CN102680619A - Colloidal metal quantitative detection technology and application thereof - Google Patents
Colloidal metal quantitative detection technology and application thereof Download PDFInfo
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- CN102680619A CN102680619A CN2011100551373A CN201110055137A CN102680619A CN 102680619 A CN102680619 A CN 102680619A CN 2011100551373 A CN2011100551373 A CN 2011100551373A CN 201110055137 A CN201110055137 A CN 201110055137A CN 102680619 A CN102680619 A CN 102680619A
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Abstract
The invention discloses a colloidal metal quantitative detection technology. The colloidal metal quantitative detection technology comprises a chromatographic column, a capture phase, samples to be detected, a detection phase and a detector, can be used for development of various analysis and detection technical products and has favorable application value and market prospect.
Description
Technical field
The present invention relates to a kind of colloidal metal quantitative measurement technology and uses thereof.
Background technology
Column chromatography technology is a technology of utilizing chromatographic column that biomolecule is separated and detected, and wherein chromatographic column is its agent structure commonly used.Chromatographic technique is divided into adsorption chromatography, Partition Chromatography, ion-exchange chromatography, gel permeation chromatography, affinity chromatography, hydrophobic chromatography etc. by the mechanism of chromatography.Adsorption chromatography is to utilize the difference of adsorbent surface to the different component absorption property, reaches the purpose of isolation identification.Partition Chromatography is to utilize the partition factor of different component between moving phase and stationary phase different, makes it to separate.Ion-exchange chromatography is to utilize the difference of different component to ion exchanger affinity, makes it to separate.Gel chromatography is to utilize the difference of some gel for the component retardation of different molecular size, makes it to separate.Affinity chromatography is to have in the single-minded interactional material a pair of, is bound up on one of them on the holder, is used for another relative material of purifying, and common is affine to enzyme and suppressant, antigen and antibody, part and acceptor etc. are arranged.
Column chromatography technology is analysis separation means and the method that separation efficiency is very high in, and it can be opened the extremely similar separating substances of the various character utmost points, and it both can be used for the Analysis and Identification of small amount of matter, can be used for the separation and purification preparation of big quantity of material again.Present this technology has been widely used in scientific research and industrial a plurality of field, is all bringing into play crucial effect in fields such as oil, chemical industry, medical and health, bio-science, environmental science, agricultural sciences.
Colloidal metal, especially collaurum are a kind of labelling techniques commonly used, are to be applied to immune detection with collaurum as the trace labelling thing, and its unique advantage is arranged, especially with simple, quick and pollution-free for being celebrated.Immune colloid gold detection technique commonly used has immune colloid gold light microscopic decoration method, immune colloid gold staining method for electron microscopy, spot immune gold percolation, colloid gold immune paper chromatography, wherein with the colloid gold immune paper chromatography, claims the test strips method again; The most commonly used; Be about to specific antigen or antibody and be fixed on the film with ribbon, colloid gold label reagent is adsorbed on the pad, be added on the sample pad of test strips one end when sample to be checked after; Move forward through capillary action; React to each other behind the colloid gold label reagent of dissolving on the pad, when moving to fixing antigen or antibody regional again, specificity takes place again with it and combines and be trapped in thing to be checked and the bond of gold marked reagent; Accumulate in to detect and be with, can be through being observed visually the colour developing result.But these methods can only be carried out qualitative and semi-quantitative analysis, can't carry out full quantitative test.Therefore, develop the full quantitative test detection technique of a kind of employing, not only easy to use, also can significantly improve work efficiency simultaneously, have important Practical significance in detection with the numerous areas of analyzing, separate.
Summary of the invention
The purpose of this invention is to provide a kind of colloidal metal quantitative measurement technology and uses thereof, particularly with the colloidal metal quantitative measurement technology of colloidal metal label as the column chromatography structure that detects phase.
For realizing above-mentioned purpose, the present invention has adopted following technical scheme.
A kind of colloidal metal quantitative measurement technology, with the colloidal metal label as the column chromatography structure that detects phase, by chromatographic column, catch phase, matter sample to be checked, detect mutually and detecting device is formed.Described detection technique has following characteristic: 1) with thing specificity junction mixture coupling capturing carrier to be checked, process and catch phase; 2) catch to be loaded into mutually in the chromatographic column and use; 3) detection is processed by colloidal metal mark thing specificity junction mixture to be checked; 4) detect the amount of captive detection phase with detecting device and then calculate the content of thing to be checked and/or detect at large obtaining and the amount of the detection phase that flows out and then calculate the content of thing to be checked with detecting device.Thing specificity junction mixture to be checked is commonly used has enzyme and suppressant, antigen and antibody, part and acceptor etc.Capturing carrier commonly used has gel particle and magnetic particle; Gel particle has the serial ion-exchange packing of ion-exchange packing and the Ago-Gel of gel filtration filler, sephadex series of the gel filtration filler of sephadex series and Ago-Gel series, hydrophobic chromatography filler, affinity chromatography filler, cellulosic filler etc.; Wherein the affinity chromatography filler is the most commonly used in technology of the present invention, and cyanogen bromide-activated Ago-Gel medium (CNBr Sepharose FF), NHS activated agarose gel media etc. are arranged.
