning, can quickly achieve quantitative detection of a PCT concentration in a sample and has the characteristics of being high in detecting flexibility, accurate and reliable in result and good in repeatability.
Description
Technical field
The present invention relates to the micro-fluidic disc of chemiluminescence of a kind of Procalcitonin. detection by quantitative and using method thereof, belong to medical immunology in-vitro diagnosis field, it is possible within very short time, realize, to the detection by quantitative of Procalcitonin. in biological sample, having simple to operate, highly sensitive, the feature of low cost.
Background technology
Procalcitonin. (PCT) is a kind of containing 116 amino acid whose glycoproteins without hormonal activity, and molecular weight is about 13kDa, by the generation of thyroid-C cell under physiological conditions, is little to be detected in the serum of healthy population.At infectious disease such as antibacterial, fungus, parasites and have whole body or/and during the inflammatory reaction of central nervous system's property, the endotoxin of pathogenic bacteria can promote the tissue beyond the thyroid such as lymphocyte and the neuroendocrine cell of thyroid-C cell, hepatic macrophages and mononuclear cell, lung and intestinal tissue to produce PCT in a large number, and causes the content in its serum to raise or persistence rising.Neonatal PCT level after birth in 1 ~ 2 day and after minority major surgery operation in patients the PCT in 1～4 day can have slight of short duration rising, but all in reaching adult normal value after downward trend day by day and Yu Santian.Check can provide definite Differential Diagnosis for the above-mentioned state of an illness for this PCT.
PCT level reflects the active degree of Systemic bacterial inflammatory reaction, and points out relevant to the size of infected organ and type, the kind of antibacterial and degree of inflammation and immunoreation situation.Though the rising of PCT level may alternatively appear in serious shock, systemic inflammatory response syndrome and multiple organ dysfunction derangement syndrome patient, as infected without antibacterial or the existence of bacterial infection focus, its level is usually less than those patients having bacterial infection focus.The clinical value of PCT detection is many-sided.The assessment of the etiological diagnosis of antibacterial, fungus, parasitic infection and pyemia sexuality dye, antibiotic clinical practice, the state of an illness and prognosis is respectively provided with obvious using value, autoimmune, allergy, viral infection and aseptic inflammation etc. are had clear and definite differential diagnosis value.
At present, the method detecting Procalcitonin. mainly has radio immunoassay, chemiluminescence immunoassay, gold colloidal colorimetry.Radio immunoassay detects length consuming time, and has the pollution of radioelement to use and be restricted；Gold colloidal colorimetry has the features such as fast and convenient, easy observation, is a kind of quickly sxemiquantitative using method；Chemiluminescence immunoassay is easy and simple to handle, high specificity, highly sensitive, and the detection time is short, is widely used at present.
Magnetic particle immunoassay technology is that carrier made by the Magnetic solid phases microgranule utilizing synthesis of polymer material certain particle size size, be coated with the method such as physical absorption, chemical coupling have specificity affinity the various immunologic active materials such as antibody or antigen, have that separating rate is fast, efficiency is high, favorable repeatability, the feature such as simple to operate, biological character and function of not affecting separated cell or other biological material, orientable motion under additional the action of a magnetic field, makes some special composition be separated, concentration or purification.
Micro-fluidic chip, as a kind of novel analysis test platform, has the advantages such as high flux, operation integrated, portable, easy, low cost, has been widely used in various fields, has shown up prominently particularly in immunoassay field.
The biological micro-fluidic chip of immunomagnetic beads is by magnetic granule technology, immunoassay is integrated into the one on micro-fluidic chip and analyzes using method, the Major Difficulties of current this comprehensive using method shows themselves in that 1) liquid is in the Based Intelligent Control of chip internal microfluidic, at present frequently with method be that multiple Micropump and micro-valve are set at chip internal so that micro-fluidic system becomes more to complicate；2) undercompounding of reaction system, causes that reaction is insufficient；3) integration degree is not high, causes that non-specific background is high.
