CN101034064A - Micro-fluidic chip and application thereof - Google Patents
Micro-fluidic chip and application thereof Download PDFInfo
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- CN101034064A CN101034064A CN 200610058759 CN200610058759A CN101034064A CN 101034064 A CN101034064 A CN 101034064A CN 200610058759 CN200610058759 CN 200610058759 CN 200610058759 A CN200610058759 A CN 200610058759A CN 101034064 A CN101034064 A CN 101034064A
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Abstract
This invention belong to capillary electrophoresis chip and quickly chemiluminescence on-line analytic technique, refer miniflow control chip and on-line chemiluminescence system, and integrate them for oneness to use in clinical multifold tumor totem timing analysis and tumor early diagnosis. Substrate and cover plate constite chip. substrate set up separating damping fluid pool (1), sample waste liquor pool (2), sample pool (3), separating waste liquor pool(4), blazing substrate Sample injection pool(5), detection window(6); separating channel(8) from sample pool to sample waste liquor pool;meeting point of Sample injection passage and separating channel is miniflow control chip decussation mouth(10);detection window lay on separating channel that between decussation mouth and separating waste liquor pool; double end of blazing substrate Sample injection passage connect with detection window and blazing substrate Sample injection pool;each pool be furnished with pole, and these pole through severalty corresponding pole to connect with high-voltage power supply.
Description
Technical field
The invention belongs to the analytical technology of capillary electrophoresis chip and the luminous coupling of rapid chemical, particularly micro-fluidic chip and online chemical luminous system, and it is become one be used for the early diagnosis of clinical kinds of tumors mark Synchronization Analysis and tumour.
Background technology
Immunoassay is to utilize antigen/antibody to react to measure a kind of high sensitivity of trace materials, the method for high selectivity, and it provides powerful measure for the clinical immunoassay of blood serum tumor markers.Usually the method for immunity of blood serum tumor markers adopts " double antibody sandwich method " mostly, as radiommunoassay, enzyme linked immunological, time-resolved fluorescence method, chemiluminescence and electrochemiluminescence method.Characteristics such as radio immunoassay disturbs and lacks because its mark is simple, highly sensitive, and range of application is wide were once becoming the most frequently used method in the clinical immunology check.But it need carry out hot operation, human body is constituted harm, limitation such as the reagent life-span is also limited, impel people constantly to explore nonradioactive labeling's immune analysis method, so a series of nonradioactive labelings' such as EIA enzyme immunoassay, fluorescence time resolution immunoassay, chemiluminescence immune assay and electrochemical immunoanalytical new method is developed in succession.These methods have greatly promoted robotization, the intellectuality and networked of weeding out the old and bring forth the new of clinical immune detection project and detection means.Yet these methods need adopt spectacle case by plate (pipe), and testing process needs the incubation of long period, repeatedly washes the detection of plate and instrumentation, and sense cycle is long, complex operation step, cost height.Although occurred automatic lmunoassays analyzer, but test item is limited, the instrument volume is bigger, and costing an arm and a leg of instrument and reagent is difficult to be extensive use of, thereby is being restricted aspect early diagnosis, prognosis monitoring and the extensive examination of malignant tumour.The step that how to simplify the operation shortens analysis time, reduces and detects cost, and the miniaturization of instrument and portability improve the accuracy and the sensitivity that detect, develops novel detecting instrument and will be very necessary with significant.The A.Manz of Switzerland has proposed micro-total analysis system in nineteen ninety, and (Micro TotalAnalysis Systems, the notion of μ-TAS) is for the solution of the problems referred to above provides a kind of new thought.
