CN102980996B - Chemiluminescence immunoassay system, as well as method and application thereof - Google Patents
Chemiluminescence immunoassay system, as well as method and application thereof Download PDFInfo
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- CN102980996B CN102980996B CN201210593263.9A CN201210593263A CN102980996B CN 102980996 B CN102980996 B CN 102980996B CN 201210593263 A CN201210593263 A CN 201210593263A CN 102980996 B CN102980996 B CN 102980996B
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Abstract
The invention discloses a chemiluminescence immunoassay system, as well as a method and an application thereof. The chemiluminescence immunoassay system comprises a fluid control unit as well as a three-dimensional motion sample feeding platform, a temperature control unit, a magnetic field control module and an optical detecting module, wherein the three-dimensional motion sample feeding platform, the temperature control unit, the magnetic field control module and the optical detecting module are connected with each other in sequence; and the fluid control unit is connected with the temperature control unit. The chemiluminescence immunoassay system disclosed by the invention has the advantages as follows: with a capillary tube as a reactor, reagent transportation and reaction are both carried out in a water-in-oil type liquid drop by using a capillary tube liquid drop technology, so that a stable reaction condition is kept, and crossed pollution between samples and reaction suppression caused by surface adsorption are effectively avoided. The chemiluminescence immunoassay system disclosed by the invention has the advantages of small size, high assay speed, high testing flux, automatic operation and flexibility in application and is suitable for analyzing single sample, batch samples and field rapid assay; medical demands of the masses can be preferably satisfied; meanwhile, the purchasing and operation cost of equipment is obviously reduced, and excellent social and economic benefits are obtained.
Description
Technical field
The invention belongs to medicine equipment and technical field of biological, particularly a kind of chemiluminescence immune assay system and analytical approach and application.
Background technology
Chemiluminescence immune assay (chemiluminescence immunoassay, CLIA) by thering is highly sensitive chemical luminescent detecting technology, combine with the immune response of high specific, for the detection analytical technology of various antigen, haptens, antibody, hormone, enzyme, fatty acid, vitamin and medicine etc.That CLIA has is highly sensitive, high specificity, reagent are cheap, stable reagent and the term of validity (6～18 months), method are stable fast, wide, the simple to operate automaticity advantages of higher of sensing range, in fields such as medicine, clinical medicine, food inspection, life science, environment and material science, be all promoted, especially in laboratory medicine field, formed the great market of chemiluminescence immune assay.
Last century, first the mid-1970s Arakawe reported CLIA, be developed so far and become ultramicron active substance detection technique a kind of maturation, advanced, it has wide range of applications, being the immune analysis method that development is at present the most ripe and be most widely used, is also current state-of-the-art label immunoassay technology.CLIA sensitivity can be exempted from method, the high several orders of magnitude of fluorescence method than enzyme, can substitute radiommunoassay and enzyme-linked immuno assay completely.Chemiluminescence immunoassay technology is approved by numerous researchists, substitutes just gradually traditional Measurement for Biotechnique.
The chemical illumination immunity analysis instrument device overwhelming majority who uses in laboratory medicine is at present main equipment, although test flux is higher, expensive and application flexibility is limited.By contrast, microminiaturized chemiluminescence immune assay system have reagent consumption low, analyze advantage quick and easy and simple to handle, be the major transformation to existing operator scheme and equipment.Therefore compare with large-scale chemiluminescence immune assay instrument, microminiaturized chemiluminescence immune assay system price is cheaper, applies more flexibly, is very suitable for outpatient service, emergency treatment and middle-size and small-size medical institutions and uses.The exploitation of this quasi-instrument be clinical examination work in the urgent need to, can effectively reduce testing cost, the Medical need that contributes to improve medical service quality and meet better broad masses of the people.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming that overcomes prior art, with not enough, provides a kind of chemiluminescence immune assay system.This chemiluminescence immune assay system is utilized kapillary drop technology, using kapillary as reactor, reagent transportation and reaction are all carried out in water-in-oil type drop, have kept the stable of reaction conditions, have effectively avoided the cross pollution between sample and the reaction that causes because of adsorption suppresses.
Another object of the present invention is to provide the analytical approach of using above-mentioned chemiluminescence immune assay system to carry out.
A further object of the present invention is to provide the application of described chemiluminescence immune assay system.