Summary of the invention
Present invention aim at providing the micro-fluidic disc of chemiluminescence of a kind of Procalcitonin. detection by quantitative and using method thereof；The present invention can realize whole blood separate (Sample pretreatment), blood plasma quantitatively, cultivate mixing, the function cleaned, can quickly realize the detection by quantitative of PCT concentration in sample, there is detection sensitivity height, result accurately and reliably, reproducible feature.
In order to achieve the above object, the technical scheme is that
A kind of chemiluminescence micro-fluidic disc of Procalcitonin. detection by quantitative, including micro-fluidic disc, described micro-fluidic disc is provided with miniflow detection unit, described miniflow detection unit includes whole blood and injects groove, described whole blood is injected groove and is connected with blood cell accumulator tank by whole blood segregated channel, and described blood cell accumulator tank transmits flow passage with blood plasma；Described blood plasma transmits runner and is connected with mixing/detecting groove top；Described mixing/detecting groove top is injected groove by the coated magnetic particle solution of pipeline and Procalcitonin monoclonal antibody and is connected；Described mixing/detecting groove top is injected groove also by pipeline with cleanout fluid and is connected；Described mixing/detecting groove side is provided with the external magnet region including Magnet；Described mixing/detecting trench bottom is connected with waste liquid tank by pipeline, and the pipeline of and described mixing/between detecting trench bottom and waste liquid tank is provided with valve；The coated magnetic particle solution of described Procalcitonin monoclonal antibody injects the bottom injecting groove by pipeline with the Procalcitonin monoclonal antibody solution of alkali phosphatase enzyme mark in groove top and connects；Described cleanout fluid injects the bottom injecting groove by pipeline with luminous substrate liquid in groove top and connects.
On described micro-fluidic disc install more than 6 and be 6 multiple miniflow detect unit.
Described micro-fluidic disc is preferably installed 12 miniflow detection unit.
A kind of using method of the micro-fluidic disc of chemiluminescence of Procalcitonin. detection by quantitative, comprise the steps: 1) micro-fluidic disc is put in detecting instrument, open automatic sample feeding device, by whole blood, Procalcitonin monoclonal antibody coated magnetic particle solution, alkali phosphatase enzyme mark Procalcitonin monoclonal antibody solution be injected separately into whole blood and inject that the coated magnetic particle solution of groove, Procalcitonin monoclonal antibody injects groove, the Procalcitonin monoclonal antibody solution of alkali phosphatase enzyme mark injects in groove；
2) manipulate micro-fluidic disc with the first acceleration to rotate, the Procalcitonin monoclonal antibody solution of coated for Procalcitonin monoclonal antibody magnetic particle solution and alkali phosphatase enzyme mark, through whole blood segregated channel separated plasma and blood cell, is sent to mixing/detecting groove by whole blood simultaneously；The coated magnetic particle solution of Procalcitonin monoclonal antibody remaines in mixing/detecting groove due to the Magnet effect in external magnet region；
3) manipulating micro-fluidic disc with the second acceleration to rotate, via blood plasma, blood plasma is transmitted runner and is sent to mixing/detecting groove, blood cell is then retained in blood cell accumulator tank；
4) manipulate micro-fluidic disc with the 3rd acceleration to rotate, and manipulated by the Magnet in external magnet region, handle the coated magnetic particle of Procalcitonin monoclonal antibody to move in mixing/detecting groove, and then make to reach between blood plasma, the Procalcitonin monoclonal antibody solution of alkali phosphatase enzyme mark, Procalcitonin monoclonal antibody coated magnetic particle solution good mixing, cultivate effect；
5) cleanout fluid is injected into cleanout fluid and injects in groove, manipulate micro-fluidic disc (13) with the 4th acceleration to rotate, cleanout fluid can be sent in mixing/detecting groove, cleanout fluid is used for the liquid replacing in mixing/detecting groove, this liquid can be transferred in waste liquid tank, and the coated magnetic particle of Procalcitonin monoclonal antibody remaines in mixing/detecting groove due to the attraction of the Magnet in external magnet region；
6) luminous substrate liquid is injected into luminous substrate liquid and injects in groove, manipulate micro-fluidic disc with the 5th acceleration to rotate, luminous substrate can be sent to mixing/detecting groove, liquid in luminous substrate displacement mixing/detecting groove, this liquid also can be transferred in waste liquid tank, and the coated magnetic particle of Procalcitonin monoclonal antibody remaines in mixing/detecting groove due to the attraction of the Magnet in external magnet region；Finally, the liquid reacted can detect in mixing/detecting groove, and according to the proportionate relationship between relative luminous intensity (RLU) and Procalcitonin. antigen concentration, detection system converts automatically, energy fast report test result, thus realizing the detection by quantitative of Procalcitonin..
