CN205910200U - Former quantitative determination's of calcitonin micro -fluidic disc of chemiluminescence - Google Patents
Former quantitative determination's of calcitonin micro -fluidic disc of chemiluminescence Download PDFInfo
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- CN205910200U CN205910200U CN201620438468.3U CN201620438468U CN205910200U CN 205910200 U CN205910200 U CN 205910200U CN 201620438468 U CN201620438468 U CN 201620438468U CN 205910200 U CN205910200 U CN 205910200U
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Abstract
The utility model discloses a former quantitative determination's of calcitonin micro -fluidic disc of chemiluminescence, including micro -fluidic disc, micro -fluidic disc on install miniflow detecting element, miniflow detecting element include that magnetic particle solution that the whole blood pours into groove, whole blood separate channel, blood cell hold up tank, plasma transport stream way, mixture / listen groove, the former monoclonal antibody peridium of calcitonin into pours into groove, washing liquid into and pours into groove, external magnet region that includes magnet, waste liquid groove, alkaline phosphatase -labeled's the former monoclonal antibody solution of calcitonin injection groove, luminescent substrate solution into and pour into the groove into. The utility model discloses can realize whole blood separation, plasma ration, cultivate mix, abluent function, can realize the quantitative determination of PCT concentration in the sample having the detectivity height fast, the result is accurate reliable, the characteristics of good reproducibility.
Description
Technical field
This utility model is related to a kind of micro-fluidic disc of chemiluminescence of Procalcitonin. detection by quantitative, belongs to medical immunology body
Outer diagnostic field, can realize the detection by quantitative to Procalcitonin. in biological sample within a very short time, have simple to operate, spirit
Sensitivity is high, the feature of low cost.
Background technology
Procalcitonin. (pct) is a kind of no hormonal activity glycoprotein containing 116 aminoacid, and molecular weight is about 13
Kda, is produced by thyroid-c cell under physiological conditions, can hardly be detected in the serum of healthy population.On antibacterial, true
Infectious disease such as bacterium, parasite when having whole body or/and the inflammatory reaction of central nervous system's property, the endotoxin of pathogenic bacteria can promote
The lymphocyte of thyroid-c cell, hepatic macrophages and mononuclear cell, lung and intestinal tissue and neuroendocrine cell
Produce pct in a large number in the tissue beyond thyroid, and lead to the content in its serum to raise or persistence rising.Neonatal
Pct level after the birth in 1 ~ 2 day and 1～4 day after minority major surgery operation in patients in pct can have slightly of short duration
Raise, but can reach adult normal value all in downward trend day by day and after Yu Santian.Check can be the above-mentioned state of an illness for this pct
Definite Differential Diagnosiss are provided.
Pct level reflects the active degree of Systemic bacterial inflammatory reaction, and point out size with infected organ and
Type, the species of antibacterial and degree of inflammation are related with immunoreation situation.Though the rising of pct level may alternatively appear in serious stopping
Gram, systemic inflammatory response syndrome and multiple organ dysfunction derangement syndrome patient, such as no bacterium infection or bacterium infection sexually transmitted disease (STD)
Stove exists, and its level is usually less than the patient that those have bacterial infection focus.The clinical value of pct detection is many.
To antibacterial, funguses, the etiological diagnosises of the sexy dye of parasitic infection and pyemia, the clinical practice of antibiotic, the state of an illness and pre-
Assessment afterwards is respectively provided with obvious using value, and autoimmune, allergy, virus infection and aseptic inflammation etc. are had clearly
Differential diagnosis value.
At present, the method for detection Procalcitonin. mainly has radio immunoassay, chemiluminescence immunoassay, gold colloidal
Colorimetry.Time-consuming for radio immunoassay detection, and the pollution having radioelement uses and is restricted；Gold colloidal colorimetric
Method have fast and convenient, easily observe the features such as, be a kind of quick sxemiquantitative using method；Chemiluminescence immunoassay is grasped
Make easy, high specificity, sensitivity is high, detection time is short, is widely used at present.
Magnetic particle immunoassay technology is to be carried using the Magnetic solid phases microgranule of synthesis of polymer material certain particle size size
Body, be coated with methods such as physical absorption, chemical couplings have specific affinity antibody or the various immunity such as antigen live
Property material, have that separating rate is fast, efficiency high, favorable repeatability, simple to operate, do not affect to be separated cell or other biological material
The features such as biological character of material and function, orientable motion under additional the action of a magnetic field, so that some special composition is separated,
Concentration or purification.
