CN102980996A - 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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- CN102980996A CN102980996A CN2012105932639A CN201210593263A CN102980996A CN 102980996 A CN102980996 A CN 102980996A CN 2012105932639 A CN2012105932639 A CN 2012105932639A CN 201210593263 A CN201210593263 A CN 201210593263A CN 102980996 A CN102980996 A CN 102980996A
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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) will have highly sensitive chemical luminescent detecting technology and combine with the immune response of high specific, be used for the detection analytical technology of various antigens, 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), stable fast, wide, the simple to operate automaticity advantages of higher of sensing range of method, all be promoted in fields such as medicine, clinical medicine, food inspection, life science, environment and material science, especially formed the great market of chemiluminescence immune assay in the laboratory medicine field.
Last century, the mid-1970s Arakawe at first reported CLIA, development has become a kind of maturation, advanced ultramicron active substance detection technique so far, it has wide range of applications, being the immune analysis method that at present development is the most ripe and be most widely used, also is present 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 fully.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 the laboratory medicine at present is main equipment, and is although the 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, uses 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 helps to improve medical service quality and satisfy better broad masses of the people.
Summary of the invention
Primary and foremost purpose of the present invention is to overcome the shortcoming of prior art with not enough, and a kind of chemiluminescence immune assay system is provided.This chemiluminescence immune assay system utilizes kapillary drop technology, with kapillary as reactor, reagent transportation and reaction are all carried out in the water-in-oil type drop, have kept the stable of reaction conditions, have effectively avoided the cross pollution between the sample and the reaction that causes because of adsorption suppresses.
Another object of the present invention is to provide the above-mentioned chemiluminescence immune assay of the utilization analytical approach that system carries out.
A further object of the present invention is to provide the application of described chemiluminescence immune assay system.
Purpose 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 with optical detecting module and are connected, and the 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 the 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 the drive motor effect, reagent or sample are transported to temperature control modules;
Described temperature control modules is provided with temperature sensor, temperature controller, relay, the cover plate that connects successively from top to bottom, contains heating aluminium sheet and the heating plate of the chimeric groove of parallel capillary pipe; Temperature sensor is arranged in the 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 in the digital signal input optical detecting module with analog signal conversion, optical detecting module feeds back to temperature controller with temperature sensor signal, the switch of temperature controller pilot relay, when temperature is lower than setting value, output current to heating plate, and when temperature is higher than setting value, stop output current;
Be provided with kapillary in the chimeric groove of described parallel capillary pipe, described kapillary can be dismantled; Cooperate the movement of three-dimensional motion sample introduction platform, kapillary can be drawn sample or the reagent of placing on the 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 the 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 the capillary sample inlet mouth with sample or reagent, cooperates multichannel syringe to realize suction or the discharge of sample or reagent;
Described fluid control module is preferably the 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, is provided with a slit between two electromagnet, and described kapillary is arranged in the slit; By input and the cut-out of control electric current, two electromagnet can provide unidirectional magnetic field, two way alternate switching field and without magnetic field state; By setting the 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 to photomultiplier, by the continuous signal collection, can be monitored optical signalling in every capillary with the chemiluminescence signal sequence delivery in the kapillary by switching at a high speed simultaneously;
Preferably, described optical detecting module comprises kapillary-optical fiber interface, input optical fibre, photoswitch, output optical fibre and the photomultiplier that connects successively;
Preferably, described temperature control modules, magnetic field control module and optical detecting module are separately fixed on the bearing support;