The structure of described chromatographic column comprises following one or more characteristic: 1) rigid conduit structure; 2) non-rigid pipeline configuration; 3) be prepacked column; 4) column structure for loading before using; 5) for being added with the pipeline configuration of switch controlling device; 6) hold back outlet and make in the post solid phase filler be easy to leave the column chromatography structure to carry out the column structure that next stage is analyzed for sample outlet end is easy to remove filter.The rigid conduit structure is common for two ends commonly used, laboratory are connected to interface or are not connected to the chromatographic column of the materials such as glass, organic glass, stainless steel of interface, also is included as the column structure that some analyzes special hard material.Non-rigid pipeline configuration refers to the pipeline configuration that can arbitrarily enclose song that materials such as plastics, rubber, silica gel are processed more, and two ends are connected to interface or are not connected to interface.The chromatographic column that the present invention's technology is adopted comprises prepacked column and the chromatographic column that uses the luggage that advances to carry; The comfort level prepacked column that consider to use will be as preferably, on prepacked column, adds simultaneously to build sample outlet end and filter and hold back outlet and be easy to be removed and make that the solid phase filler is easy to leave the column chromatography structure in the post.Its pipeline configuration of chromatographic column that the present invention technology is adopted also is preferred object, and the privileged site that chromatographic stuffing is fixed on pipeline when detecting carries out, and analyzes to be discharged from pipeline after accomplishing, and carries out new analyzing and testing again.
Described capturing carrier in catching mutually comprises and including but not limited to: 1) gel particle; 2) magnetic particle.Wherein gel particle is multiple column chromatography filler, affinity chromatography filler commonly used, and magnetic particle like antibody, antigen etc., carries out the magnetic particle of mark for being applicable to thing specificity junction mixture to be checked.
Described matter sample to be checked is to derive from the sample that human body and animal body can carry out the sample of medical diagnosis on disease and health detection and be used for environment, Pharmaceutical Analysis, food and technical analysis.
Described colloidal metal comprises collaurum commonly used and other colloidal metal developer, like electroselenium, collargol, electrocuprol, CI.
The used detection method of described detecting device includes but not limited to: when 1) detection to be detected was mutually for liquid phase, directly the degree of depth with colorimetric method for determining liquid phase color to be detected detected; When 2) detection to be detected is liquid phase mutually, analyses technology through ply of paper colloidal metal in the liquid phase to be checked is adsorbed onto on the paper material, form the colour developing band, the degree of depth of measuring the formed colour developing band of liquid phase to be detected with densimetry detects.
Described detection technique comprises following one or more step: 1) with thing specificity junction mixture coupling capturing carrier to be checked, process and catch phase; 2) will catch and be loaded into mutually in the column chromatography structure; 3) with colloidal metal mark thing specificity junction mixture to be checked, process the detection phase; The sample to be checked that 4) will contain thing to be checked is loaded on the column chromatography structure, flows through then and catches phase, and thing to be checked is hunted down; 5) clean with cleaning fluid and catch Xiang Shangwei by combination and residual thing to be checked and sample to be checked thereof in the chromatographic column; 6) will detect and be loaded on mutually on the column chromatography structure, and flow through then and catch phase, and detect and combine with captive thing specificity to be checked, phase-thing to be checked-detection phase compound is caught in formation, and then the Acquisition Detection phase; 7) directly detect at large obtaining and the amount of the detection phase that flows out and then calculate the content of thing to be checked; 8) with the captive detection of eluent wash-out mutually with detect compound mutually, detect mutually with detection mutually compound amount so that calculate the content of thing to be checked.
Described detection technique; It is characterized in that uniting and use multiple colloidal metal color signal amplifying technique to improve detection sensitivity, comprise that immunogold silver staining amplifying technique, biotin-avidin amplifying technique, the plain amplifying technique of biotin-strepto-affinity, SA combine amplifying technique.
The application of described detection technique in multiple testing product exploitation.Multi-field testing products such as environment, clinical, Pharmaceutical Analysis, food and technical analysis are arranged.
[beneficial effect]
1) the invention property column chromatography analytical technology and colloidal gold technique are organically combined, designed column chromatography collaurum detection technique, obviously improved the accuracy that colloidal gold technique detects, realize the purpose of the full detection by quantitative of collaurum.