In described detection process, micro-fluidic disc rotation rotating ratio is: the first rotating speed: the second rotating speed: the 3rd rotating speed: the 4th rotating speed: five rotating speeds=11:16:5:18:10；First acceleration: the second acceleration: the 3rd acceleration: the 4th acceleration: five acceleration=11:10:12:8:16.
The invention has the beneficial effects as follows: the present invention can realize whole blood separation (Sample pretreatment), blood plasma is quantitative, cultivates the function mixing, cleaning, can quickly realize the detection by quantitative of PCT concentration in sample, there is detection sensitivity height, result accurately and reliably, reproducible feature.
Accompanying drawing explanation
Fig. 1 is the structural representation of the micro-fluidic disc of chemiluminescence of the present invention a kind of Procalcitonin. detection by quantitative；
Fig. 2 is that in Fig. 1, on micro-fluidic disc, single miniflow detects the enlarged diagram of unit.
Detailed description of the invention
Embodiment 1: the detection by quantitative of Procalcitonin.
A kind of chemiluminescence micro-fluidic disc of the Procalcitonin. detection by quantitative of the present embodiment, as shown in Figure 1, 2, including micro-fluidic disc 13, described micro-fluidic disc 13 is provided with 12 miniflow detection unit, described each miniflow detection unit includes whole blood and injects groove 1, described whole blood is injected groove 1 and is connected with blood cell accumulator tank 3 by whole blood segregated channel 2, and described blood cell accumulator tank 3 transmits runner 4 with blood plasma and connects；Described blood plasma transmits runner 4 and is connected with mixing/detecting groove 7 top；Described mixing/detecting groove 7 top is injected groove 9 by the coated magnetic particle solution of pipeline and Procalcitonin monoclonal antibody and is connected；Described mixing/detecting groove 7 top is injected groove 10 also by pipeline with cleanout fluid and is connected；Described mixing/detecting groove 7 side is provided with the external magnet region 8 including Magnet；Connected with waste liquid tank 6 by pipeline bottom described mixing/detecting groove 7, and the pipeline bottom described mixing/detecting groove 7 and between waste liquid tank 6 is provided with valve 5；The coated magnetic particle solution of described Procalcitonin monoclonal antibody injects the bottom injecting groove 11 by pipeline with the Procalcitonin monoclonal antibody solution of alkali phosphatase enzyme mark in groove 9 top and connects；Described cleanout fluid injects the bottom injecting groove 12 by pipeline with luminous substrate liquid in groove 10 top and connects.
1. Procalcitonin. solution preparation
1) preparation of Procalcitonin. series of calibration product: be series of calibration product by Procalcitonin. sterling (Hai Tai biotechnology (Shanghai) Co., Ltd.) dilution with hyclone, its calibration object concentration range is 0～50ng/mL.