Micro-fluidic chip as a kind of new analysis test platform, have high flux, integrated, portable, easy to operate,
Inexpensive the advantages of, it is widely used in various fields, especially show up prominently in immunoassay field.
The biological micro-fluidic chip of immunomagnetic beadses is by magnetic granule technology, and immunoassay is integrated on micro-fluidic chip one
Kind of analysis using method, the Major Difficulties of this at present comprehensive using method show themselves in that 1) liquid is in chip internal miniflow
Dynamic Based Intelligent Control, at present frequently with method be, in chip internal, multiple Micropumps and micro-valve are set so that micro-fluidic system becomes
Obtain and more complicate；2) undercompounding of reaction system, leads to react insufficient；3) integration degree is not high, leads to non-
Specificity background is high.
Utility model content
This utility model purpose there are provided a kind of micro-fluidic disc of chemiluminescence of Procalcitonin. detection by quantitative；This reality
Separated (Sample pretreatment), blood plasma quantitation, cultivated the function of mixing, cleaning with the new whole blood that enables, can quickly realize sample
The detection by quantitative of middle pct concentration, it is high to have a detection sensitivity, result accurately and reliably, reproducible feature.
In order to achieve the above object, the technical solution of the utility model is:
A kind of micro-fluidic disc of chemiluminescence of Procalcitonin. detection by quantitative, including micro-fluidic disc, described is micro-fluidic
Miniflow detector unit is provided with disc, described miniflow detector unit includes whole blood injection groove, described whole blood injection groove leads to
Cross whole blood separation channel to be connected with blood cell accumulator tank, described blood cell accumulator tank transmits flow passage with blood plasma；Described blood plasma passes
Runner is sent to be connected with mixing/detecting groove top；Pipeline and Procalcitonin monoclonal antibody are passed through in described mixing/detecting groove top
Coated magnetic particle solution injection groove connection；Described mixing/detecting groove top is connected with cleanout fluid injection groove also by pipeline；
Described mixing/detecting groove side is provided with the external magnet region including Magnet；Described mixing/detecting trench bottom passes through pipe
Road is connected with waste liquid tank, and the pipeline of and described mixing/between detecting trench bottom and waste liquid tank is provided with valve；Described fall
The coated magnetic particle solution of the former monoclonal antibody of calcium element injects the Procalcitonin. that pipeline and alkali phosphatase enzyme mark are passed through in groove top
Monoclonal antibody solution injects the bottom connection of groove；Described cleanout fluid injection groove top is passed through pipeline and is injected with luminous substrate liquid
The bottom connection of groove.
The miniflow detector unit of installation more than 6 and the multiple for 6 on described micro-fluidic disc.
12 miniflow detector units are preferably installed on described micro-fluidic disc.
The beneficial effects of the utility model are: this utility model enables whole blood, and separately (Sample pretreatment), blood plasma are fixed
Amount, cultivation mixing, the function of cleaning, can quickly realize the detection by quantitative of pct concentration in sample, have detection sensitivity height,
Result accurately and reliably, reproducible feature.
Brief description
Fig. 1 is a kind of structural representation of the micro-fluidic disc of chemiluminescence of this utility model Procalcitonin. detection by quantitative；
Fig. 2 is the enlarged diagram of single miniflow detector unit on micro-fluidic disc in Fig. 1.
Specific embodiment
Embodiment 1: the detection by quantitative of Procalcitonin.
A kind of micro-fluidic disc of chemiluminescence of Procalcitonin. detection by quantitative of the present embodiment, as shown in Figure 1, 2, including micro-
Stream is manipulated stock quotations piece 13, and described micro-fluidic disc 13 is provided with 12 miniflow detector units, described each miniflow detector unit
Inject groove 1 including whole blood, described whole blood injection groove 1 is connected with blood cell accumulator tank 3 by whole blood separation channel 2, described blood cell
Accumulator tank 3 transmits runner 4 with blood plasma and connects；Described blood plasma transmission runner 4 is connected with mixing/detecting groove 7 top；Described is mixed
Close/detect groove 7 top to connect by pipeline and Procalcitonin monoclonal antibody coated magnetic particle solution injection groove 9；Described
Mixing/detecting groove 7 top is injected groove 10 also by pipeline and cleanout fluid and is connected；In described mixing/detecting groove 7 side is provided with
External magnet region 8 containing Magnet；Described mixing/detecting groove 7 bottom is connected with waste liquid tank 6 by pipeline, and described mixing
Close/detect, on the pipeline between groove 7 bottom and waste liquid tank 6, valve 5 is installed；Described Procalcitonin monoclonal antibody is coated
Magnetic particle solution injects the Procalcitonin monoclonal antibody solution injection groove 11 that pipeline and alkali phosphatase enzyme mark are passed through in groove 9 top
Bottom connection；Described cleanout fluid is injected groove 10 top and is connected by the bottom that pipeline injects groove 12 with luminous substrate liquid.