Object of the present invention is achieved through the following technical solutions: a kind of chemiluminescence immune assay system, comprises three-dimensional motion sample introduction platform, temperature control modules, magnetic field control module, fluid control module and optical detecting module; Three-dimensional motion sample introduction platform, temperature control modules, magnetic field control module are connected successively with optical detecting module, and fluid control module is connected with temperature control modules;
Described three-dimensional motion sample introduction platform is comprised of sample/reagent bearing tray, displacement platform, leading screw and drive motor; Sample/reagent bearing tray is arranged at the top of displacement platform, and displacement platform is arranged on leading screw, and drive motor is arranged at the top of leading screw; Displacement platform can be done along leading screw three-dimensional mobile under drive motor orders about, and displacement platform and sample/reagent bearing tray can carry out three-dimensional motion under drive motor effect, and reagent or sample are transported to temperature control modules;
Described temperature control modules is provided with temperature sensor, temperature controller, relay, the cover plate connecting successively from top to bottom, heating aluminium sheet and the heating plate that contains the chimeric groove of parallel capillary pipe; Temperature sensor is arranged in heating aluminium sheet, and temperature sensor is connected with temperature controller by wire, and relay is connected with temperature sensor, temperature controller respectively; Temperature control unit is arranged at the left side of sample/reagent bearing tray; Temperature controller is converted to simulating signal in digital signal input optical detecting module, optical detecting module feeds back to temperature controller by temperature sensor signal, the switch of temperature controller pilot relay, when temperature outputs current to heating plate during lower than setting value, and stop output current during higher than setting value when temperature;
In the described chimeric groove of parallel capillary pipe, be provided with kapillary, described kapillary can be dismantled; Coordinate the movement of three-dimensional motion sample introduction platform, kapillary can be drawn sample or the reagent of placing on three-dimensional motion sample introduction platform;
Described material capillaceous is preferably plastics, glass or quartz;
Described internal diameter capillaceous is preferably 100～1000 microns, and length is preferably 10～50cm;
Described temperature sensor is preferably PT-100 temperature sensor;
Described heating aluminium sheet is preferably the heating aluminium sheet that contains 2～96 chimeric grooves of parallel capillary pipe;
Described heating plate is preferably Peltier (Peltier) heating plate;
Described fluid control module is multichannel syringe, and multichannel syringe is connected with the end of described parallel capillary pipe; Three-dimensional motion sample introduction platform is delivered to capillary sample inlet mouth by sample or reagent, coordinates multichannel syringe to realize suction or the discharge of sample or reagent;
Described fluid control module is preferably hyperchannel micro syringe;
Described magnetic field control module is arranged at the left side of temperature control modules, comprises two electromagnet that independently, stack up and down, between two electromagnet, is provided with a slit, and described kapillary is arranged in slit; By controlling input and the cut-out of electric current, two electromagnet can provide unidirectional magnetic field, two way alternate switching field and without magnetic field state; By setting electromagnet current, input, can control location, the unidirectional or bidirectional-movement of magnetic bead in kapillary, thereby realize magnetic bead location, immune compatible reaction and the reaction of magnetic immunochemiluminescence;
Described optical detecting module is arranged at the left side of electromagnet, comprises optical fiber, photoswitch and photomultiplier, and optical fiber is connected with photoswitch, and photoswitch is connected with photomultiplier by simple optical fiber, and optical fiber is corresponding vertical coupled one by one with described kapillary; Photoswitch switches by high speed, and the chemiluminescence signal sequence delivery in kapillary, to photomultiplier, is gathered by continuous signal, can monitor optical signalling in every capillary simultaneously;
Preferably, described optical detecting module comprises kapillary-optical fiber interface, input optical fibre, photoswitch, output optical fibre and the photomultiplier connecting successively;
Preferably, described temperature control modules, magnetic field control module and optical detecting module are separately fixed on bearing support;
Displacement platform and sample/reagent bearing tray can carry out three-dimensional motion along leading screw under drive motor effect, and reagent or sample are transported to capillary sample inlet mouth, coordinate multichannel syringe to realize suction or the discharge of sample or reagent; By controlling input and the cut-out of electric current, two electromagnet can provide unidirectional magnetic field, two way alternate switching field and without magnetic field state, by setting the input of electromagnet current, location, the unidirectional or bidirectional-movement of magnetic bead in kapillary be can control, thereby magnetic bead location, immune compatible reaction and the reaction of magnetic immunochemiluminescence realized; Photoswitch switches by high speed, and the chemiluminescence signal sequence delivery in kapillary, to photomultiplier, is gathered by continuous signal, can monitor optical signalling in every capillary simultaneously;
