CN101021530B - Automatic channel resolution chemiluminescent multicomponent immunodetection system and analytical method - Google Patents
Automatic channel resolution chemiluminescent multicomponent immunodetection system and analytical method Download PDFInfo
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Abstract
A multicomponent immune detection system and the analyzing method of the auto channel resolution chemical radiation. The tube of the warm cultivating system is set with the magnetic ball and the mixing rod which is set on the magnetic stirring apparatus; the transforming system is made up of the peristaltic pump, the connecting pipe and the multiposition valve. The signal resolution collecting system is made up of the detecting admittance, the light door, the magnet and the chemical radiation detector. The method is to fix the multicomponent encrusting antibody on the ball surface to react with the sample and the tracing antibody labeled by the alkaline phosphatase to form the sandwich immune compound; after washing the uncombining residue enzyme labeled antibody by the buffer, it is mixed with the chemical radiation substrate solution, the detection channel is blocked by the light door to detect the component concentration in different channel, then the ball is washed out to the next process. The method has the high speed, low cost and high sensitivity, so it is proper for the environment monitor, the clinic diagnose and the food safety.
Description
One, technical field
The present invention is a channel resolution multicomponent chemical luminescence immunoassay technology, relate to the immune analysis method that closely detects various ingredients in the same sample simultaneously, especially in conjunction with chemiluminescence and Flow Analysis Technique, and use the multi-component immunity analytical method of magnetic bead as the immune response carrier; The invention still further relates to the robotization detecting instrument system that is used for the multicomponent chemical luminescence immunoassay.
Two, background technology
Immunoassay since its selectivity height, advantage such as detectability is low, universality good, instrument is simple have a wide range of applications in fields such as clinical diagnosis, environmental monitoring, food security, Pharmaceutical Analysis and Micro biological Tests.In practical application area, often need to measure the content of various ingredients in the complex sample, as in medical diagnosis on disease, the Conjoint Analysis of multiple biomarker provides strong foundation for diagnosis.And for example in environmental monitoring, the concentration index of multiple pesticide and herbicide in the frequent monitoring of environmental water body simultaneously is with comprehensive evaluation environmental pollution level.
Measure the content of various ingredients in the complex system, the parallel single component analytic approach of many traditionally employings, promptly each analysis process is only measured wherein a kind of component concentration, and repeatedly parallel this flow process of execution obtains all required component concentrations at last.This analytical model required time is long, and reagent consumption is many, and labor capacity is excessive.The multi-component immunity analytical technology of rising in recent years can be in single analysis process simultaneously or closely realize the detection of various ingredients simultaneously, have that analysis throughput height, required time are short, sample consumption less, outstanding advantage such as analysis cost is low.Present multi-component immunity analytical technology can roughly be divided into two classes, and the first kind is the multi-tracer pattern, and its ultimate principle is with different tracer-labelling different components, realizes the identification of different component by the signal of distinguishing different labels.As in fluorescence and absorption spectrophotometry detection, discerning with wavelength; Discern with operating potential in the electrochemical process; Both wavelength available identification in the time-resolved fluorescence method, discern also available die-away time.Pattern based on multi-tracer has a very big problem, and different exactly labels often has distinct optimum analysis condition, is respectively 5-7,8-10 and 6-8 as the optimum pH value of horseradish peroxidase, alkaline phosphatase and galactase.Unite in same analysis system simply and use multiple label, can only select a compromise property condition in the optimum analysis condition of different labels, this has caused the reduction of analytical effect.Label is many more, and condition selects difficulty big more, therefore
Great majority all are no more than two (Kricka LJ.Multianalyte testing.Clin.Chem.1992 based on the number of components that analytical approach is surveyed of multi-tracer pattern at present; 38:327).Simultaneously, though different labels can wavelength or parameter such as current potential differentiate, it is often limited that but it differentiates efficient, signal overlap problem sometimes very outstanding (Goldman ER, Clapp AR, Anderson GP, Uyeda HT, Mauro JM, Medintz IL, et al.Multiplexed toxin analysisusing four colors of quantum dot fluororeagents.Anal.Chem.2004; 76:684).Multi-tracer pattern maximum be problematic in that available tracer is limited.Though the detection means of immunoassay is a lot, but really have higher sensitivity and better stability in every kind of means, only two or three kind of the tracer of in real work, being used widely, this has just limited quantity (the Mastichiadis C of the component that can detect simultaneously greatly, Kakabakos SE, Christofidis I, Koupparis MA, Willetts C, Misiakos K.Simultaneous determination of pesticides using a four-band disposable optical capillary immunosensor.Anal.Chem.2002; 74:6064).The second class multi-component immunity analytical technology is the spatial discrimination pattern, be at the fixing corresponding immunoreagent of different component of the zones of different of immune reactor, the immune response of different component is taken place in the different spatial of reactor, detect with array detector then, realize multi-component detection simultaneously.This pattern is more common in Array Method, and its transducing mode comprises optics (chemiluminescence, fluorescence, ultraviolet-visible absorbance) and galvanochemistry (ampere, electric capacity) method.This pattern needs expensive array detector (Fu ZF such as charge coupled cell (CCD) detecting device and multi-channel electrochemical workstation usually, Liu H, Ju HX.Flow-through multianalyte chemiluminescent immunosensing system with designed substrate zone-resolved technique for sequential detection of tumor markers.Anal.Chem.2006; 78:6999).