2) preparation of the coated magnetic particle solution of Procalcitonin monoclonal antibody:
Procalcitonin monoclonal antibody (Hai Tai biotechnology (Shanghai) Co., Ltd.) is placed in bag filter, dialyzed overnight in PBS；Prepare BNHS solution with PBS, every mg antibody adds 15 μ LBNHS(biotin-N-hydroxy succinimine ester (Li Rui bio tech ltd, Shanghai) solution, mix homogeneously, room temperature reaction 30min；The antibody that labelling is good is placed in bag filter, dialyzed overnight in PBS.The Streptavidin magnetic particles of 2.0 μm (Guangzhou Da Yucheng trade Co., Ltd) are added magnetic field, stands 5min, pour out supernatant, clean 3 times with PBS, and suspend with this buffer；Pour out supernatant；Every mL magnetic particle suspension adds the antibody that 1mg labelling is good, mix homogeneously, room temperature reaction 20min；With containing 5% the Tris-HCl buffer solution for cleaning 3 times of bovine serum albumin；With containing 0.5% bovine serum albumin and 0.02%Proclin300(preservative, Li Rui bio tech ltd, Shanghai) PBS resuspended, obtain the coated magnetic particle solution of Procalcitonin monoclonal antibody.
3) preparation of the Procalcitonin monoclonal antibody solution of alkali phosphatase enzyme mark: with PB buffer Sulfo-SMCC solution, every mg alkali phosphatase adds 0.1mLSulfo-SMCC(4-(N-maleimidomehyl) hexamethylene-1-carboxylic acid sulfonic group butanimide ester sodium salt, Li Rui bio tech ltd, Shanghai) solution, mix homogeneously, room temperature reaction 30min, dialyses in PB buffer；With PB buffer 2-imino group thiol heptane hydrochloride saline solution, every mg antibody adds 0.1mL2-imino group thiol heptane hydrochloride saline solution, mix homogeneously, room temperature reaction 30min, dialyses in pPB buffer；By the alkali phosphatase after above-mentioned dialysis and antibody mix homogeneously, room temperature reaction 120min；Collect albumen by protein purification system (AKTApurifier100), add the glycerol of 50%, obtain the Procalcitonin. antibody-solutions of alkali phosphatase enzyme mark.
4) preparation of the Chemoluminescent substrate of alkaline phosphatase enzyme catalysis: add 2-amino-2-methyl-1-propanol, AMPPD(1 in Tris-HCl buffer, 2-dioxy thiacyclohexane derivant, Guangzhou Da Yucheng trade Co., Ltd) and luminescence enhancer (AP substrate reinforcing agent, Li Rui bio tech ltd, Shanghai).
5) preparation of cleanout fluid: add the polysorbas20 of 0.02% and the sodium chloride of 15% in Tris-HCl buffer.
2. the using method of the micro-fluidic disc of chemiluminescence of a kind of Procalcitonin. detection by quantitative of the present embodiment, comprises the steps:
1) making of standard curve: take 30 μ LPCT serial standards (concentration is 0ng/mL, 0.1ng/mL, 0.5ng/mL, 1ng/mL, 10ng/mL, 50ng/mL) and add the coated magnetic particle solution injection groove 9 of Procalcitonin monoclonal antibody of the micro-fluidic disc in the present embodiment, micro-fluidic disc 13 is placed in supporting detecting instrument.Each sample is replication five times respectively.Necessary instrument is according to the proportionate relationship between RLU and PCT concentration, and automatic Fitting calculates PCT concentration, takes five times and measures meansigma methods, drawing standard curve.
2) micro-fluidic disc 13 is put in necessary instrument, open automatic sample feeding device, can by whole blood 150 μ L, Procalcitonin monoclonal antibody coated magnetic particle solution 30 μ L, alkali phosphatase enzyme mark Procalcitonin monoclonal antibody solution 20 μ L be injected into whole blood and inject that the coated magnetic particle solution of groove 1, Procalcitonin monoclonal antibody injects groove 9, the Procalcitonin monoclonal antibody solution of alkali phosphatase enzyme mark injects in groove 11.
3) with 4,500RPM (acceleration a=1,500RPM/s) manipulate disc to rotate 50 seconds, can at whole blood segregated channel 2 separated plasma and blood cell, and the Procalcitonin monoclonal antibody solution of coated for Procalcitonin monoclonal antibody magnetic particle solution and alkali phosphatase enzyme mark is sent to mixing/detecting groove 7, the coated magnetic particle solution of Procalcitonin monoclonal antibody the attraction of Magnet in mat external magnet region 8 can remain in mixing/detecting groove 7 and (can avoid being washed to waste liquid tank).