The analytical approach of using above-mentioned chemiluminescence immune assay system to carry out, comprises the following steps:
(1) in kapillary, introduce successively 5 μ L mineral oil, 2 μ L magnetic bead suspensions and 5 μ L sample to be checked, liquid section migrates to behind temperature controlled region with 0.1～5cm/s, magnetic bead suspension and sample to be checked carry out immune response, and immunoreactive condition is 20～40 ℃ of reaction 1～10min;
(2) the liquid section in step (1) completes after immune response, with 0.1～5cm/s, moves to field regions, starts the electromagnet of kapillary top, and magnetic field intensity is preferably 150mT, and magnetic bead is fixed on capillary wall;
(3) start micro syringe, discharge after the solution in kapillary, in kapillary, introduce 10 μ L cleansing solutions, 20～40 ℃ of washing magnetic beads, discharge cleansing solution, repeated washing 2 times; Meanwhile, start magnetic field control module, magnetic field intensity is preferably 150mT, with 1～100Hz frequency oscillation;
(4) in kapillary, introduce the detection antibody that is marked with horseradish peroxidase or alkaline phosphatase, hatch 1～10min for 20～40 ℃, on magnetic bead, form antibody-Ag-Ab or Ag-Ab-antibody complex; Meanwhile, start magnetic field control module, magnetic field intensity is preferably 150mT, with 1～100Hz frequency oscillation;
(5) start micro syringe, discharge after the solution in kapillary, in kapillary, introduce 10 μ L cleansing solutions, 20～40 ℃ of washing magnetic beads, discharge cleansing solution, repeated washing 2 times; Meanwhile, start magnetic field control module, magnetic field intensity is preferably 150mT, with 1～100Hz frequency oscillation;
(6) in kapillary, introduce the reactant liquor that 5 μ L contain chemical luminous substrate, after this reactant liquor contacts with magnetic bead, in the catalysis of horseradish peroxidase or alkaline phosphatase, issue biochemical luminescence-producing reaction, reaction conditions is 25～40 ℃ of reaction 0.5～20min; Meanwhile, start magnetic field control module, magnetic field intensity is preferably 150mT, with 1～100Hz frequency oscillation;
(7) the liquid section in step (6) completes after chemiluminescence reaction, with 0.1～5cm/s, migrates to detection zone; Chemiluminescence signal is by collecting fiber and transfer to photomultiplier and detect;
In step (1):
Described magnetic bead surfaces is fixed with capture antibody or capture antigen;
Described magnetic bead suspension adopts following methods to prepare: magnetic bead particles is scattered in phosphate buffer, and every milliliter of phosphate buffer contains 0.1～0.5g magnetic bead particles, stirs, and obtains magnetic bead suspension;
Preferably, every milliliter of phosphate buffer contains 0.25g magnetic bead particles;
Described phosphate buffer is preferably pH7.2,0.1mol/L Na
2hPO
4aqueous solution;
Described temperature controlled region refers to that kapillary is arranged in the region of temperature control modules;
Field regions described in step (2) refers to that kapillary is arranged in the region of magnetic field control module;
Cleansing solution described in step (3) and (5) is preferably pH7.2,0.1mol/L Na
2hPO
4aqueous solution;
In step (6):
Described chemical luminous substrate is luminol/reinforcing agent or 3-(2-spiral diamantane)-4-methoxyl-4-(3-phosphorus oxygen acyl)-phenyl-1,2-dioxane;
Described reactant liquor is luminol-hydrogen peroxide mixed liquor of volume ratio 1:1 or the 3-of volume ratio 1:1 (2-spiral diamantane)-4-methoxyl-4-(3-phosphorus oxygen acyl)-phenyl-1,2-dioxane-hydrogen peroxide mixed liquor.
Detection zone described in step (7) refers to that kapillary is arranged in the region of optical detecting module;
The described chemiluminescence immune assay system based on kapillary drop technology can be applicable in the fields such as medicine, clinical medicine, food inspection, life science, environment and material science.
Invention mechanism of the present invention is:
Chemiluminescence immune assay system based on kapillary drop technology is a kind of microminiaturized kapillary drop chemiluminescence immune assay system based on micro-total analysis system theory.This system forms water-in-oil type drop in parallel kapillary, by means of the transportation of drop and controlling of magnetic bead and complete the sequence of operations that antigen-antibody immunoassay is relevant.Specifically, this analytic system is utilized hydrophobic property capillaceous, in kapillary, successively introduce oil phase and aqueous phase solution, during the operation of liquid section, under surface tension effects, oil phase infiltrates tube wall, because water and oil phase exist relative motion, the position of water in service moves forward relatively, forms complete water-in-oil type drop; The cross-contamination issue that the formation of water-in-oil type drop has effectively been avoided evaporation and brought by adsorption.By introducing successively the drop that contains various sample and control so that external magnetic field is auxiliary in kapillary, in kapillary, complete a series of processes such as sample introduction, antigen-antibody combination, wash-out, luminescence-producing reaction and optical signalling detection.