Current immuno analytical method overall process commonly used needs repeatedly application of sample, incubation usually, washes plate and reaction, carries out instrument detecting at last.Operation mostly is manual and finishes, and labor capacity is big, and required time is many more than 2 hours, is not suitable for the high flux express-analysis.Flow Analysis Technique has favorable reproducibility, the automaticity height, and advantage such as analysis speed is fast is to realize one of the most effective means of high throughput analysis.And chemiluminescence analysis is fast-developing in recent years analytical technology, and its instrument is cheap, and is easy and simple to handle, environmental friendliness, and be one of at present the sensitiveest analytical technology, be particularly suitable for the detection of trace materials.
Three, summary of the invention
The objective of the invention is: based on the spatial discrimination pattern, be carrier,, provide a kind of automatic channel resolution chemiluminescent multicomponent immune analysis method in conjunction with chemiluminescence detection and Flow Analysis Technique with the immunomagnetic beads; Another object of the present invention also is to provide a cover automatic channel resolution chemiluminescent multicomponent immunodetection system.
The objective of the invention is to realize by following technical scheme:
A kind of automatic channel resolution chemiluminescent multicomponent immunodetection system, it is characterized in that this system is made of incubation system, solution transmission system, signal resolution acquisition system and computing machine, wherein the incubation system is made up of with magnetic stirring apparatus (9) the glass test tube (6) of 1 to 6 built-in magnetic bead (7) and miniature stirrer (8), and glass test tube (6) is placed on the magnetic stirring apparatus (9); The solution transmission system is made up of multi-channel peristaltic pump (11), connecting pipe (10) and 1 to 6 multi-position valve (5), connecting pipe (10) is connected respectively to first inlet (1), second inlet (2), the 3rd inlet (3) of multi-position valve (5) through multi-channel peristaltic pump (11), first inlet (1) leads to incubation system transmissions bead suspension by connecting pipe (10), second inlet (2) is by connecting pipe (10) transmission chemical luminous substrate liquid (19), and the 3rd inlet (3) is by connecting pipe (10) transmission dcq buffer liquid (18); The signal resolution acquisition system is made up of with chemiluminescence detector (15) 1 to 6 sense channel (12), 1 to 6 tubulose optical gate (13), magnet (14), the inlet of sense channel (12) is connected with the outlet (4) at multi-position valve (5) center, connecting pipe is derived the raffinate in the sense channel (12), this raffinate is discharged from raffinate outlet (16), the top of sense channel (12) is placed magnet (14) below and is placed chemiluminescence detector (15), and solution transmission system and chemiluminescence detector (15) are controlled by computing machine (17).
The rotating speed of above-mentioned multi-channel peristaltic pump (11) and flow rate of liquid are to automatically adjust by computing machine (17).
Above-mentioned multi-position valve (5) can be realized the switching of different streams by rotating multi-position valve, on the multi-position valve (5) first inlet (1), second inlet (2), the 3rd inlet (3) communicate with the multi-position valve outlet (4) at center respectively, and the rotation of multi-position valve (5) is by computing machine (17) control automatically.