1. Procalcitonin. solution is prepared
1) preparation of Procalcitonin. series of calibration product: with hyclone by Procalcitonin. sterling (extra large peptide biotechnology (on
Sea) company limited) it is diluted to series of calibration product, its calibration object concentration range is 0～50 ng/ml.
2) preparation of Procalcitonin monoclonal antibody coated magnetic particle solution:
Procalcitonin monoclonal antibody (Hai Tai biotechnology (Shanghai) Co., Ltd.) is placed in bag filter, delays in pbs
Rush dialyzed overnight in liquid；With pbs buffer bnhs solution, in every mg antibody, add 15 μ l bnhs(biotin-n- hydroxyls
Succinimine ester (Shanghai Li Rui bio tech ltd) solution, mix homogeneously, room temperature reaction 30min；Good for labelling is resisted
Body is placed in bag filter, dialyzed overnight in pbs buffer.By 2.0 μm of Streptavidin magnetic particles, (Guangzhou reaches in one-tenth trade
Easily company limited) plus magnetic field, stand 5min, pour out supernatant, with pbs buffer solution for cleaning 3 times, and hanged with this buffer
Floating；Pour out supernatant；The good antibody of 1mg labelling, mix homogeneously, room temperature reaction 20min is added in every ml magnetic particle suspension；With
The tris-hcl buffer solution for cleaning of the bovine serum albumin containing 5% 3 times；With containing 0.5% bovine serum albumin and 0.02%
Proclin300(preservative, Shanghai Li Rui bio tech ltd) pbs buffer resuspended, obtain Procalcitonin. monoclonal
Antibody coated magnetic particle solution.
3) preparation of the Procalcitonin monoclonal antibody solution of alkali phosphatase enzyme mark: with pb buffer sulfo-
Smcc solution, adds 0.1ml sulfo-smcc(4- (n- maleimidomehyl) hexamethylene -1- carboxylic in every mg alkali phosphatase
Sour sulfonic group butanimide ester sodium salt, Shanghai Li Rui bio tech ltd) solution, mix homogeneously, room temperature reaction
30min, dialyses in pb buffer；With pb buffer 2- imino group thiol heptane hydrochloride saline solution, add in every mg antibody
0.1ml 2- imino group thiol heptane hydrochloride saline solution, mix homogeneously, room temperature reaction 30min, dialyses in p pb buffer；Will be upper
State the alkali phosphatase after dialysis and antibody mix homogeneously, room temperature reaction 120min；By protein purification system (akta
Purifier100) collect albumen, add 50% glycerol, obtain the Procalcitonin. antibody-solutions of alkali phosphatase enzyme mark.
4) preparation of the Chemoluminescent substrate of alkaline phosphatase enzyme catalysiss: in tris-hcl buffer add 2- amino-
2- methyl isophthalic acid-propanol, amppd(1,2- dioxy thiacyclohexane derivant, Guangzhou Da Yucheng trade Co., Ltd) and luminescence enhancer
(ap substrate reinforcing agent, Shanghai Li Rui bio tech ltd).
5) preparation of cleanout fluid: add 0.02% polysorbas20 and 15% sodium chloride 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, including
Following steps:
1) making of standard curve: (concentration is 0ng/ml, 0.1ng/ml, 0.5ng/ to take 30 μ l pct serial standards
Ml, 1ng/ml, 10ng/ml, 50ng/ml) add the Procalcitonin monoclonal antibody of the micro-fluidic disc in the present embodiment to be coated
Magnetic particle solution injection groove 9, micro-fluidic disc 13 is placed in supporting detecting instrument.Each sample replication five respectively
Secondary.Necessary instrument calculates pct concentration according to the proportionate relationship between rlu and pct concentration, automatic Fitting, takes five surveys
Allocate average, draw standard curve.
2) micro-fluidic disc 13 is put in necessary instrument, open automatic sample feeding device, can be by whole blood 150 μ l, fall calcium
Plain former monoclonal antibody coated magnetic particle solution 30 μ l, the Procalcitonin monoclonal antibody solution of alkali phosphatase enzyme mark
20 μ l are injected into whole blood injection groove 1, Procalcitonin monoclonal antibody coated magnetic particle solution injection groove 9, alkali phosphatase
In the Procalcitonin monoclonal antibody solution injection groove 11 of labelling.