Heating plate is fixed on heating aluminium sheet below, is inlaid with temperature sensor, in order to feed back temperature of heating plate in heating aluminium sheet.Temperature sensor is connected with temperature controller, and temperature controller is converted into digital data transmission to computing machine by simulating signal; Computer control software utilize pid algorithm (proportional/integral/derivative algorithm) according to temperature sense feedback signal to temperature controller, by temperature controller pilot relay, thereby whether regulate electrical current transports to heating plate.In kapillary, drop enters temperature controlled region, can realize fast temperature and change.
Chemiluminescence signal detects the Weak-luminescence detecting device that adopts the system integration.For realizing the dynamic monitoring of drop chemiluminescence signal in parallel capillary pipe, the present invention adopts a kind of detection method based on optical fiber-photoswitch.Every capillary is provided with optical fiber, for gathering chemiluminescence signal and being transferred to photomultiplier, detects.Photoswitch input end is connected with input optical fibre, and its output terminal is connected with output optical fibre, and output optical fibre is transferred to photomultiplier by luminous signal.During detection signal, photoswitch carries out high speed to signals collecting optical fiber and switches, and makes input end parallel signal gather optical fiber and is communicated with output terminal optical fiber successively.
The principle schematic of immunochemiluminometry is as shown in Figure 2: the first step, by mineral oil, be marked with identification antibody (or antigen) magnetic bead suspension and sample introduce successively kapillary, in motion, form water-in-oil type drop, identification antibody (or antigen) and antigen to be detected (or antibody) combination; Second step, when liquid section moves to field regions, magnetic bead is hunted down, and then gets rid of waste liquid; The 3rd step, the liquid section that just mineral oil and containing detects antibody is introduced kapillary successively, forms water-in-oil type drop in motion, detects antibody and antigen to be detected (or antibody) combination when drop moves to field regions.After completing association reaction, get rid of waste liquid; The 4th step, just mineral oil is introduced kapillary successively with the liquid section that contains cleansing solution, forms water-in-oil type drop in motion, when drop moves to field regions, will in conjunction with detecting antibody elution, then not discharge kapillary; The 5th step, just mineral oil is introduced kapillary successively with the liquid section that contains enzyme luminous substrate, in motion, form water-in-oil type drop, the enzymatic enzyme luminous substrate that detects mark on antibody when drop moves to field regions is luminous, luminescence-producing reaction finishes rear liquid section and continues to move to detector area, and in drop, luminous signal is gathered by detecting device.
The present invention has following advantage and effect with respect to prior art:
(1) immunochemiluminometry system provided by the invention has advantages of that volume is little, analysis speed fast, test flux is high and operation automation, its applying flexible, be suitable for the analysis of single sample and batch sample, be particularly suitable for the analysis of field quick detection.This system is for actual demand and design and development, there is function advantage integrated and simple and efficient to handle, the purchase and the operating cost that have significantly reduced this kind equipment, contribute to improve medical service quality, can meet better broad masses of the people's Medical need.
(2) chemiluminescence immune analysis method based on kapillary drop technology provided by the invention, reagent transportation and reaction are all carried out in water-in-oil type drop, keep the stable of reaction conditions, effectively avoided the cross pollution between sample and the reaction that causes because of adsorption suppresses.
(3) analytical approach provided by the invention is usingd standard capillary as reactor, and not only reagent consumption is little, and with low cost, effectively reduces the cost that instrument is produced and moved, and has good society and economic benefit.
Accompanying drawing explanation
Fig. 1 is the structural representation of the chemiluminescence immune assay system of embodiment 1.Wherein: the heating aluminium sheet that 1a drive motor, 1b displacement platform (horizontal component), 2 samples/reagent bearing tray, 3 bearing supports, 4 parallel capillary pipes, 5 heating plates, 6 contain the chimeric groove of kapillary, 7 cover plates, 8 temperature sensors, 9 bidirectional electromagnets, 10 hyperchannel micro syringes, 11 kapillaries-optical fiber interface, 12 input optical fibres, 13 photoswitches, 14 output optical fibres, 15 photomultipliers, 16 temperature controllers, 17 relays.
Fig. 2 is the principle schematic of immunochemiluminometry.