Above-mentioned magnet (14) is used for holding back suspending liquid magnetic bead particles (7).
Above-mentioned tubulose optical gate (13) is movably, is used for occlusion detection passage (12), realizes the resolution of luminous signal in the different passages.
A kind of analytical approach of automatic channel resolution chemiluminescent multicomponent immunodetection system, its analytical procedure is as follows:
1. the antibody sandwich magnetic bead (7) with sample, component to be measured joins in the glass test tube (6) of incubation system with alkali phosphatase enzyme mark antibody, stirs incubation under room temperature, forms immune sandwich complex;
2. the valve position mouth is switched to first inlet (1), with peristaltic pump (11) bead suspension is flowed into multi-position valve outlet (4) through connecting pipe (10) by first inlet (1) and feed sense channel (12) again, hold back magnetic bead (7) with magnet (14), the residue raffinate is discharged from raffinate outlet (16);
3. the valve position mouth is switched to the 3rd inlet (3), feed dcq buffer liquid (18) through connecting pipe (10), clean the unconjugated immunoreagent of magnetic bead (7) surface adsorption with peristaltic pump (11);
4. the valve position mouth is switched to second inlet (2), feed chemical luminous substrate liquid (19) through connecting pipe (10), remove the magnetic field of magnet (14), cause chemiluminescence reaction with peristaltic pump (11);
5. detect the chemiluminescence in the single passage (12), and cover all the other sense channels (12), draw the concentration of institute's detected components in this single passage, and in kind obtain the concentration of each passage institute detected components with tubulose optical gate (13);
6. the valve position mouth is switched to the 3rd inlet (3), feed dcq buffer liquid (18) with peristaltic pump (11) through connecting pipe (10), magnetic bead (7) is discharged from sense channel (10), finish full testing process, and enter next analysis cycle, the unified used magnetic bead (7) of collecting is handled to reuse with the regeneration damping fluid.
Above-mentioned steps dcq buffer liquid (18) 3. is the 0.01M phosphate buffer, and pH 7.4, contain 0.05% Tween-20.
The chemical luminous substrate liquid (19) of above-mentioned steps described in 4. is CSPD.
The regeneration damping fluid of above-mentioned steps described in 6. is 0.1M glycocoll-hydrochloride buffer, and pH 2.2.
Three inlets, first inlet (1) on the above-mentioned multi-position valve (5), second inlet (2), the 3rd inlet (3) can communicate with the outlet (4) at center respectively by switching valve position, realize the switching of different streams.The teflon that above-mentioned connecting pipe (10) available internal diameter is 0.8 millimeter is made.
The used immune response carrier of above-mentioned immunoassay system is an immunomagnetic beads, pan coating determinand corresponding antibody.A whole set of detection system and whole analytic process comprise that incubation, sample introduction, flushing, detection and discharge magnetic bead all carry out robotization control by computing machine, record the chemiluminescence signal value and also export from computing machine.
The principle of work of this detection system:
This detection system is based on traditional spatial discrimination pattern, in conjunction with flow analysis and channel resolution technology, can measure multiple material in an analysis process.Distinguish the capture antibody of covalent bond different component as the immune response carrier in the magnetic bead surfaces of functionalization; After fixedly the magnetic bead of different antibodies and sample and corresponding enzyme labelled antibody mix, at room temperature stir incubation, form the sandwich immunoassay compound; Hold back magnetic bead with magnet behind the sense channel that the corresponding bead suspension feeding of different component is parallel; With dcq buffer liquid flush away binding immunoassay reagent not; After rinsing well, feed chemical luminous substrate (CSPD), it is luminous to produce extensive chemical; The resolution collection of chemiluminescence signal is finished by optical gate, when promptly detecting the chemiluminescence of one of them passage, covers all the other sense channels with the tubulose optical gate, can obtain each components contents after writing down each sense channel luminous respectively; After mensuration is finished, feed dcq buffer liquid and discharge magnetic bead, to enter the next circulation of measuring; Handle with the regeneration damping fluid the unified back of collecting of the magnetic bead of discharging, and can realize the use repeatedly of immunomagnetic beads.