2) the present invention all is controlled at the adsorption separation process of sample to be checked on the chromatographic column, has simplified running program, has made things convenient for use, has reduced the detection cost.
3) the present invention adopts the column chromatography adsorption separation technology, has simplified the complexity in the instrument preparation designs and produces greatly, the design of lowering apparatus and production cost.
Therefore, technology of the present invention has great importance and good prospects for application to improving existing chemiluminescence detection technology.
Embodiment]
Through following practical implementation instance, can further understand the present invention, but following instance not to qualification of the present invention.
Embodiment 1,The present invention's technology and existing chemiluminescence detection technology for detection result's comparison:
Experiment material: the NHS activated agarose gel media, horseradish peroxidase-labeled antihuman hemoglobin monoclonal antibody, colloid gold label antihuman hemoglobin monoclonal antibody, luminol of prepackage chromatographic column, the antihuman hemoglobin polyclonal antibody mark of the NHS activated agarose gel media 50ul of antihuman hemoglobin polyclonal antibody mark is housed, to iodophenol, urea peroxide, chemiluminescence detector, spectrophotometer, human hemoglobin solution.
Experimental technique: get the human hemoglobin solution of concentration known, with PBS solution dilution configuration 100ng, 300 ng, 700 ng, 1ug, 3ug, 7ug human hemoglobin solution and 10ng, 30 ng, 70 ng, 100ng, 300ng, 700ng human hemoglobin solution.Other gets the healthy subjects whole blood, and the deionized water haemolysis that adds equivalent discharges haemoglobin, carries out 1000 times of dilutions with PBS again and is used for technical measurement of the present invention, carries out 10000 times of dilutions with PBS again and is used for chemiluminescence mensuration.Technical measurement 100ng of the present invention, 300 ng, 700 ng, 1ug, 3ug, 7ug human hemoglobin solution and drawing standard curve are measured the healthy subjects haemoglobin then, and calculate HC.Chemiluminescence is measured 10ng, 30 ng, 70 ng, 100ng, 300ng, 700ng human hemoglobin solution and drawing standard curve, measures the healthy subjects haemoglobin then, and calculates HC.Get 42 test tubes, be divided into technology groups of the present invention and existing chemiluminescence detection technology groups.Each sample is made 3 parallel pipes.
Existing chemiluminescence detection technology groups, every pipe adds the NHS activated agarose gel media 50ul of antihuman hemoglobin polyclonal antibody mark, adds human hemoglobin solution and each 50ul of healthy subjects hemoglobin solutions to be measured of concentration known more respectively; 37 ℃ jolted incubation 30 minutes (37 ℃ of experiment confirms jolt incubation be optimum reacting time in 30 minutes) before, and 3000 change, divided centrifugal 3 minutes, abandon supernatant; Add PBS 300ul, mixing, 3000 change, divided centrifugal 3 minutes; Abandon supernatant, add horseradish peroxidase-labeled antihuman hemoglobin monoclonal antibody 50ul, 37 ℃ jolted incubation 30 minutes (experiment confirm jolt incubation for 37 ℃ be optimum reacting time in 30 minutes) before; 3000 change, divided centrifugal 3 minutes, abandon supernatant, add PBS 300ul; Mixing, 3000 change, divided centrifugal 3 minutes, abandon supernatant; Shift gel media to glow cup, add the 100ul luminol, to the luminous substrate working fluid of configurations such as iodophenol and urea peroxide, when reaction is carried out 2 minutes; Record 6 seconds of luminous quantity, calculation sample content.
Technology groups of the present invention is got the prepackage chromatographic column of the NHS activated agarose gel media 50ul that antihuman hemoglobin polyclonal antibody mark is housed, adds human hemoglobin solution and each 50ul of healthy subjects hemoglobin solutions to be measured of concentration known respectively, collects effluent; Add PBS damping fluid 100ul, collect and the merging effluent, merge effluent and go up appearance twice again, about 2 minutes altogether; Add PBS damping fluid 300ul again and clean, the abandoned stream fluid adds colloid gold label antihuman hemoglobin monoclonal antibody 50ul, collects effluent; Add PBS damping fluid 100ul, collect and the merging effluent, merge effluent and go up appearance again twice; About 2 minutes altogether, collect the merging effluent, shift the merging effluent to cuvette; Carry out colorimetric with spectrophotometer, the light absorption value (OD) at record 520nm place, calculation sample content.The prepackage chromatographic column is used 300ul PBS buffer solution for cleaning again, divides with 150ul affinity elution liquid then and carries out wash-out three times, and collect eluent; Shift eluent to cuvette; Carry out colorimetric with spectrophotometer, the light absorption value (OD) at record 520nm place, calculation sample content.