Micro-total analysis system is a frontier interdisciplinary, and it utilizes combining of micro electronmechanical processing (MEMS) technology and biotechnology, and the function of assay laboratory is integrated on the chip.Therefore, micro-total analysis system is also referred to as " chip lab ".According to chip structure and principle of work, micro-total analysis system can be divided into two big classes: micro-array chip (Microarray chip) and micro-fluidic chip (Microfluidicchip).The micro-array chip technology, it is a kind of passive type chip technology, it is the surface with the probe stationary chip of testing sample, form array structure, but it is also relative longer on general higher and detection time while that it detects cost, have nonspecific interference, false positive and false negative appear in testing result easily.Micro-fluidic chip is a kind of active chip technology as second generation chip technology, has solved the above shortcoming of array chip, has also inherited its high-throughout characteristics (micro-fluidic chip also can be made array format, forms array electrophoresis) simultaneously.Capillary array electrophoresis has been brought into play enormous function in the order-checking of human genome.Along with the arrival in proteomics epoch, the micro-fluidic chip technology will be brought into play bigger effect.With respect to traditional immunoassay, have following advantage based on the immunoassay system of micro-fluidic chip: (1) consumption sample amount is few; (2) simple to operate, step is simplified greatly; (3) can analyze a plurality of samples simultaneously; (4) sensitivity is higher.
Summary of the invention
Purpose one of the present invention is to provide a kind of micro-fluidic chip.
The micro-fluidic chip that a further object of the present invention provides purpose one is used for the early diagnosis of clinical kinds of tumors mark Synchronization Analysis and tumour, use this micro-fluidic chip can carry out the quick separated in synchronization and the detection of kinds of tumors mark, simple to operate, whole detection is with low cost.
Micro-fluidic chip of the present invention is that integrated separation and online chemiluminescence detection are one, form (as shown in Figure 1) by substrate and cover plate, on substrate, be provided with separation buffer liquid pool 1, sample waste liquid pool 2, testing sample pond 3, separate waste liquid pool 4, luminous substrate sample inlet pool 5, detection window 6; Be connected by microchannel between described each pond; Wherein microchannel is divided into testing sample sample intake passage 7, split tunnel 8, luminous substrate sample intake passage 9;
Described split tunnel 8 is simple straight channel, by testing sample pond 3 to separating waste liquid pool 4; The two ends of the sample intake passage 7 of described testing sample are respectively split tunnel 8 and testing sample pond 3, and testing sample sample intake passage 7 is the cross junction 10 of micro-fluidic chip with the joint of split tunnel 8; Detection window 6 is on the cross junction 10 of micro-fluidic chip and the split tunnel 8 that separates between the waste liquid pool 4; The two ends of described luminous substrate sample intake passage 9 are the split tunnel 8 and the luminous substrate sample inlet pool 5 at joint detection window 6 places respectively.The inside of described separation buffer liquid pool 1, sample waste liquid pool 2, testing sample pond 3, separation waste liquid pool 4, luminous substrate sample inlet pool 5 is separately installed with electrode, and these electrodes link to each other with high-voltage power supply by each self-corresponding electrode respectively.
The sample intake passage 9 of described luminous substrate is connected with split tunnel is vertical, is easy to form sample and substrate and effectively mixes and reaction.
The manufacturing materials of described micro-fluidic chip is selected from one or more any multipolymer in transparent superpolymer, polystyrene, Polyvinylchloride, phenolics, epoxy resin, the polyurethane such as (substrate and cover plate) glass, polymethylmethacrylate, dimethyl silicone polymer.
The type of drive of all passages on the micro-fluidic chip of the present invention all is the electric osmose input mode, and form is simple, unified, and it is integrated to be easy to high density.
The capillary array electrophoresis that forms with online chemiluminescence detection based on above single channel electrophoretic separation separates and the chip system of the cold CCD scanning imagery of chemiluminescence can be carried out high-throughout detection.
Micro-fluidic chip of the present invention is used for the early diagnosis of clinical kinds of tumors mark Synchronization Analysis and tumour, uses this micro-fluidic chip can carry out the quick separated in synchronization and the detection of kinds of tumors mark.
The present invention adopts efficiently fast that luminous substrate is common reagent luminol or APS-5 (U.S. Lumigen company), and wherein the structure of APS-5 is as follows:
Their corresponding respectively enzyme labeling thing horseradish peroxidase (HRP) and alkaline phosphatases (ALP) commonly used are used for the early diagnosis of clinical tumor; The corresponding horseradish peroxidase of luminol wherein, the corresponding alkaline phosphatase detection system of APS-5.