Displacement platform and sample/reagent bearing tray can carry out three-dimensional motion along leading screw under the drive motor effect, reagent or sample are transported to the capillary sample inlet mouth, cooperates multichannel syringe to realize suction or the discharge of sample or reagent; Input and cut-out by the control 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 to photomultiplier, by the continuous signal collection, can be monitored optical signalling in every capillary with the chemiluminescence signal sequence delivery in the kapillary by switching at a high speed simultaneously;
The analytical approach of using above-mentioned chemiluminescence immune assay system to carry out may further comprise the steps:
(1) in kapillary, introduces successively 5 μ L mineral oil, 2 μ L magnetic bead suspensions and 5 μ L sample to be checked, after the liquid section migrates to 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) after the liquid section in the step (1) is finished immune response, move to field regions with 0.1～5cm/s, start the electromagnet of kapillary top, magnetic field intensity is preferably 150mT, and magnetic bead is fixed on the capillary wall;
(3) start micro syringe, behind the solution in the discharge kapillary, introduce 10 μ L cleansing solutions in kapillary, 20～40 ℃ of washing magnetic beads are discharged cleansing solution, repeated washing 2 times; Simultaneously, start the 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 ℃, form antibody-Ag-Ab or Ag-Ab-antibody complex at magnetic bead; Simultaneously, start the magnetic field control module, magnetic field intensity is preferably 150mT, with 1～100Hz frequency oscillation;
(5) start micro syringe, behind the solution in the discharge kapillary, introduce 10 μ L cleansing solutions in kapillary, 20～40 ℃ of washing magnetic beads are discharged cleansing solution, repeated washing 2 times; Simultaneously, start the magnetic field control module, magnetic field intensity is preferably 150mT, with 1～100Hz frequency oscillation;
(6) introduce the reactant liquor that 5 μ L contain chemical luminous substrate in the kapillary, this reactant liquor is with after magnetic bead contacts, and issues biochemical luminescence-producing reaction in the catalysis of horseradish peroxidase or alkaline phosphatase, and reaction conditions is 25～40 ℃ and reacts 0.5～20min; Simultaneously, start the magnetic field control module, magnetic field intensity is preferably 150mT, with 1～100Hz frequency oscillation;
(7) after the liquid section in the step (6) is finished chemiluminescence reaction, migrate to detection zone with 0.1～5cm/s; Chemiluminescence signal is by collecting fiber and transfer to photomultiplier and detect;
In the 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 the phosphate buffer, and every milliliter of phosphate buffer contains 0.1～0.5g magnetic bead particles, stirs, and obtains the magnetic bead suspension;
Preferably, every milliliter of phosphate buffer contains the 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 zone of temperature control modules;
Field regions described in the step (2) refers to that kapillary is arranged in the zone 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 the step (6):
Described chemical luminous substrate is luminol/reinforcing agent or 3-(2-spiral diamantane)-4-methoxyl-4-(3-phosphorus oxygen acyl)-phenyl-1, the 2-dioxane;
Described reactant liquor is the luminol-hydrogen peroxide mixed liquor of volume ratio 1:1 or the 3-(2-spiral diamantane) of volume ratio 1:1-4-methoxyl-4-(3-phosphorus oxygen acyl)-phenyl-1,2-dioxane-hydrogen peroxide mixed liquor.
Detection zone described in the step (7) refers to that kapillary is arranged in the zone of optical detecting module;
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:
A kind of microminiaturized kapillary drop chemiluminescence immune assay system based on the micro-total analysis system theory based on the chemiluminescence immune assay system of kapillary drop technology.This system forms the water-in-oil type drop in parallel kapillary, finish the relevant sequence of operations of antigen-antibody immunoassay by means of the transportation of drop and controlling of magnetic bead.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 there are relative motion in water and oil phase, 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 in kapillary, introducing successively the drop contain various sample and with the auxiliary control in external magnetic field, in kapillary, finishing a series of processes such as sample introduction, antigen-antibody combination, wash-out, luminescence-producing reaction and optical signalling detection.
Heating plate is fixed in heating aluminium sheet below, is inlaid with temperature sensor in the heating aluminium sheet, in order to feed back temperature of heating plate.Temperature sensor is connected with temperature controller, and temperature controller is converted into digital data transmission to computing machine with simulating signal; Computer control software utilize pid algorithm (proportional/integral/derivative algorithm) according to the temperature sense feedback signal to temperature controller, by the temperature controller pilot relay, thereby whether regulate electrical current transports to heating plate.Drop enters temperature controlled region in the kapillary, can realize that fast temperature changes.