In the analytic process, all solution enter analytic system by the connecting pipe on peristaltic pump transmission, realize the switching of different streams by rotating multi-position valve, and overall process is carried out sequencing by computing machine and controlled automatically, and signal is also exported from computing machine.
The present invention has proposed channel resolution multi-component immunity analytical system in conjunction with Flow Analysis Technique and chemiluminescence detection, detects multiple material in a flow process.With respect to other multi-component immunity analytical methods, have following characteristics:
(1) simple to operate, the total analysis process is all finished in current system, carries out sequencing with computing machine and controls automatically, and manual operations is few, need not skilled operating personnel.
(2) lack analysis time, overall process comprises that application of sample, incubation, flushing and detection only need 18 minutes, are not only at present one of multi-component immunity analytical method the most fast, also greatly faster than common single component immune analysis method.
(3) instrument and equipment is simple, and is with low cost, need not the array detector of costliness commonly used in the multi-component immunity analytical, and whole analytic system is made up of peristaltic pump, multidigit selection valve, teflon connecting pipe, magnet and the chemiluminescence detector of low value.
(4) magnetic bead of antibody sandwich can use with regeneration damping fluid repeated regeneration, compares with the immune analysis method of routine, has saved expensive coated antibody greatly, has further reduced analysis cost.
(5) because its detecting pattern is extremely sensitive enzymatic chemiluminescence reaction, and this method can be measured the sample of extremely low concentration, satisfy most analyze demands.
(6) owing to adopted the Flow Analysis Technique of robotization, feasible operation technique by operating personnel greatly reduces with the different individual differences that cause of custom, and the reappearance of method greatly improves than traditional manual manipulation method, helps formulating relevant criterion.
The structural representation of Fig. 1 automatic channel resolution multicomponent chemical luminescence immunoassay detection system
Four, description of drawings
1-leads to the incubation system, and luminous substrate is learned in 2-Tonghua, and 3-leads to dcq buffer liquid, the outlet of 4-multi-position valve, the 5-multi-position valve, 6-glass test tube, 7-magnetic bead, the miniature stirrer of 8-, 9-magnetic stirring apparatus, 10-connecting pipe, the 11-peristaltic pump, 12-sense channel, 13-tubulose optical gate, 14-magnet, 15-chemiluminescence detector, the outlet of 16-raffinate, the 17-computing machine, 18-dcq buffer liquid, 19-chemical luminous substrate liquid.
Five, embodiment
This detection system is made of incubation system, solution transmission system, signal resolution acquisition system and computing machine.Detect for three components, the incubation system is 0.8 centimetre by three diameters, highly is 2 centimetres, and built-in length is that the glass test tube and the magnetic stirring apparatus of 0.5 centimetre miniature stirrer formed, and glass test tube places on the magnetic stirring apparatus; The solution transmission system is made up of the multi-position valve of a continuously adjustable multi-channel peristaltic pump of rotating speed, three three inlets, one outlets and the teflon connecting pipe that some internal diameters are 0.8 millimeter, first inlet (1) of multi-position valve leads to the corresponding incubation of each component system, second inlet (2) leads to chemical luminous substrate liquid, and the 3rd inlet (3) leads to dcq buffer liquid; The signal resolution acquisition system is made up of three sense channels, three removable tubulose optical gates, a chemiluminescence detector and a magnet, sense channel is that internal diameter is 1 millimeter, length is 4 centimetres quartz ampoule, be placed on the magnet below, the chemiluminescence detector top, there is movably black tubulose optical gate on the quartz ampoule side.Analysis system all carries out robotization control by computing machine.
The concrete analysis process is as shown in table 1, and all analytical procedures are carried out sequencing by computing machine and controlled automatically, record chemiluminescence signal and export from computing machine.The unified magnetic bead of collecting antibody sandwich after analytical procedure is finished is immersed in glycocoll-hydrochloride buffer of pH 2.2 10 minutes, and with 0.01M phosphate buffer (pH 7.4) washing 5 times, to reach the reusable purpose of regeneration.
With three kinds of important tumor markers: alpha-fetoprotein (AFP), carcinomebryonic antigen (CEA) are example with cancer antigen 125 (CA125), and the application of this automatic channel resolution chemiluminescent multicomponent immune analysis method is described.