4) manipulating disc with 1,500RPM (acceleration a=500RPM/s) to rotate 20 seconds, via blood plasma, blood plasma can be transmitted runner 4 and be sent to mixing/detecting groove 7, blood cell is then retained in blood cell accumulator tank 3.
5) with 1,300RPM (acceleration a=2,500RPM/s) manipulates disc and rotates 40 seconds, manipulates by the Magnet in external magnet region 8, the coated magnetic particle of Procalcitonin monoclonal antibody can be handled mobile in mixing/detecting groove 7, and then reach good mixing/cultivation effect.
6) cleanout fluid 60 μ L is injected into cleanout fluid and injects in groove 10, with 3,600RPM (acceleration a=3,600RPM/s) manipulate micro-fluidic disc 13 and rotate 22 seconds, cleanout fluid can be sent to mixing/detecting groove 7, liquid in the replaceable mixing of cleanout fluid/detecting groove 7, this liquid can be transferred into waste liquid tank 6, and the coated magnetic particle of Procalcitonin monoclonal antibody can remain in mixing/detecting groove 7 by the attraction of the Magnet in external magnet region 8.
7) luminous substrate liquid 40 μ L is injected into luminous substrate liquid and injects in groove 12, with 2,700RPM (acceleration a=1,150RPM/s) manipulate disc to rotate 16 seconds, luminous substrate can be sent to mixing/detecting groove 7, liquid in the replaceable mixing of luminous substrate/detecting groove 7, this liquid can be transferred into waste liquid tank, and the coated magnetic particle of Procalcitonin monoclonal antibody can remain in mixing/detecting groove 7 by the attraction of the Magnet in external magnet region 8.Finally, the liquid reacted can detect at mixing/detecting groove 7, and according to the proportionate relationship between relative luminous intensity (RLU) and Procalcitonin. antigen concentration, detection system can convert automatically, can fast report test result, thus realizing the detection by quantitative of Procalcitonin..
8) result shows: adopting the method for the invention detection sensitivity is 0.05ng/mL, and detection range is 0.05～100ng/mL, and interassay coefficient of variation is less than 10%, and variation within batch coefficient is less than 10%.
The present embodiment can realize whole blood separate (Sample pretreatment), blood plasma quantitatively, cultivate mixing, the function cleaned, can quickly realize the detection by quantitative of PCT concentration in sample, there is detection sensitivity height, result accurately and reliably, reproducible feature.
Embodiment 2: magnetic particle particle size is screened
The particle diameter of magnetic microsphere is little, and specific surface area is big, and active group is contained on surface, therefore coupling capacity is big, but magnetic microsphere is undersized is unfavorable for that Magnet is collected, the oversize hybrid reaction that is unfavorable in disc, therefore carries out magnetic particle size selection.
Other experiment condition is referring to embodiment 1, and the size of magnetic particle granule carries out according to below scheme.
Select respectively 0.1 μm, 0.5 μm, 1.0 μm, 1.5 μm, 2.0 μm, 3.0 μm, 5.0 μm magnetic particle marker Procalcitonin monoclonal antibody antibody of particle size.Adopt magnetic size through preferred permanent magnet, fixed magnet height during detection.
Experimental result is as follows:
Magnetic particle grain size strengthens successively from 0.1 μm, 0.5 μm, 1.0 μm, 1.5 μm, 2.0 μm, 3.0 μm, and 3.0 μm of interference strengthen, and 5.0 μm start to reduce, and signal value is minimum, and comprehensive 2.0 μm of signals of each factor are the strongest, disturb minimum.