Chemiluminescence signal detects the Weak-luminescence detecting device that adopts the system integration.For realizing the dynamic monitoring of drop chemiluminescence signal in the parallel capillary pipe, the present invention adopts a kind of detection method based on optical fiber-photoswitch.Every capillary is provided with optical fiber, is used for gathering chemiluminescence signal and being transferred to photomultiplier detecting.The 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 with luminous signal.During detection signal, photoswitch carries out high speed to signals collecting optical fiber and switches, and makes the 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, with mineral oil, be marked with identification antibody (or antigen) magnetic bead suspension and sample introduce successively kapillary, form the water-in-oil type drop in the motion, identification antibody (or antigen) and antigen to be detected (or antibody) combination; Second step, when the liquid section moved to field regions, magnetic bead was hunted down, and then got rid of waste liquid; In the 3rd step, just mineral oil and contain the liquid section that detects antibody and introduce successively kapillary forms the water-in-oil type drop, detection antibody and antigen to be detected (or antibody) combination when drop moves to field regions in the motion.Get rid of waste liquid after finishing association reaction; In the 4th step, just mineral oil is introduced kapillary successively with the liquid section that contains cleansing solution, forms the water-in-oil type drop in the motion, will then not discharge kapillary in conjunction with detecting antibody elution when drop moves to field regions; The 5th step, just mineral oil is introduced kapillary successively with the liquid section that contains the enzyme luminous substrate, form the water-in-oil type drop in the motion, the enzymatic enzyme luminous substrate that detects mark on the antibody when drop moves to field regions is luminous, the liquid section continued to move to detector area after luminescence-producing reaction finished, and luminous signal is gathered by detecting device in the drop.
The present invention has following advantage and effect with respect to prior art:
(1) immunochemiluminometry provided by the invention system have that volume is little, analysis speed fast, the advantage of test flux height 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, has function advantage integrated and simple and efficient to handle, significantly reduce purchase and the operating cost of this kind equipment, helped to improve medical service quality, can satisfy 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 the water-in-oil type drop, keep the stable of reaction conditions, effectively avoided the cross pollution between the sample and the reaction that causes because of adsorption suppresses.
(3) as reactor, not only reagent consumption is little, and with low cost with standard capillary for analytical approach provided by the invention, effectively reduces the cost of instrument production and operation, has good society and economic benefit.
Description of drawings
Fig. 1 is the structural representation of the chemiluminescence immune assay system of embodiment 1.Wherein: 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 heating aluminium sheet, 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 of the chimeric groove of kapillary.
Fig. 2 is the principle schematic of immunochemiluminometry.
Fig. 3 is the optical detecting module detailed construction synoptic 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 synoptic diagram of chemiluminescence immune assay system, and wherein: 4 kapillaries, 5 heating plates, 6 contain heating aluminium sheet, 7 cover plates, 8PT-100 temperature sensor, 16 temperature controllers, 17 relays, 22 computing machines of the chimeric groove of kapillary.
Fig. 5 is the chemiluminescence detection signal of blood serum sample TNF Beta in 12 passage polytetrafluoroethylene capillaries of embodiment 2.
Fig. 6 is the chemiluminescence detection signal of blood serum sample C hepatitis virus antigen in 2 channel glass kapillaries of embodiment 3.
Fig. 7 is the chemiluminescence detection signal of blood serum sample TNF Beta in 96 passage quartz capillaries of embodiment 4.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.
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 with optical detecting module and are connected, and the 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 control;
Be provided with parallel capillary pipe 4 in the chimeric groove of kapillary; 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 being provided with a slit, and parallel capillary pipe 4 is arranged in the slit;
Described fluid control module is that 96 passage micro syringes, 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 the bearing support 3;
Displacement platform and sample/reagent bearing tray can carry out three-dimensional motion along leading screw under the drive motor effect, reagent or sample are transported to the capillary sample inlet mouth, cooperates multichannel syringe to realize suction or the discharge of sample or reagent; Input and cut-out by the control 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 to photomultiplier, by the continuous signal collection, can be monitored optical signalling in every capillary with the chemiluminescence signal sequence delivery in the kapillary by switching at a high speed simultaneously.