Fig. 3 is the optical detecting module detailed structure schematic diagram of chemiluminescence immune assay system, wherein: 4 kapillaries, 12 input optical fibres, 13 photoswitches, 14 output optical fibres, 18 photoswitch openings, 19 utilizing emitted light optical filters, 20 convex lens, 21 pin holes, 15 photomultipliers.
Fig. 4 is the temperature control principle schematic diagram of chemiluminescence immune assay system, wherein: the heating aluminium sheet that 4 kapillaries, 5 heating plates, 6 contain the chimeric groove of kapillary, 7 cover plates, 8PT-100 temperature sensor, 16 temperature controllers, 17 relays, 22 computing machines.
Fig. 5 is the blood serum sample TNF Beta of the embodiment 2 chemiluminescence detection signal in 12 passage polytetrafluoroethylene capillaries.
Fig. 6 is the blood serum sample C hepatitis virus antigen of the embodiment 3 chemiluminescence detection signal in 2 channel glass kapillaries.
Fig. 7 is the blood serum sample TNF Beta of the embodiment 4 chemiluminescence detection signal in 96 passage quartz capillaries.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1
As shown in Figure 1, a kind of chemiluminescence immune assay system, comprises three-dimensional motion sample introduction platform, temperature control modules, magnetic field control module, fluid control module and optical detecting module; Three-dimensional motion sample introduction platform, temperature control modules, magnetic field control module are connected successively with optical detecting module, and fluid control module is connected with temperature control modules;
Described three-dimensional motion sample introduction platform is comprised of sample/reagent bearing tray 2, displacement platform 1b, leading screw and drive motor 1a; Sample/reagent bearing tray 2 is arranged at the top of displacement platform 1b, displacement platform 1b motion under drive motor 1a controls;
Cover plate 7, the heating aluminium sheet 6 that contains 96 chimeric grooves of parallel capillary pipe and Peltier (Peltier) heating plate 5 that described temperature control modules is provided with PT-100 temperature sensor 8, temperature controller 16, relay 17, connects successively from top to bottom; PT-100 temperature sensor 8 is arranged in heating aluminium sheet 6, and PT-100 temperature sensor 8 is connected with temperature controller 16 by wire, and relay 17 is connected with PT-100 temperature sensor 8, temperature controller 16 respectively; Temperature control modules is arranged at the left side of sample/reagent bearing tray 2;
In the chimeric groove of kapillary, be provided with parallel capillary pipe 4; Material capillaceous is plastics, glass or quartz; Internal diameter capillaceous is 100～1000 microns, and length is 10～50cm;
Parallel capillary pipe 4 can be dismantled;
Described magnetic field control module is arranged at the left side of temperature control modules, comprises between two 9, two electromagnet of electromagnet that independently, stack up and down and is provided with a slit, and parallel capillary pipe 4 is arranged in slit;
Described fluid control module is that 96 passage micro syringe 10,96 passage micro syringes 10 are connected with the end of kapillary 4;
Described optical detecting module is arranged at the left side of electromagnet 9, comprise the input optical fibre 12, photoswitch 13, output optical fibre 14, utilizing emitted light optical filter 16, convex lens 17, pin hole 18 and the photomultiplier 15 that connect successively, input optical fibre 12 is connected with output optical fibre 15 by opening 14; Input optical fibre 12 kapillaries 4 are corresponding vertical coupled one by one;
Described temperature control modules, magnetic field control module and optical detecting module are separately fixed on bearing support 3;
Displacement platform and sample/reagent bearing tray can carry out three-dimensional motion along leading screw under drive motor effect, and reagent or sample are transported to capillary sample inlet mouth, coordinate multichannel syringe to realize suction or the discharge of sample or reagent; By controlling input and the cut-out of electric current, two electromagnet can provide unidirectional magnetic field, two way alternate switching field and without magnetic field state, by setting the input of electromagnet current, location, the unidirectional or bidirectional-movement of magnetic bead in kapillary be can control, thereby magnetic bead location, immune compatible reaction and the reaction of magnetic immunochemiluminescence realized; Photoswitch switches by high speed, and the chemiluminescence signal sequence delivery in kapillary, to photomultiplier, is gathered by continuous signal, can monitor optical signalling in every capillary simultaneously.