Interpretation of result: when magnetic particle is smaller, specific surface area is relatively big, and the biotinylated molecular weight of surface institute load is big, can be well dispersed in solution simultaneously, but it is big to ensure that magnetic microsphere is fully collected required magnetic field intensity.Magnetic field force suffered by magnetic bead is it cannot be guaranteed that it is fully collected in the present embodiment, causes that part effectively magnetic bead runs off in cleaning process, thus causing that final detected signal value is not high.When magnetic grain diameter is bigger, specific surface area is little, the mark rate of surface biomolecules is relatively low, under the same terms, magnetic particle institute is magnetic field force induced, and scattered magnetic bead can be collected fully greatly, but it is owing to very easily settling, cause between biomolecule, reacting insufficient, thus weakening luminous signal.Considering, particle diameter is that 1.0 μm to 3.0 μm magnetic microsphere effects are better, and therefore in embodiments of the invention, the magnetic microsphere effect of 2.0 μm is best.
Claims (5)
1. the micro-fluidic disc of the chemiluminescence of a Procalcitonin. detection by quantitative, it is characterized in that: include micro-fluidic disc (13), described micro-fluidic disc (13) is provided with miniflow detection unit, described miniflow detection unit includes whole blood and injects groove (1), described whole blood is injected groove (1) and is connected with blood cell accumulator tank (3) by whole blood segregated channel (2), and described blood cell accumulator tank (3) is transmitted runner (4) with blood plasma and connected；Described blood plasma transmits runner (4) and is connected with mixing/detecting groove (7) top；Described mixing/detecting groove (7) top is injected groove (9) by the coated magnetic particle solution of pipeline and Procalcitonin monoclonal antibody and is connected；Described mixing/detecting groove (7) top is injected groove (10) also by pipeline with cleanout fluid and is connected；Described mixing/detecting groove (7) side is provided with the external magnet region (8) including Magnet；Described mixing/detecting groove (7) bottom is connected with waste liquid tank (6) by pipeline, and is provided with valve (5) on the pipeline bottom described mixing/detecting groove (7) and between waste liquid tank (6)；The coated magnetic particle solution of described Procalcitonin monoclonal antibody injects the bottom injecting groove (11) by pipeline with the Procalcitonin monoclonal antibody solution of alkali phosphatase enzyme mark in groove (9) top and connects；Described cleanout fluid injects the bottom injecting groove (12) by pipeline with luminous substrate liquid in groove (10) top and connects.
2. the micro-fluidic disc of chemiluminescence of a kind of Procalcitonin. detection by quantitative as claimed in claim 1, it is characterised in that: described micro-fluidic disc (13) is upper install more than 6 and be 6 the miniflow of multiple detect unit.
3. the micro-fluidic disc of chemiluminescence of a kind of Procalcitonin. detection by quantitative as claimed in claim 2, it is characterised in that: described micro-fluidic disc (13) is upper installs 12 miniflow detection unit.
4. the using method of the micro-fluidic disc of chemiluminescence of a Procalcitonin. detection by quantitative as claimed in claim 1, it is characterized in that, comprise the steps: 1) micro-fluidic disc (13) is put in detecting instrument, open automatic sample feeding device, by whole blood, the coated magnetic particle solution of Procalcitonin monoclonal antibody, the Procalcitonin monoclonal antibody solution of alkali phosphatase enzyme mark is injected separately into whole blood and injects groove (1), the coated magnetic particle solution of Procalcitonin monoclonal antibody injects groove (9), the Procalcitonin monoclonal antibody solution of alkali phosphatase enzyme mark injects in groove (11)；