The analytical approach that the chemiluminescence immune assay system of utilization embodiment 1 carries out may further comprise the steps:
(1) in 12 parallel polytetrafluoroethylene capillaries, introduces 5 μ L mineral oil, 2 μ L magnetic bead suspensions and 5 μ L blood serum sample to be checked successively; Used capillary pipe length 10cm, 1000 microns of internal diameters; Used magnetic bead surfaces is fixed with anti-tumor necrosis factor Beta antibody (anti-TNF Beta, mouse-anti people), and magnetic bead particles is dispersed in the phosphate buffer, and the quality percent by volume is 0.25g/mL; Control liquid section migrates to temperature controlled region with 1cm/s speed, and 37 ℃ kept 2 minutes;
(2) move to field regions when drop, start kapillary top electromagnet (magnetic field intensity 150mT), magnetic bead is fixed on capillary wall;
(3) control micro syringe effluent discharge; The control micro syringe is introduced cleansing solution in the kapillary, and (composition is 0.1mol/L Na
2HPO
4, pH7.2), 10 μ L, 37 ℃ of washing magnetic beads 2 times; This process starts the magnetic field control module, and magnetic field intensity is 150mT, with the 10Hz frequency oscillation;
(4) in kapillary, introduce the detection antibody (sheep anti mouse) that is marked with horseradish peroxidase, hatched 2 minutes, and formed antibody-antigen-antibody complex at magnetic bead for 37 ℃; This process starts the magnetic field control module, and magnetic field intensity is 150mT, with the 10Hz frequency oscillation;
(5) (composition is 0.1mol/L Na again to introduce cleansing solution in the kapillary
2HPO
4, pH7.2), 10 μ L, 37 ℃ of washing magnetic beads 2 times; This process starts the magnetic field control module, and magnetic field intensity is 150mT, with the 10Hz frequency oscillation;
(6) in kapillary, introduce reactant liquor (1:1 luminol/reinforcing agent-superoxol) the 5 μ L that contain chemical luminous substrate, until it with after magnetic bead contacts, catalysis at horseradish peroxidase issues biochemical luminescence-producing reaction, and reaction conditions is 37 ℃, and the reaction time is 2 minutes; This process starts the magnetic field control module, and magnetic field intensity is 150mT, with the 10Hz frequency oscillation;
(7) control liquid section is moved 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 when the reactant liquor section arrives detection zone, can detect the chemiluminescence signal in 12 capillaries as shown in Figure 5 simultaneously.
The analytical approach that the chemiluminescence immune assay system of utilization embodiment 1 carries out may further comprise the steps:
(1) in 2 parallel glass kapillaries, introduces 5 μ L mineral oil, 2 μ L magnetic bead suspensions and 5 μ L blood serum sample to be checked successively; Used capillary pipe length 50cm, 500 microns of internal diameters; Used magnetic bead surfaces is fixed with C hepatitis virus antigen, and magnetic bead particles is dispersed in the phosphate buffer, and the quality percent by volume is 0.1g/mL; Control liquid section migrates to temperature controlled region with 1cm/s speed, 20 ℃ of lower maintenances 10 minutes;
(2) move to field regions when drop, start kapillary top electromagnet (magnetic field intensity 150mT), magnetic bead is fixed on capillary wall;
(3) control micro syringe effluent discharge; The control micro syringe is introduced cleansing solution in the kapillary, and (composition is 0.1mol/L Na
2HPO
4, pH7.2), 10 μ L, 37 ℃ of washing magnetic beads 2 times; This process starts the magnetic field control module, and magnetic field intensity is 150mT, with the 1Hz frequency oscillation;
(4) in kapillary, introduce the detection antibody (goat-anti people) that is marked with alkaline phosphatase, hatched 2 minutes, and formed Ag-Ab-antibody complex at magnetic bead for 37 ℃; This process starts the magnetic field control module, and magnetic field intensity is 150mT, with the 100Hz frequency oscillation;
(5) (composition is 0.1mol/L Na again to introduce cleansing solution in the kapillary
2HPO
4, pH7.2), 10 μ L, 37 ℃ of washing magnetic beads 2 times; This process starts the magnetic field control module, and magnetic field intensity is 150mT, with the 1Hz frequency oscillation;