Being used for immunoassay of the present invention uses the specific antibody of micro-fluidic chip can be anti-protein antibodies, anti-nucleic acid antibody or antihormones small molecular antibody.
The present invention has simple chip structure, makes simply, and the separation efficiency height, the detection sensitivity height, low being easy to of early diagnosis analytic system cost that this micro-fluidic chip is used for clinical tumor promoted.
Description of drawings
Fig. 1. the structural representation of the micro-fluidic chip of the embodiment of the invention 1.
Fig. 2. the AFP of the embodiment of the invention 1 and AFP-mAb-ALP chip electrophoresis separating spectrum.
Fig. 3. the alpha-fetoprotein chip electrophoresis separating spectrum of the embodiment of the invention 1.
Fig. 4. the working curve that the peak height according to AFP-mAb-ALP separation detection peak of the embodiment of the invention 1 obtains.
Fig. 5. the carcinomebryonic antigen chip electrophoresis separating spectrum of the embodiment of the invention 2.
Fig. 6. the working curve that the peak height according to CEA-mAb-ALP separation detection peak of the embodiment of the invention 2 obtains.
Fig. 7. the serum chip electrophoresis separating spectrum that contains alpha-fetoprotein and carcinomebryonic antigen of the embodiment of the invention 3.
Reference numeral
1. separation buffer liquid pool 2. sample waste liquid pools 3. testing sample ponds
4. separate waste liquid pool 5. luminous substrate sample inlet pools 6. detection windows
7. testing sample sample intake passage 8. split tunnels 9. luminous substrate sample intake passages
10. the cross junction of micro-fluidic chip
Embodiment
Embodiment 1:
Chip design and making
(1) makes micro-fluidic chip by structural design shown in Figure 1.
On glass substrate, use diamond head to get the separation buffer liquid pool 1 of 1～3mm, sample waste liquid pool 2, testing sample pond 3 respectively, separate waste liquid pool 4, luminous substrate sample inlet pool 5; By wide 100 μ m, the microchannel of dark 30 μ m (the rotten liquid BOE solution of carving of faintly acid glass, wet etching obtained in 1 hour) is connected between each pond; Wherein testing sample pond 3 is to the straight-through split tunnel 8 long 4cm of being that separate waste liquid pool 4; Sample intake passage 7 by testing sample between testing sample pond 3 and the split tunnel 8 is connected, and the joint that testing sample sample intake passage 7 communicates with split tunnel 8 is the cross junction 10 of micro-fluidic chip; One detection window 6 is on the cross junction 10 of micro-fluidic chip and the split tunnel 8 that separates between the waste liquid pool 4; Between the split tunnel 8 at detection window 6 places and luminous substrate sample inlet pool 5, be connected by luminous substrate sample intake passage 9.
The inside of described separation buffer liquid pool 1, sample waste liquid pool 2, testing sample pond 3, separation waste liquid pool 4, luminous substrate sample inlet pool 5 is separately installed with electrode, and these electrodes link to each other with high-voltage power supply by each self-corresponding electrode respectively.Cover plate with a micro-fluidic chip covers on the substrate then.