2) manipulate micro-fluidic disc (13) with the first acceleration to rotate, the Procalcitonin monoclonal antibody solution of coated for Procalcitonin monoclonal antibody magnetic particle solution and alkali phosphatase enzyme mark, through whole blood segregated channel (2) separated plasma and blood cell, is sent to mixing/detecting groove (7) by whole blood simultaneously；The coated magnetic particle solution of Procalcitonin monoclonal antibody remaines in mixing/detecting groove (7) due to the Magnet effect in external magnet region (8)；
3) manipulating micro-fluidic disc (13) with the second acceleration to rotate, via blood plasma, blood plasma is transmitted runner (4) and is sent to mixing/detecting groove (7), blood cell is then retained in blood cell accumulator tank (3)；
4) manipulate micro-fluidic disc (13) with the 3rd acceleration to rotate, and manipulated by the Magnet in external magnet region (8), handle the coated magnetic particle of Procalcitonin monoclonal antibody to move in mixing/detecting groove, and then make to reach between blood plasma, the Procalcitonin monoclonal antibody solution of alkali phosphatase enzyme mark, Procalcitonin monoclonal antibody coated magnetic particle solution good mixing, cultivate effect；
5) cleanout fluid is injected into cleanout fluid and injects in groove (10), manipulate micro-fluidic disc (13) with the 4th acceleration to rotate, cleanout fluid can be sent in mixing/detecting groove (7), cleanout fluid is used for the liquid replacing in mixing/detecting groove (7), this liquid can be transferred in waste liquid tank (6), and the coated magnetic particle of Procalcitonin monoclonal antibody remaines in mixing/detecting groove due to the attraction of the Magnet in external magnet region (8)；
6) luminous substrate liquid is injected into luminous substrate liquid and injects in groove (12), manipulate micro-fluidic disc (13) with the 5th acceleration to rotate, luminous substrate can be sent to mixing/detecting groove (7), liquid in luminous substrate displacement mixing/detecting groove (7), this liquid also can be transferred in waste liquid tank (6), and the coated magnetic particle of Procalcitonin monoclonal antibody remaines in mixing/detecting groove (7) due to the attraction of the Magnet in external magnet region (8)；Finally, the liquid reacted can detect in mixing/detecting groove (7), and according to the proportionate relationship between relative luminous intensity and Procalcitonin. antigen concentration, detection system converts automatically, energy fast report test result, thus realizing the detection by quantitative of Procalcitonin..
5. the using method of the micro-fluidic disc of chemiluminescence of a kind of Procalcitonin. detection by quantitative as claimed in claim 4, it is characterized in that, in described detection process, micro-fluidic disc (13) rotational speed ratio is: the first rotating speed: the second rotating speed: the 3rd rotating speed: the 4th rotating speed: five rotating speeds=11:16:5:18:10；First acceleration: the second acceleration: the 3rd acceleration: the 4th acceleration: five acceleration=11:10:12:8:16.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107655878A (en) * | 2017-09-01 | 2018-02-02 | 北京华科泰生物技术有限公司 | For the micro-fluidic chemiluminescence detection system for the magnetic particle for detecting thyroid function |
| CN108732339A (en) * | 2017-04-19 | 2018-11-02 | 光宝电子(广州)有限公司 | Runner design and its detection method for multiple reaction biological detection |
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| CN112147199A (en) * | 2020-08-31 | 2020-12-29 | 济南大学 | Based on CoS2/MoS2Preparation of microfluidic electrochemical sensor of-Pd |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3799742A (en) * | 1971-12-20 | 1974-03-26 | C Coleman | Miniaturized integrated analytical test container |
| US20110201101A1 (en) * | 2006-09-05 | 2011-08-18 | Samsung Electronics Co., Ltd. | Centrifugal force-based microfluidic device for protein detection and microfluidic system including the same |