(6) in kapillary, introduce the reactant liquor (3-(2-spiral diamantane)-4-methoxyl-4-(3-phosphorus oxygen acyl)-phenyl-1 that contains chemical luminous substrate, the 2-dioxane, AMPPD) 5 μ L, until it with after magnetic bead contacts, catalysis at alkaline phosphatase issues biochemical luminescence-producing reaction, reaction conditions is 25 ℃, and the reaction time is 20 minutes; This process starts the magnetic field control module, and magnetic field intensity is 150mT, with the 100Hz frequency oscillation;
(7) control liquid section is moved 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 when the reactant liquor section arrives detection zone, can detect the chemiluminescence signal in 2 capillaries as shown in Figure 6 simultaneously.
The analytical approach that the chemiluminescence immune assay system of utilization embodiment 1 carries out may further comprise the steps:
(1) in 96 parallel quartz capillaries, introduces 5 μ L mineral oil, 2 μ L magnetic bead suspensions and 5 μ L blood serum sample to be checked successively; Used capillary pipe length 25cm, 100 microns of internal diameters; Used magnetic bead surfaces is fixed with anti-TNF Beta antibody, the mouse-anti people, and magnetic bead particles is dispersed in the phosphate buffer, and the quality percent by volume is 0.5g/mL; Control liquid section migrates to temperature controlled region with 1cm/s speed, and 40 ℃ kept 1 minute;
(2) move to field regions when drop, start kapillary top electromagnet (magnetic field intensity 150mT), magnetic bead is fixed on capillary wall;
(3) control micro syringe effluent discharge; The control micro syringe is introduced cleansing solution in the kapillary, and (composition is 0.1mol/L Na
2HPO
4, pH7.2), 10 μ L, 37 ℃ of washing magnetic beads 2 times; This process starts the magnetic field control module, and magnetic field intensity is 150mT, with the 1Hz frequency oscillation;
(4) in kapillary, introduce the detection antibody (anti-human TNF Beta, sheep anti mouse) that is marked with horseradish peroxidase, hatched 2 minutes, and formed antibody-antigen-antibody complex at magnetic bead for 37 ℃; This process starts the magnetic field control module, and magnetic field intensity is 150mT, with the 1Hz frequency oscillation;
(5) (composition is 0.1mol/L Na again to introduce cleansing solution in the kapillary
2HPO
4, pH7.2), 10 μ L, 37 ℃ of washing magnetic beads 2 times; This process starts the magnetic field control module, and magnetic field intensity is 150mT, with the 1Hz frequency oscillation;
(6) in kapillary, introduce reactant liquor (1:1 luminol/reinforcing agent-superoxol) the 5 μ L that contain chemical luminous substrate, until it with after magnetic bead contacts, catalysis at horseradish peroxidase issues biochemical luminescence-producing reaction, and reaction conditions is 40 ℃, and the reaction time is 30 seconds; This process starts the magnetic field control module, and magnetic field intensity is 150mT, with the 1Hz frequency oscillation;
(7) control liquid section is moved 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 when the reactant liquor section arrives detection zone, can detect the chemiluminescence signal in 96 capillaries as shown in Figure 7 simultaneously.
Above-described embodiment is the better 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 the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within 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 with optical detecting module and are connected, and the 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 the 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 that connects successively from top to bottom, contains heating aluminium sheet and the heating plate of the chimeric groove of parallel capillary pipe; Temperature sensor is arranged in the 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; Be provided with kapillary in the chimeric groove of described parallel capillary pipe;
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, is provided with a slit between two electromagnet, and described kapillary is arranged in the 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 according to claim 1 system, it is characterized in that: described material capillaceous is plastics, glass or quartz.