(2) the tumor markers alpha-fetoprotein (AFP) with liver cancer is an example, and this albumen is strand glycoprotein, the about 65～70KD of molecular weight.The serum and the mAb-ALP that at first will contain AFP react (37 ℃ of incubation 30min) afterwards, get its reacted solution and separate.Select the borax soln of 3.75mM for use, 20mMNaCl, 0.01%Tween 20 (pH=9.68) is as the buffer solution system that separates.The sample introduction electric field is 300V/cm, and the separation electric field is 400V/cm, and the sample introduction electric field of chemical luminous substrate is 400V/cm.Employing is based on the input mode of " time ": at first testing sample is put into testing sample pond 3, in order dissociating buffer put into separation buffer liquid pool 1, sample waste liquid pool 2 then, separated waste liquid pool 4; Because the microchannel size is all in the micron order scope, solution in each sample cell can enter under the effect of REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power automatically in the microchannel that links to each other separately and (enter in the microchannel if accelerate solution, to be full of pairing solution except that each solution pool separating waste liquid pool earlier, on the separation waste liquid pool, add negative pressure, make the solution in each solution pool enter institute's communication channel, after arriving in the separation waste liquid pool under the effect of microchannel solution in negative pressure, will separate waste liquid pool at last and be full of dissociating buffer).Open testing sample pond electrode and separate the waste liquid pool electrode, sample waste liquid pool electrode and separation buffer liquid pool electrode are suspended, sample introduction 5 seconds, testing sample enter the cross junction 10 of chip through sample intake passage 7 from testing sample pond 3; Open separation buffer liquid pool electrode, luminous substrate sample inlet pool electrode then and separate the waste liquid pool electrode, testing sample pond electrode and sample waste liquid pool electrode are suspended, testing sample enters detection window 6 and detects through split tunnel 8 electrophoretic separation; Sample area band after testing finally enters the separation waste liquid pool under electricity drives.Obtain the chip electrophoresis collection of illustrative plates as shown in Figure 2.A is mAb-ALP separation detection peak, and B is the separation detection peak of AFP-mAb-ALP immune complex.The separation of AFP tumor markers is finished in 3 minutes with detection.Keeping changing the concentration (2.09mIU/L of AFP under the constant situation of other condition; 6.14mIU/L; 19.72mIU/L; 60.21mIU/L; 200.81mIU/L), carry out separation detection and can obtain as shown in Figure 3 separating spectrum, obtain working curve (coefficient R=0.993) according to the peak height at AFP-mAb-ALP separation detection peak as Fig. 4.Detection sensitivity is 0.6ng/nL.
Embodiment 2:
Chip design and making are with embodiment 1.
Mark carcinomebryonic antigen (CEA) with straight colon cancer is an example, and this tumor markers molecular weight is about 180KD.The serum and the mAb-ALP that at first will contain CEA react (37 ℃ of incubation 30min) afterwards, get its reacted solution and separate.Select the borax soln of 3.75mM for use, 20mM NaCl, 0.01%Tween 20 (pH=9.68) is as the buffer solution system that separates.The sample introduction electric field is 300V/cm, and the separation electric field is 400V/cm, and the sample introduction electric field of chemical luminous substrate is 400V/cm.Employing is based on the input mode of " time ": open testing sample pond electrode and separate the waste liquid pool electrode, sample waste liquid pool electrode and separation buffer liquid pool electrode are suspended, sample introduction 5 seconds, testing sample enter the cross junction 10 of chip through sample intake passage 7 from testing sample pond 3; Open separation buffer liquid pool electrode, luminous substrate sample inlet pool electrode then and separate the waste liquid pool electrode, testing sample pond electrode and sample waste liquid pool electrode are suspended, testing sample enters detection window 6 and detects through split tunnel 8 electrophoretic separation.Obtain the chip electrophoresis collection of illustrative plates as shown in Figure 5.The 150s separation detection to be mAb-ALP, what obtain at 250s is the separation detection peak of CEA-mAb-ALP immune complex.Concentration (the 80mIU/L of corresponding CEA; 200mIU/L; 1000mIU/L; 2000mIU/L) obtain working curve (coefficient R=0.999) as Fig. 6 according to the peak height at CEA-mAb-ALP separation detection peak.Detection sensitivity is 0.3ng/nL.
Embodiment 3:
Chip design and making are with embodiment 1.