3) with 4,500 rpm (acceleration a=1,500 rpm/s) manipulation disc rotates 50 seconds, can separate in whole blood
Channel 2 separated plasma and blood cell, and by coated for Procalcitonin monoclonal antibody magnetic particle solution and alkali phosphatase enzyme mark
Procalcitonin monoclonal antibody solution is sent to mixing/detecting groove 7, and Procalcitonin monoclonal antibody coated magnetic particle solution can
The attraction of the Magnet in mat external magnet region 8 remaines in mixing/detecting groove 7 and (can avoid being washed to waste liquid tank).
4) with 1,500 rpm (acceleration a=500 rpm/s) manipulation disc rotates 20 seconds, can be by blood plasma via blood
Slurry transmission runner 4 is sent to mixing/detecting groove 7, and blood cell is then retained in blood cell accumulator tank 3.
5) with 1,300 rpm (acceleration a=2,500 rpm/s) manipulation disc rotates 40 seconds, by external magnet
Magnet manipulation in region 8, can manipulate the coated magnetic particle of Procalcitonin monoclonal antibody and move in mixing/detecting groove 7, enter
And reach good mixing/cultivate effect.
6) by cleanout fluid 60 μ l be injected into cleanout fluid injection groove 10 in, with 3,600 rpm (acceleration a=3,600
Rpm/s) the micro-fluidic disc 13 of manipulation rotates 22 seconds, cleanout fluid can be sent to mixing/detecting groove 7, and cleanout fluid is replaceable mixed
Close/detect the liquid in groove 7, this liquid can be transferred into waste liquid tank 6, and the coated magnetic particle of Procalcitonin monoclonal antibody can mat
Remained in 7 in mixing/detecting groove by the attraction of the Magnet in external magnet region 8.
7) luminous substrate liquid 40 μ l is injected in luminous substrate liquid injection groove 12, with 2,700 rpm (acceleration a=
1,150 rpm/s) manipulate disc rotation 16 seconds, luminous substrate can be sent to mixing/detecting groove 7, luminous substrate is replaceable
Liquid in mixing/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 of reaction can be mixed
Close/detect groove 7 to be detected, according to the proportionate relationship between relative luminous intensity (rlu) and Procalcitonin. antigen concentration, detect
System can convert automatically, can fast report test result, thus realizing the detection by quantitative of Procalcitonin..
8) result shows: is 0.05 ng/ml using method detection sensitivity described in the utility model, detection range is
0.05～100ng/ml, interassay coefficient of variation is less than 10%, and variation within batch coefficient is less than 10%.
The present embodiment enables whole blood and separates (Sample pretreatment), blood plasma quantitation, cultivates mixing, the function of cleaning, can be soon
Speed realizes the detection by quantitative of pct concentration in sample, and it is high to have a detection sensitivity, result accurately and reliably, reproducible feature.
Embodiment 2: magnetic particle particle size screening
The particle diameter of magnetic microsphere is little, and specific surface area is big, and active group is contained on surface, therefore is coupled capacity greatly, but magnetic microsphere
Undersized be unfavorable for that Magnet is collected, oversized be unfavorable for hybrid reaction in disc, therefore carry out magnetic particle size selection.
Other experiment conditions are carried out according to below scheme referring to embodiment 1, the size of magnetic particle granule.
Particle size is selected to be 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.Magnetic size is adopted through preferred permanent magnet, fixing magnetic during detection
Ferrum height.
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,
3.0 μm of interference increase, and 5.0 μm start to reduce, and signal value is minimum, and comprehensive each 2.0 μm of signals of factor are the strongest, and interference is
Little.
Interpretation of result: when magnetic particle size is less, specific surface area is larger, the biotinylated molecular weight that surface is loaded is big, simultaneously
Can be well dispersed in solution, but the magnetic field intensity needed for magnetic microsphere to be ensured fully is collected is big.In the present embodiment
In due to the magnetic field force suffered by magnetic bead it cannot be guaranteed that it is fully collected, lead to partly effectively magnetic bead to run off in cleaning process,
Thus leading to final detected signal value not high.When magnetic grain diameter is larger, specific surface area is little, the mark rate of surface biomolecules
Relatively low, under the same terms, magnetic particle institute is magnetic field force induced big, and scattered magnetic bead can sufficiently be collected, but its by
In easily settling, lead between biomolecule, react insufficient, thus weakening luminous signal.Consider, particle diameter is 1.0
μm to 3.0 μm of magnetic microsphere effects preferably, 2.0 μm of magnetic microsphere effect in embodiment therefore of the present utility model
Best.