| CN102472739A (en) * | 2010-01-29 | 2012-05-23 | 三星电子株式会社 | Centrifugal micro-fluidic device and method for detecting analytes from liquid specimen |
| US20130034912A1 (en) * | 2011-08-03 | 2013-02-07 | Unist Academy-Industry Research Corporation | Centrifugal force-based microfluidic device available for reliability verification, and analyzing method using the same |
| CN103223357A (en) * | 2012-01-26 | 2013-07-31 | 三星电子株式会社 | Microfluidic device and control method thereof |
| CN103323605A (en) * | 2013-06-18 | 2013-09-25 | 杭州普施康生物科技有限公司 | Micro-fluidic chip for glycosylated hemoglobin immunodetection |
| CN104597266A (en) * | 2013-10-31 | 2015-05-06 | 逢甲大学 | Centrifugal-type detection platform and operating process thereof |
| CN105203775A (en) * | 2015-10-26 | 2015-12-30 | 深圳华迈兴微医疗科技有限公司 | Magnetic particulate chemiluminescent micro-fluidic chip for quantitatively detecting procalcitonin |
| CN205910200U (en) * | 2016-05-13 | 2017-01-25 | 绍兴普施康生物科技有限公司 | Former quantitative determination's of calcitonin micro -fluidic disc of chemiluminescence |
-
2016
- 2016-05-13 CN CN201610323118.7A patent/CN105785054A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3799742A (en) * | 1971-12-20 | 1974-03-26 | C Coleman | Miniaturized integrated analytical test container |
| US20110201101A1 (en) * | 2006-09-05 | 2011-08-18 | Samsung Electronics Co., Ltd. | Centrifugal force-based microfluidic device for protein detection and microfluidic system including the same |
| CN102472739A (en) * | 2010-01-29 | 2012-05-23 | 三星电子株式会社 | Centrifugal micro-fluidic device and method for detecting analytes from liquid specimen |
| US20130034912A1 (en) * | 2011-08-03 | 2013-02-07 | Unist Academy-Industry Research Corporation | Centrifugal force-based microfluidic device available for reliability verification, and analyzing method using the same |
| CN103223357A (en) * | 2012-01-26 | 2013-07-31 | 三星电子株式会社 | Microfluidic device and control method thereof |
| CN103323605A (en) * | 2013-06-18 | 2013-09-25 | 杭州普施康生物科技有限公司 | Micro-fluidic chip for glycosylated hemoglobin immunodetection |
| CN104597266A (en) * | 2013-10-31 | 2015-05-06 | 逢甲大学 | Centrifugal-type detection platform and operating process thereof |
| CN105203775A (en) * | 2015-10-26 | 2015-12-30 | 深圳华迈兴微医疗科技有限公司 | Magnetic particulate chemiluminescent micro-fluidic chip for quantitatively detecting procalcitonin |
| CN205910200U (en) * | 2016-05-13 | 2017-01-25 | 绍兴普施康生物科技有限公司 | Former quantitative determination's of calcitonin micro -fluidic disc of chemiluminescence |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108732339A (en) * | 2017-04-19 | 2018-11-02 | 光宝电子(广州)有限公司 | Runner design and its detection method for multiple reaction biological detection |
| CN108732339B (en) * | 2017-04-19 | 2021-04-13 | 光宝电子(广州)有限公司 | Flow channel device for multiple reaction biological detection and detection method thereof |
| CN107655878A (en) * | 2017-09-01 | 2018-02-02 | 北京华科泰生物技术有限公司 | For the micro-fluidic chemiluminescence detection system for the magnetic particle for detecting thyroid function |
| CN107655878B (en) * | 2017-09-01 | 2019-03-15 | 北京华科泰生物技术股份有限公司 | For detecting the micro-fluidic chemiluminescence detection system of the magnetic particle of thyroid function |
| CN108982824A (en) * | 2018-05-11 | 2018-12-11 | 石家庄禾柏生物技术股份有限公司 | A kind of reagent disc test device |
| CN108982824B (en) * | 2018-05-11 | 2023-11-10 | 石家庄禾柏生物技术股份有限公司 | Reagent disk testing device |
| CN109374884A (en) * | 2018-12-24 | 2019-02-22 | 四川沃文特生物技术有限公司 | A kind of PCT concentration detection kit and preparation method thereof |
| CN109374884B (en) * | 2018-12-24 | 2021-10-22 | 四川沃文特生物技术有限公司 | PCT concentration detection kit and preparation method thereof |
| CN110508335A (en) * | 2019-03-27 | 2019-11-29 | 广州万孚生物技术股份有限公司 | Micro-fluidic chip and vitro detection device containing the micro-fluidic chip |
| CN112147199A (en) * | 2020-08-31 | 2020-12-29 | 济南大学 | Based on CoS2/MoS2Preparation of microfluidic electrochemical sensor of-Pd |
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