3. chemiluminescence immune assay according to claim 1 system, it is characterized in that: described internal diameter capillaceous is 100～1000 microns, length is 10～50cm.
4. chemiluminescence immune assay according to claim 1 system, it 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 according to claim 1 system, it is characterized in that: described optical detecting module comprises kapillary-optical fiber interface, input optical fibre, photoswitch, output optical fibre and the photomultiplier that connects successively.
6. chemiluminescence immune assay according to claim 1 system, it is characterized in that: described fluid control module is the hyperchannel micro syringe.
7. chemiluminescence immune assay according to claim 1 system, it is characterized in that: described temperature control modules, magnetic field control module and optical detecting module are separately fixed on the bearing support.
8. the analytical approach of using each described chemiluminescence immune assay system of claim 1～7 to carry out is characterized in that may further comprise the steps:
(1) in kapillary, introduces successively 5 μ L mineral oil, 2 μ L magnetic bead suspensions and 5 μ L sample to be checked, after the liquid section migrates to 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) after the liquid section in the step (1) is finished immune response, move to field regions with 0.1～5cm/s, start the electromagnet of kapillary top, magnetic field intensity is 150mT, and magnetic bead is fixed on the capillary wall;
(3) start micro syringe, behind the solution in the discharge kapillary, introduce 10 μ L cleansing solutions in kapillary, 20～40 ℃ of washing magnetic beads are discharged cleansing solution, repeated washing 2 times; Simultaneously, start the 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 ℃, form antibody-Ag-Ab or Ag-Ab-antibody complex at magnetic bead; Simultaneously, start the magnetic field control module, magnetic field intensity is 150mT, with 1～100Hz frequency oscillation;
(5) start micro syringe, behind the solution in the discharge kapillary, introduce 10 μ L cleansing solutions in kapillary, 20～40 ℃ of washing magnetic beads are discharged cleansing solution, repeated washing 2 times; Simultaneously, start the magnetic field control module, magnetic field intensity is 150mT, with 1～100Hz frequency oscillation;
(6) introduce the reactant liquor that 5 μ L contain chemical luminous substrate in the kapillary, this reactant liquor is with after magnetic bead contacts, and issues biochemical luminescence-producing reaction in the catalysis of horseradish peroxidase or alkaline phosphatase, and reaction conditions is 25～40 ℃ and reacts 0.5～20min; Simultaneously, start the magnetic field control module, magnetic field intensity is 150mT, with 1～100Hz frequency oscillation;
(7) after the liquid section in the step (6) is finished chemiluminescence reaction, migrate to detection zone with 0.1～5cm/s; Chemiluminescence signal is by collecting fiber and transfer to photomultiplier and detect.
9. the chemiluminescence immune assay according to claim 8 system analytical approach of carrying out is characterized in that:
In the 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 the phosphate buffer, and every milliliter of phosphate buffer contains 0.1～0.5g magnetic bead particles, stirs, and obtains the magnetic bead suspension;
Cleansing solution described in step (3) and (5) is pH7.2,0.1mol/L Na
2HPO
4Aqueous solution;
In the step (6):
Described chemical luminous substrate is luminol/reinforcing agent or 3-(2-spiral diamantane)-4-methoxyl-4-(3-phosphorus oxygen acyl)-phenyl-1, the 2-dioxane;
Described reactant liquor is the luminol-hydrogen peroxide mixed liquor of volume ratio 1:1 or the 3-(2-spiral diamantane) of volume ratio 1:1-4-methoxyl-4-(3-phosphorus oxygen acyl)-phenyl-1,2-dioxane-hydrogen peroxide mixed liquor.
10. the application of each described chemiluminescence immune assay system of claim 1～7 is characterized in that: described chemiluminescence immune assay system uses in medicine, clinical medicine, food inspection, life science, environment or material science.
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