Embodiment 2
The analytical approach that the chemiluminescence immune assay system of utilization embodiment 1 is carried out, comprises the following steps:
(1) in 12 parallel polytetrafluoroethylene capillaries, introduce 5 μ L mineral oil, 2 μ L magnetic bead suspensions and 5 μ L blood serum sample to be checked successively; Capillary pipe length 10cm used, 1000 microns of internal diameters; Magnetic bead surfaces used is fixed with anti-tumor necrosis factor Beta antibody (anti-TNF Beta, mouse-anti people), and magnetic bead particles is dispersed in phosphate buffer, and quality percent by volume is 0.25g/mL; Control liquid section and migrate to temperature controlled region with 1cm/s speed, 37 ℃ keep 2 minutes;
(2) when drop moves to field regions, start kapillary top electromagnet (magnetic field intensity 150mT), magnetic bead is fixed on to capillary wall;
(3) control micro syringe effluent discharge; (composition is 0.1mol/L Na toward introducing cleansing solution in kapillary to control micro syringe
2hPO
4, pH7.2), 10 μ L, 37 ℃ of washing magnetic beads 2 times; This process starts magnetic field control module, and magnetic field intensity is 150mT, with 10Hz frequency oscillation;
(4) in kapillary, introduce the detection antibody (sheep anti mouse) that is marked with horseradish peroxidase, hatch 2 minutes, on magnetic bead, form antibody-antigen-antibody complex for 37 ℃; This process starts magnetic field control module, and magnetic field intensity is 150mT, with 10Hz frequency oscillation;
(5) toward introducing cleansing solution in kapillary, (composition is 0.1mol/L Na again
2hPO
4, pH7.2), 10 μ L, 37 ℃ of washing magnetic beads 2 times; This process starts magnetic field control module, and magnetic field intensity is 150mT, with 10Hz frequency oscillation;
(6) in kapillary, introduce reactant liquor (1:1 luminol/reinforcing agent-superoxol) the 5 μ L that contain chemical luminous substrate, after it contacts with magnetic bead, catalysis at horseradish peroxidase issues biochemical luminescence-producing reaction, and reaction conditions is 37 ℃, and the reaction time is 2 minutes; This process starts magnetic field control module, and magnetic field intensity is 150mT, with 10Hz frequency oscillation;
(7) controlling liquid section moves to detection zone with 1cm/s speed; Chemiluminescence signal is by the collecting fiber of kapillary below and transfer to photomultiplier and detect;
Testing result as shown in Figure 5, when reactant liquor section arrives detection zone, can detect the chemiluminescence signal in 12 capillaries simultaneously.
Embodiment 3
The analytical approach that the chemiluminescence immune assay system of utilization embodiment 1 is carried out, comprises the following steps:
(1) in 2 parallel glass kapillaries, introduce 5 μ L mineral oil, 2 μ L magnetic bead suspensions and 5 μ L blood serum sample to be checked successively; Capillary pipe length 50cm used, 500 microns of internal diameters; Magnetic bead surfaces used is fixed with C hepatitis virus antigen, and magnetic bead particles is dispersed in phosphate buffer, and quality percent by volume is 0.1g/mL; Control liquid section and migrate to temperature controlled region with 1cm/s speed, keep 10 minutes at 20 ℃;
(2) when drop moves to field regions, start kapillary top electromagnet (magnetic field intensity 150mT), magnetic bead is fixed on to capillary wall;
(3) control micro syringe effluent discharge; (composition is 0.1mol/L Na toward introducing cleansing solution in kapillary to control micro syringe
2hPO
4, pH7.2), 10 μ L, 37 ℃ of washing magnetic beads 2 times; This process starts magnetic field control module, and magnetic field intensity is 150mT, with 1Hz frequency oscillation;
(4) in kapillary, introduce the detection antibody (goat-anti people) that is marked with alkaline phosphatase, hatch 2 minutes, on magnetic bead, form Ag-Ab-antibody complex for 37 ℃; This process starts magnetic field control module, and magnetic field intensity is 150mT, with 100Hz frequency oscillation;
(5) toward introducing cleansing solution in kapillary, (composition is 0.1mol/L Na again
2hPO
4, pH7.2), 10 μ L, 37 ℃ of washing magnetic beads 2 times; This process starts magnetic field control module, and magnetic field intensity is 150mT, with 1Hz frequency oscillation;
(6) in kapillary, introduce reactant liquor (3-(2-spiral diamantane)-4-methoxyl-4-(3-phosphorus oxygen acyl)-phenyl-1 of containing chemical luminous substrate, 2-dioxane, AMPPD) 5 μ L, after it contacts with magnetic bead, catalysis at alkaline phosphatase issues biochemical luminescence-producing reaction, reaction conditions is 25 ℃, and the reaction time is 20 minutes; This process starts magnetic field control module, and magnetic field intensity is 150mT, with 100Hz frequency oscillation;
(7) controlling liquid section moves to detection zone with 0.1cm/s speed; Chemiluminescence signal is by the collecting fiber of kapillary below and transfer to photomultiplier and detect;
Testing result as shown in Figure 6, when reactant liquor section arrives detection zone, can detect the chemiluminescence signal in 2 capillaries simultaneously.