The immune response carrier is the magnetic bead of activated carboxylic, and bag is by mouse monoclonal AFP antibody, mouse monoclonal CEA antibody and mouse monoclonal CA 125 antibody, with bovine serum albumin(BSA) sealing residual activity site respectively.Tracer antibody is the sheep polyclone AFP of alkali phosphatase enzyme mark, CEA and CA 125 antibody.
Flow process as shown in table 1 respectively adds 20 microlitre samples in three test tubes of incubation system, and adds antibody sandwich magnetic bead and each 20 microlitre of enzyme labelled antibody of AFP, CEA and CA 125 respectively, stirs incubation 10 minutes under the room temperature.Multi-position valve is switched to valve position 1, bead suspension is fed three sense channels, hold back magnetic bead with magnet after, raffinate flows out from passage.Multi-position valve is switched to valve position 3, feed dcq buffer liquid continuously with the flow velocity of 0.4mL/min, flushing magnetic bead 1 minute and 50 seconds, flush away is binding immunoassay reagent not.Multi-position valve is switched to valve position 2, in three passages, feed chemical luminous substrate 30 microlitres respectively, remove magnetic field, cause chemiluminescence reaction.React after 5 minutes, cover the corresponding sense channel of CEA and CA 125, detect the chemiluminescence in the AFP passage, obtain the concentration of AFP with optical gate; Cover the CEA sense channel corresponding with optical gate, detect the chemiluminescence in CA 125 passages, obtain the concentration of CA 125 with AFP; Cover the corresponding sense channel of AFP and CA 125 with optical gate, detect the chemiluminescence in the CEA passage, obtain the concentration of CEA.Valve position is switched to valve position 3, fed washing lotion continuously 0.5 minute, magnetic bead is discharged from passage with the flow velocity of 1.0mL/min.So far finish whole analysis process.The magnetic bead of being discharged is unified collect after, be immersed in glycocoll-HCl damping fluid of pH 2.2 10 minutes, and with 0.01M phosphate buffer (pH 7.4) washing 5 times, to reach the reusable purpose of regeneration.
Claims (9)
1. automatic channel resolution chemiluminescent multicomponent immunodetection system, it is characterized in that this system is made of incubation system, solution transmission system, signal resolution acquisition system and computing machine, wherein the incubation system is made up of with magnetic stirring apparatus (9) the glass test tube (6) of 1 to 6 built-in magnetic bead (7) and miniature stirrer (8), and glass test tube (6) is placed on the magnetic stirring apparatus (9); The solution transmission system is made up of multi-channel peristaltic pump (11), connecting pipe (10) and 1 to 6 multi-position valve (5), connecting pipe (10) is connected respectively to first inlet (1), second inlet (2), the 3rd inlet (3) of multi-position valve (5) through multi-channel peristaltic pump (11), first inlet (1) leads to incubation system transmissions bead suspension by connecting pipe (10), second inlet (2) is by connecting pipe (10) transmission chemical luminous substrate liquid (19), and the 3rd inlet (3) is by connecting pipe (10) transmission dcq buffer liquid (18); The signal resolution acquisition system is made up of with chemiluminescence detector (15) 1 to 6 sense channel (12), 1 to 6 tubulose optical gate (13), magnet (14), the inlet of sense channel (12) is connected with the outlet (4) at multi-position valve (5) center, connecting pipe is derived the raffinate in the sense channel (12), this raffinate is discharged from raffinate outlet (16), the top of sense channel (12) is placed magnet (14) below and is placed chemiluminescence detector (15), and wherein solution transmission system and chemiluminescence detector (15) are by computing machine (17) control automatically.
2. detection system according to claim 1 is characterized in that the rotating speed of described multi-channel peristaltic pump (11) and flow rate of liquid are to automatically adjust by computing machine (17).
3. detection system according to claim 1, it is characterized in that described multi-position valve (5) can realize the switching of different streams by rotating multi-position valve, on the multi-position valve (5) first inlet (1), second inlet (2), the 3rd inlet (3) communicate with the multi-position valve outlet (4) at center respectively, and the rotation of multi-position valve (5) is controlled by computing machine (17).