The serum and corresponding with it the respectively mAb-ALP of AFP serum that at first will contain CEA react (37 ℃ of incubation 30min) afterwards, then two kinds of solution are mixed, and get its mixed solution and separate.Select the borax soln of 3.75mM for use, 20mM NaCl, 0.01%Tween 20 (pH=9.68) is as the buffer solution system that separates.The sample introduction electric field is 300V/cm, and the separation electric field is 400V/cm, and the sample introduction electric field of chemical luminous substrate is 400V/cm.Employing is based on the input mode of " time ": open testing sample pond electrode and separate the waste liquid pool electrode, sample waste liquid pool electrode and separation buffer liquid pool electrode are suspended, sample introduction 5 seconds, testing sample enter the cross junction 10 of chip through sample intake passage 7 from testing sample pond 3; Open separation buffer liquid pool electrode, luminous substrate sample inlet pool electrode then and separate the waste liquid pool electrode, testing sample pond electrode and sample waste liquid pool electrode are suspended, testing sample enters detection window 6 and detects through split tunnel 8 electrophoretic separation.Obtain the chip electrophoresis collection of illustrative plates as shown in Figure 7.As can be seen from the figure: the separation detection peak correspondence of 35s be the AFP labelled antibody, the separation detection peak correspondence of 110s be the immune complex of AFP, the separation detection peak correspondence of 150s be the labelled antibody of CEA, the 250s correspondence be the CEA immune complex.Peak height characterizes corresponding content, and can be found by the working curve (Fig. 4 and Fig. 5) of AFP and CEA.
Claims (5)
1. micro-fluidic chip, be that integrated separation and online chemiluminescence detection are one, the type of drive of all passages on the micro-fluidic chip all is the electric osmose input mode, it is characterized in that: described micro-fluidic chip is made up of substrate and cover plate, is provided with separation buffer liquid pool (1), sample waste liquid pool (2), testing sample pond (3), separation waste liquid pool (4), luminous substrate sample inlet pool (5), detection window (6) on substrate; Be connected by microchannel between described each pond; Wherein microchannel is divided into testing sample sample intake passage (7), split tunnel (8), luminous substrate sample intake passage (9);
Described split tunnel (8) by testing sample pond (3) to separating waste liquid pool (4); The two ends of the sample intake passage of described testing sample (7) are respectively sample waste liquid pool (2) and separation buffer liquid pool (1), and testing sample sample intake passage (7) is the cross junction (10) of micro-fluidic chip with the joint of split tunnel (8); On the cross junction (10) that detection window (6) is positioned at micro-fluidic chip and the split tunnel (8) that separates between the waste liquid pool (4); Split tunnel (8) and luminous substrate sample inlet pool (5) that the two ends difference joint detection windows (6) of described luminous substrate sample intake passage (9) are located;
The inside of described separation buffer liquid pool (1), sample waste liquid pool (2), testing sample pond (3), separation waste liquid pool (4), luminous substrate sample inlet pool (5) is separately installed with electrode, and these electrodes link to each other with high-voltage power supply by each self-corresponding electrode respectively.
2. micro-fluidic chip according to claim 1 is characterized in that: the sample intake passage of described luminous substrate (9) is connected with split tunnel is vertical.
3. micro-fluidic chip according to claim 1, it is characterized in that: the material of described micro-fluidic chip is selected from glass, polymethylmethacrylate, a kind of in the dimethyl silicone polymer, or one or more any multipolymer in the polystyrene, Polyvinylchloride, phenolics, epoxy resin, polyurethane.
4. according to the purposes of each described micro-fluidic chip of claim 1～3, it is characterized in that: described micro-fluidic chip is used for the early diagnosis of clinical kinds of tumors mark Synchronization Analysis and tumour.
5. according to the purposes of each described micro-fluidic chip of claim 1～3, it is characterized in that: described micro-fluidic chip can carry out the quick separated in synchronization and the detection of kinds of tumors mark.