Embodiment 4
The analytical approach that the chemiluminescence immune assay system of utilization embodiment 1 is carried out, comprises the following steps:
(1) in 96 parallel quartz capillaries, introduce 5 μ L mineral oil, 2 μ L magnetic bead suspensions and 5 μ L blood serum sample to be checked successively; Capillary pipe length 25cm used, 100 microns of internal diameters; Magnetic bead surfaces used is fixed with anti-TNF Beta antibody, mouse-anti people, and magnetic bead particles is dispersed in phosphate buffer, and quality percent by volume is 0.5g/mL; Control liquid section and migrate to temperature controlled region with 1cm/s speed, 40 ℃ keep 1 minute;
(2) when drop moves to field regions, start kapillary top electromagnet (magnetic field intensity 150mT), magnetic bead is fixed on to capillary wall;
(3) control micro syringe effluent discharge; (composition is 0.1mol/L Na toward introducing cleansing solution in kapillary to control micro syringe
2hPO
4, pH7.2), 10 μ L, 37 ℃ of washing magnetic beads 2 times; This process starts magnetic field control module, and magnetic field intensity is 150mT, with 1Hz frequency oscillation;
(4) in kapillary, introduce the detection antibody (anti-human TNF Beta, sheep anti mouse) that is marked with horseradish peroxidase, hatch 2 minutes, on magnetic bead, form antibody-antigen-antibody complex for 37 ℃; This process starts magnetic field control module, and magnetic field intensity is 150mT, with 1Hz frequency oscillation;
(5) toward introducing cleansing solution in kapillary, (composition is 0.1mol/L Na again
2hPO
4, pH7.2), 10 μ L, 37 ℃ of washing magnetic beads 2 times; This process starts magnetic field control module, and magnetic field intensity is 150mT, with 1Hz frequency oscillation;
(6) in kapillary, introduce reactant liquor (1:1 luminol/reinforcing agent-superoxol) the 5 μ L that contain chemical luminous substrate, after it contacts with magnetic bead, catalysis at horseradish peroxidase issues biochemical luminescence-producing reaction, and reaction conditions is 40 ℃, and the reaction time is 30 seconds; This process starts magnetic field control module, and magnetic field intensity is 150mT, with 1Hz frequency oscillation;
(7) controlling liquid section moves to detection zone with 5cm/s speed; Chemiluminescence signal is by the collecting fiber of kapillary below and transfer to photomultiplier and detect;
Testing result as shown in Figure 7, when reactant liquor section arrives detection zone, can detect the chemiluminescence signal in 96 capillaries simultaneously.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (10)
1. a chemiluminescence immune assay system, is characterized in that comprising three-dimensional motion sample introduction platform, temperature control modules, magnetic field control module, fluid control module and optical detecting module; Three-dimensional motion sample introduction platform, temperature control modules, magnetic field control module are connected successively with optical detecting module, and fluid control module is connected with temperature control modules;
Described three-dimensional motion sample introduction platform is comprised of sample/reagent bearing tray, displacement platform, leading screw and drive motor; Sample/reagent bearing tray is arranged at the top of displacement platform, and displacement platform is arranged on leading screw, and drive motor is arranged at the top of leading screw;
Described temperature control modules is provided with temperature sensor, temperature controller, relay, the cover plate connecting successively from top to bottom, heating aluminium sheet and the heating plate that contains the chimeric groove of parallel capillary pipe; Temperature sensor is arranged in heating aluminium sheet, and temperature sensor is connected with temperature controller by wire, and relay is connected with temperature sensor, temperature controller respectively; Temperature control modules is arranged at the left side of sample/reagent bearing tray; In the chimeric groove of described kapillary, be provided with kapillary;
Described fluid control module is multichannel syringe, and multichannel syringe is connected with described end capillaceous;
Described magnetic field control module is arranged at the left side of temperature control modules, comprises two electromagnet that independently, stack up and down, between two electromagnet, is provided with a slit, and described kapillary is arranged in slit;
Described optical detecting module is arranged at the left side of electromagnet, comprises optical fiber, photoswitch and photomultiplier, and optical fiber is connected with photoswitch, and photoswitch is connected with photomultiplier by simple optical fiber, and optical fiber is corresponding vertical coupled one by one with described kapillary.
2. chemiluminescence immune assay system according to claim 1, is characterized in that: described material capillaceous is plastics, glass or quartz.