Experimental result:Existing techniques was accomplished experimental period 80 minutes; Measure 3.11ug/50ul as a result, effluent detection technique of the present invention was accomplished experimental period 15 minutes, measured 3.09ug/50ul as a result; The eluent detection technique deadline is 18 minutes; Measure 3.26ug/50ul as a result, two kinds of experimental technique gained are basically identical as a result, but completion experimental period of the present invention is significantly shorter than existing techniques.
Claims (10)
1. colloidal metal quantitative measurement technology is characterized in that: described detection technique is as the column chromatography structure that detects phase, by chromatographic column, catch phase, matter sample to be checked, detect mutually and detecting device is formed with the colloidal metal label.
2. according to claim 1 or 2 described detection techniques, have following characteristic:
1) with thing specificity junction mixture coupling capturing carrier to be checked, processes and catch phase;
2) catch to be loaded into mutually in the chromatographic column and use;
3) detection is processed by colloidal metal mark thing specificity junction mixture to be checked;
4) detect the amount of captive detection phase with detecting device and then calculate the content of thing to be checked and/or detect at large obtaining and the amount of the detection phase that flows out and then calculate the content of thing to be checked with detecting device.
3. according to the arbitrary described detection technique of claim 1-2, it is characterized in that: the structure of described chromatographic column comprises following one or more characteristic:
1) rigid conduit structure;
2) non-rigid pipeline configuration;
3) be prepacked column;
4) column structure for loading before using;
5) for being added with the pipeline configuration of switch controlling device;
6) hold back outlet and be easy to be removed and make in the post solid phase filler be easy to leave the column chromatography structure to carry out the column structure that next stage is analyzed for sample outlet end filters.
4. according to the arbitrary described detection technique of claim 1-3, it is characterized in that: described capturing carrier in catching mutually comprises and including but not limited to:
1) gel particle;
2) magnetic particle.
5. according to the arbitrary described detection technique of claim 1-4, it is characterized in that: described matter sample to be checked is to derive from the sample that human body and animal body can carry out the sample of medical diagnosis on disease and health detection and be used for environment, Pharmaceutical Analysis, food and technical analysis.
6. according to the arbitrary described detection technique of claim 1-5, it is characterized in that: described colloidal metal comprises collaurum, electroselenium, collargol, electrocuprol, CI.
7. according to the arbitrary described detection technique of claim 1-6, it is characterized in that: the used detection method of described detecting device includes but not limited to:
When 1) detection to be detected was liquid phase mutually, directly the degree of depth with colorimetric method for determining liquid phase color to be detected detected;
When 2) detection to be detected is liquid phase mutually, analyses technology through ply of paper colloidal metal in the liquid phase to be checked is adsorbed onto on the paper material, form the colour developing band, the degree of depth of measuring the formed colour developing band of liquid phase to be detected with densimetry detects.
8. according to the arbitrary described detection technique of claim 1-7, it is characterized in that: described detection technique comprises following one or more step:
1) with thing specificity junction mixture coupling capturing carrier to be checked, processes and catch phase;
2) will catch and be loaded into mutually in the column chromatography structure;
3) with colloidal metal mark thing specificity junction mixture to be checked, process the detection phase;
The sample to be checked that 4) will contain thing to be checked is loaded on the column chromatography structure, flows through then and catches phase, and thing to be checked is hunted down;
5) clean with cleaning fluid and catch Xiang Shangwei by combination and residual thing to be checked and sample to be checked thereof in the chromatographic column;
6) will detect and be loaded on mutually on the column chromatography structure, and flow through then and catch phase, and detect and combine with captive thing specificity to be checked, phase-thing to be checked-detection phase compound is caught in formation, and then the Acquisition Detection phase;
7) directly detect at large obtaining and the amount of the detection phase that flows out and then calculate the content of thing to be checked;
8) with the captive detection of eluent wash-out mutually with detect compound mutually, detect mutually with detection mutually compound amount so that calculate the content of thing to be checked.
9. according to the arbitrary described detection technique of claim 1-8; It is characterized in that uniting and use multiple colloidal metal color signal amplifying technique to improve detection sensitivity, comprise that immunogold silver staining amplifying technique, biotin-avidin amplifying technique, the plain amplifying technique of biotin-strepto-affinity, SA combine amplifying technique.
10. the application of the arbitrary described detection technique of claim 1-9 in multiple testing product exploitation.
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| CN103869061A (en) * | 2012-11-25 | 2014-06-18 | 常州博闻迪医药科技有限公司 | Filtering detection technique device and application thereof |
| CN112485427A (en) * | 2020-10-26 | 2021-03-12 | 绍兴梅奥心磁医疗科技有限公司 | Immune quantitative detection device, detection method and application of tumor marker |
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