Claims (3)
1. a kind of Procalcitonin. detection by quantitative the micro-fluidic disc of chemiluminescence it is characterised in that: include micro-fluidic disc (13),
Miniflow detector unit is provided with described micro-fluidic disc (13), described miniflow detector unit includes whole blood injection groove (1),
Described whole blood injection groove (1) is connected with blood cell accumulator tank (3) by whole blood separation channel (2), described blood cell accumulator tank (3) and
Blood plasma transmission runner (4) connection；Described blood plasma transmission runner (4) is connected with mixing/detecting groove (7) top；Described mixing/
Detecting groove (7) top is passed through pipeline and is connected with Procalcitonin monoclonal antibody coated magnetic particle solution injection groove (9)；Described
Mixing/detecting groove (7) top is connected with cleanout fluid injection groove (10) also by pipeline；Described mixing/detecting groove (7) side
The external magnet region (8) including Magnet is installed；Pipeline is passed through with waste liquid tank (6) even in described mixing/detecting groove (7) bottom
Logical, the pipeline of and described mixing/between detecting groove (7) bottom and waste liquid tank (6) is provided with valve (5)；Described fall calcium
The coated magnetic particle solution of the former monoclonal antibody of element injects the Procalcitonin. that pipeline and alkali phosphatase enzyme mark are passed through in groove (9) top
Monoclonal antibody solution injects the bottom connection of groove (11)；Described cleanout fluid injection groove (10) top is passed through pipeline and is lighted
Substrate solution injects the bottom connection of groove (12).
2. as claimed in claim 1 a kind of Procalcitonin. detection by quantitative the micro-fluidic disc of chemiluminescence it is characterised in that: institute
The upper installation of micro-fluidic disc (13) more than 6 stated and the miniflow detector unit of the multiple for 6.
3. as claimed in claim 2 a kind of Procalcitonin. detection by quantitative the micro-fluidic disc of chemiluminescence it is characterised in that: institute
Micro-fluidic disc (13) the 12 miniflow detector units of upper installation stated.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105785054A (en) * | 2016-05-13 | 2016-07-20 | 绍兴普施康生物科技有限公司 | Chemical-luminescent microfluidic disk for quantitative detection of procalcitonin and using method thereof |
| CN107091936A (en) * | 2017-06-14 | 2017-08-25 | 绍兴普施康生物科技有限公司 | Chemiluminescence immunoassay disc and its method of work based on microflow control technique |
| CN108519373A (en) * | 2018-04-27 | 2018-09-11 | 广州万孚生物技术股份有限公司 | A kind of chemiluminescence micro-fluidic chip and the analytical instrument containing it |
-
2016
- 2016-05-13 CN CN201620438468.3U patent/CN205910200U/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105785054A (en) * | 2016-05-13 | 2016-07-20 | 绍兴普施康生物科技有限公司 | Chemical-luminescent microfluidic disk for quantitative detection of procalcitonin and using method thereof |
| CN107091936A (en) * | 2017-06-14 | 2017-08-25 | 绍兴普施康生物科技有限公司 | Chemiluminescence immunoassay disc and its method of work based on microflow control technique |
| CN107091936B (en) * | 2017-06-14 | 2018-12-25 | 绍兴普施康生物科技有限公司 | Chemiluminescence immunoassay disc and its working method based on microflow control technique |
| CN108519373A (en) * | 2018-04-27 | 2018-09-11 | 广州万孚生物技术股份有限公司 | A kind of chemiluminescence micro-fluidic chip and the analytical instrument containing it |
| CN108519373B (en) * | 2018-04-27 | 2024-03-15 | 广州万孚生物技术股份有限公司 | Chemiluminescence micro-fluidic chip and analysis instrument comprising same |
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Address after: Room 408, building C, scientific research building, No. 398, mahuan Road, Lihai Town, Binhai New City, Shaoxing City, Zhejiang Province Patentee after: Zhejiang pushkang Biotechnology Co., Ltd Address before: 312366 Zhejiang city in Shaoxing Province, the coastal city of Shaoxing Ma Huanlu No. 398 Branch Center Patentee before: SHAOXING PUSHKANG BIOTECHNOLOGY Co.,Ltd. |
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