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| CN101796419A (en) * | 2007-07-23 | 2010-08-04 | 数字生物科技株式会社 | Module for detecting analytes in fluids and chip having the same |
| WO2010135852A1 (en) * | 2009-05-27 | 2010-12-02 | 西门子公司 | Capillary electrophoresis chip, apparatus and method suitable for online application |
| CN101126765B (en) * | 2007-10-09 | 2011-02-16 | 中国科学院理化技术研究所 | Microfluid sample boat |
| CN101692047B (en) * | 2009-10-27 | 2011-10-05 | 浙江大学 | Microfluidic chip for capillary electrophoresis separation and chemiluminescence detection |
| CN102899246A (en) * | 2012-10-10 | 2013-01-30 | 凯晶生物科技(苏州)有限公司 | Dynamic PCR (Polymerase Chain Reaction) and CE (capillary electrophoresis) functional integrated micro-fluidic chip of microcavity |
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| CN101796419A (en) * | 2007-07-23 | 2010-08-04 | 数字生物科技株式会社 | Module for detecting analytes in fluids and chip having the same |
| CN101796419B (en) * | 2007-07-23 | 2013-06-26 | 纳诺恩科技有限公司 | Module for detecting analytes in fluids and chip having the same |
| CN101126765B (en) * | 2007-10-09 | 2011-02-16 | 中国科学院理化技术研究所 | Microfluid sample boat |
| CN101609088B (en) * | 2008-06-16 | 2013-06-12 | 索尼株式会社 | Micro-fluidic chip and flow sending method in micro-fluidic chip |
| WO2010135852A1 (en) * | 2009-05-27 | 2010-12-02 | 西门子公司 | Capillary electrophoresis chip, apparatus and method suitable for online application |
| CN101692047B (en) * | 2009-10-27 | 2011-10-05 | 浙江大学 | Microfluidic chip for capillary electrophoresis separation and chemiluminescence detection |
| CN102899246A (en) * | 2012-10-10 | 2013-01-30 | 凯晶生物科技(苏州)有限公司 | Dynamic PCR (Polymerase Chain Reaction) and CE (capillary electrophoresis) functional integrated micro-fluidic chip of microcavity |
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| CN103529195B (en) * | 2013-10-24 | 2014-08-06 | 山东大学 | Detection method applied to measurement of trace target materials |
| CN104166008A (en) * | 2014-08-11 | 2014-11-26 | 江苏大学 | Automatic synchronous sample introduction method and device for micro-fluidic chip |
| CN104166008B (en) * | 2014-08-11 | 2016-03-02 | 江苏大学 | A kind of micro-fluidic chip automatic synchronization sample injection method and device |
| CN104316680A (en) * | 2014-11-04 | 2015-01-28 | 张晓杰 | Detection device of helicobacter pylori and application method thereof |
| CN104316680B (en) * | 2014-11-04 | 2016-08-17 | 张晓杰 | The detection device of a kind of helicobacter pylori and using method thereof |
| CN104360060A (en) * | 2014-11-14 | 2015-02-18 | 国家纳米科学中心 | Detection method for specific antibodies IgM of mycoplasma pneumonia and influenza viruses based on micro-fluidic chip |
| CN104360060B (en) * | 2014-11-14 | 2016-11-02 | 国家纳米科学中心 | A kind of mycoplasma pneumoniae based on micro-fluidic chip and the detection method of influenza virus specific antibody IgM |
| CN104597232A (en) * | 2014-12-03 | 2015-05-06 | 中国科学院理化技术研究所 | Captured-antibody competition sandwich immunodetection method capable of extending detection scope and biosensor |
| CN106238109A (en) * | 2016-07-13 | 2016-12-21 | 厦门大学 | The micro-fluidic chip of a kind of methamphetamine hydrochloride in Raman detection hair and using method thereof |
| CN106238109B (en) * | 2016-07-13 | 2018-03-27 | 厦门大学 | A kind of micro-fluidic chip and its application method for being used for methamphetamine in Raman detection hair |
| CN107702973A (en) * | 2017-09-08 | 2018-02-16 | 深圳市太赫兹科技创新研究院有限公司 | A kind of whole blood blood plasma piece-rate system and method |
| CN109030608A (en) * | 2018-07-06 | 2018-12-18 | 中国科学院合肥物质科学研究院 | Zwitterion based on minor effect genes is synchronous to be detected and isolated system and method |
| CN109884305A (en) * | 2019-03-06 | 2019-06-14 | 无锡壹闪生物科技有限公司 | The luminous micro-fluidic chip of homogeneous chemistry and its detection method |
| CN111089888A (en) * | 2019-12-03 | 2020-05-01 | 广州视源电子科技股份有限公司 | Analytical equipment, and electrophoresis separation method and device based on microfluidic chip |
| CN114137196A (en) * | 2021-12-07 | 2022-03-04 | 世纪亿康（天津）医疗科技发展有限公司 | Reagent card for blood detection |
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