4. detection system according to claim 1 is characterized in that described magnet (14) is used for holding back suspending liquid magnetic bead particles (7).
5. detection system according to claim 1 is characterized in that described tubulose optical gate (13) is movably, is used for occlusion detection passage (12), realizes the resolution of luminous signal in the different passages.
6. analytical approach with the described automatic channel resolution chemiluminescent multicomponent immunodetection system of claim 1, its analytical procedure is as follows:
1. the antibody sandwich magnetic bead (7) with sample, component to be measured joins in the glass test tube (6) of incubation system with alkali phosphatase enzyme mark antibody, stirs incubation under room temperature, forms immune sandwich complex;
2. the valve position mouth is switched to first inlet (1), with peristaltic pump (11) bead suspension is flowed into multi-position valve outlet (4) through first inlet (1) of connecting pipe (10) by multi-position valve (5) and feed sense channel (12) again, hold back magnetic bead (7) with magnet (14), the residue raffinate is discharged from raffinate outlet (16);
3. the valve position mouth is switched to the 3rd inlet (3), feed dcq buffer liquid (18) through connecting pipe (10), clean the unconjugated immunoreagent of magnetic bead (7) surface adsorption with peristaltic pump (11);
4. the valve position mouth is switched to second inlet (2), feed chemical luminous substrate liquid (19) through connecting pipe (10), remove the magnetic field of magnet (14), cause chemiluminescence reaction with peristaltic pump (11);
5. detect the chemiluminescence in the single passage (12), mobile tubulose optical gate (13) covers all the other sense channels (12), draws the concentration of institute's detected components in this single passage, and in kind obtains the concentration of each passage institute detected components;
6. the valve position mouth is switched to the 3rd inlet (3), feed dcq buffer liquid (18) with peristaltic pump (11) through connecting pipe (10), magnetic bead (7) is discharged from sense channel (12), finish full testing process, and enter next analysis cycle, the unified used magnetic bead (7) of collecting is handled to reuse with the regeneration damping fluid.
7. analytical approach according to claim 6 is characterized in that at the dcq buffer liquid (18) of step described in 3. be the 0.01M phosphate buffer, and pH 7.4, contain 0.05% Tween-20.
8. analytical approach according to claim 6 is characterized in that at the chemical luminous substrate liquid (19) of step described in 4. be CSPD.
9. analytical approach according to claim 6 is characterized in that at the regeneration damping fluid of step described in 6. be 0.1M glycocoll-hydrochloride buffer, and pH 2.2.
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| CN2007100210743A CN101021530B (en) | 2007-03-26 | 2007-03-26 | Automatic channel resolution chemiluminescent multicomponent immunodetection system and analytical method |
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| CN101545902B (en) * | 2008-03-24 | 2014-01-08 | 江苏省肿瘤医院 | Automatic sampling distinguishing chemiluminescent multi-component immunological detection system and analysis method of same |
| CN101672841B (en) * | 2008-09-09 | 2013-05-08 | 北京万德高科技发展有限公司 | Detection instrument and detection method for biological sample |
| CN101865912B (en) * | 2009-04-14 | 2014-05-14 | 南京大学 | Fast chemiluminescence immune detection system and analysis method |
| CN101694491B (en) * | 2009-10-20 | 2013-01-02 | 上海理工大学 | Minitype liquid flux distributor used for multi-passage biochemical analyzer |
| CN102213724A (en) * | 2010-04-09 | 2011-10-12 | 中国科学院电子学研究所 | Method for quickly detecting trace protein based on chemoluminescence |
| CN102980996B (en) * | 2012-12-31 | 2014-09-10 | 广州市第一人民医院 | Chemiluminescence immunoassay system, as well as method and application thereof |
| CN103018441A (en) * | 2012-12-31 | 2013-04-03 | 西南大学 | Multicomponent immunoassay method based on time-resolved chemiluminescence |
| CN109085346B (en) * | 2018-09-18 | 2023-06-09 | 天津博硕科技有限公司 | Electrochemical immunity analyzer and analysis method thereof |
| CN112630430B (en) * | 2020-11-16 | 2021-08-27 | 北京美联泰科生物技术有限公司 | Kit for quantitatively detecting UCHL-1 and application thereof |
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