3. chemiluminescence immune assay system according to claim 1, is characterized in that: described internal diameter capillaceous is 100～1000 microns, and length is 10～50cm.
4. chemiluminescence immune assay system according to claim 1, is characterized in that: described heating aluminium sheet is the heating aluminium sheet that contains 2～96 chimeric grooves of parallel capillary pipe.
5. chemiluminescence immune assay system according to claim 1, is characterized in that: described optical detecting module comprises kapillary-optical fiber interface, input optical fibre, photoswitch, output optical fibre and the photomultiplier connecting successively.
6. chemiluminescence immune assay system according to claim 1, is characterized in that: described fluid control module is hyperchannel micro syringe.
7. chemiluminescence immune assay system according to claim 1, is characterized in that: described temperature control modules, magnetic field control module and optical detecting module are separately fixed on bearing support.
8. the analytical approach that the chemiluminescence immune assay system described in utilization claim 1～7 any one is carried out, is characterized in that comprising the following steps:
(1) in kapillary, introduce successively 5 μ L mineral oil, 2 μ L magnetic bead suspensions and 5 μ L sample to be checked, liquid section migrates to behind temperature controlled region with 0.1～5cm/s, magnetic bead suspension and sample to be checked carry out immune response, and immunoreactive condition is 20～40 ℃ of reaction 1～10min;
(2) the liquid section in step (1) completes after immune response, with 0.1～5cm/s, moves to field regions, starts the electromagnet of kapillary top, and magnetic field intensity is 150mT, and magnetic bead is fixed on capillary wall;
(3) start micro syringe, discharge after the solution in kapillary, in kapillary, introduce 10 μ L cleansing solutions, 20～40 ℃ of washing magnetic beads, discharge cleansing solution, repeated washing 2 times; Meanwhile, start magnetic field control module, magnetic field intensity is 150mT, with 1～100Hz frequency oscillation;
(4) in kapillary, introduce the detection antibody that is marked with horseradish peroxidase or alkaline phosphatase, hatch 1～10min for 20～40 ℃, on magnetic bead, form antibody-Ag-Ab or Ag-Ab-antibody complex; Meanwhile, start magnetic field control module, magnetic field intensity is 150mT, with 1～100Hz frequency oscillation;
(5) start micro syringe, discharge after the solution in kapillary, in kapillary, introduce 10 μ L cleansing solutions, 20～40 ℃ of washing magnetic beads, discharge cleansing solution, repeated washing 2 times; Meanwhile, start magnetic field control module, magnetic field intensity is 150mT, with 1～100Hz frequency oscillation;
(6) in kapillary, introduce the reactant liquor that 5 μ L contain chemical luminous substrate, after this reactant liquor contacts with magnetic bead, in the catalysis of horseradish peroxidase or alkaline phosphatase, issue biochemical luminescence-producing reaction, reaction conditions is 25～40 ℃ of reaction 0.5～20min; Meanwhile, start magnetic field control module, magnetic field intensity is 150mT, with 1～100Hz frequency oscillation;
(7) the liquid section in step (6) completes after chemiluminescence reaction, with 0.1～5cm/s, migrates to detection zone; Chemiluminescence signal is by collecting fiber and transfer to photomultiplier and detect.
9. the analytical approach that chemiluminescence immune assay system according to claim 8 is carried out, is characterized in that:
In step (1):
Described magnetic bead surfaces is fixed with capture antibody or capture antigen;
Described magnetic bead suspension adopts following methods to prepare: magnetic bead particles is scattered in phosphate buffer, and every milliliter of phosphate buffer contains 0.1～0.5g magnetic bead particles, stirs, and obtains magnetic bead suspension;
Cleansing solution described in step (3) and (5) is pH7.2,0.1mol/L Na
2hPO
4aqueous solution;
In step (6):
Described chemical luminous substrate is luminol/reinforcing agent or 3-(2-spiral diamantane)-4-methoxyl-4-(3-phosphorus oxygen acyl)-phenyl-1,2-dioxane;
Described reactant liquor is luminol-hydrogen peroxide mixed liquor of volume ratio 1:1 or the 3-of volume ratio 1:1 (2-spiral diamantane)-4-methoxyl-4-(3-phosphorus oxygen acyl)-phenyl-1,2-dioxane-hydrogen peroxide mixed liquor.
10. the application of the chemiluminescence immune assay system described in claim 1～7 any one, is characterized in that: described chemiluminescence immune assay system is applied in medicine, clinical medicine, food inspection, life science, environment or material science.
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