CN101349690A - Unlimited flux magnetic microsphere quantitative determination system and uses in biomedicine thereof - Google Patents

Unlimited flux magnetic microsphere quantitative determination system and uses in biomedicine thereof Download PDF

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CN101349690A
CN101349690A CNA2007103069757A CN200710306975A CN101349690A CN 101349690 A CN101349690 A CN 101349690A CN A2007103069757 A CNA2007103069757 A CN A2007103069757A CN 200710306975 A CN200710306975 A CN 200710306975A CN 101349690 A CN101349690 A CN 101349690A
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magnetic microsphere
impedance
magnetic
different
diagnosis
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王占科
雷万生
于永洲
祝仲珍
柴长春
冯青青
熊晓平
胡扬涛
潘小清
王志
肖群
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Priority to PCT/CN2008/000433 priority patent/WO2009082866A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se

Abstract

The invention relates to an impedance variation unlimited flux diagnosis magnetic microsphere and a process for preparation, a detection method and the usage in biomedicine thereof. Various magnetic microspheres with different impedances are adopted to code the species of various matters under test in the program, namely unlimited flux. Light signal intensity on the surface of the magnetic microsphere represents the content of measured matters, various matters under test in a sample can be simultaneously detected by using a device which can simultaneously detect magnetic microsphere impedance and magnetic microsphere surface light signals and by means of software analysis system, which satisfies the demands of increasingly developing clinical diagnosis and biomedical research. The program belongs to a novel technical platform which is established on the base of immunological reaction and nucleic acid molecular hybridization reaction, which is a technical innovation in the field of biomedicine, and is widely used in the biomedical research fields such as diagnosis of diseases, drug effect judgment, genomics, proteomics and the like.

Description

Unlimited flux magnetic microsphere quantitative determination system and in biomedicine purposes
Affiliated technical field
The invention belongs to biomedical technology industry development and uses thereof field.Be specifically related to that the different impedances diagnosis of countless versions magnetic microsphere is made, the different test substance kinds of the different impedance magnetic microspheres coding of countless versions countless versions, diagnosis magnetic microsphere detection method and pick-up unit thereof with and purposes in diagnosing the illness, judge biomedical sectors such as curative effect of medication and genomics, proteomics.
Background technology
Be based upon the detection technique on the antigen-antibody reaction basis at present, comprise enzyme linked immunosorbent assay (ELISA) technology, radioimmunoassay technique, electrochemiluminescent immunoassay technology, colloid gold immune technology, immunofluorence technic, electrochemiluminescent immunoassay technology, protein biochip technology and bioprotein sensing technology etc.
Be based upon the technical detection technique of nucleic acid molecules base pairing principle, comprise Northern blot technology, Southern blot technology and hybridization in situ technique and PCR technology (round pcr) etc.
We are that example generally has the following disadvantages with the elisa technique: 1. be that a kind of reagent can only be measured a project.HBsAg can only be measured such as the reagent of measuring hepatitis b virus s antigen (HBsAg), and anti-HBs (HbsAb) can not be measured simultaneously.Hospital laboratory or the each experiment of scientific research technician can only be measured an index, detect the related substances of certain disease, will carry out a large amount of repetitive operation (as HBV markers series), the cost great amount of manpower, material resources and resource increase inspection cost.2. be relatively difficulty of quality control.We can not measure hole to each of the assay plate of a collection of kit, carry out quality check one by one.Quality control department can only carry out random test from a collection of ELISA mensuration is pulled, this is difficult to guarantee accuracy of sample measurement result.This also is that elisa technique is difficult to measure blood sugar as liquid reagent, the reason that quality is effectively supervised.3. be accurate quantitative comparison difficulty, sensitivity is not high, complicated operation.Elisa technique finally to measure the hole liquid color depth as positive interpretation standard, owing to be subjected to the influence of developing time, makes the result be difficult to accomplish accurately quantitatively.This technical measurement step is loaded down with trivial details in addition, needs the time long, has shortcomings such as personal error.
Except elisa technique, the also each have their own shortcoming and defect of other technologies, though the technology that has is quick, can only be qualitative, can not be quantitative; Though the technology that has can be quantitative, it is very high to detect cost, and is difficult to carry out quality control.Solid biologic chip (comprising protein and fund chip) technology simultaneously can be measured thousands of kinds of materials, but generally is used for qualitative (judging negative and positive) and is difficult to use in quantitative measurement.
Be based upon the liquid chip technology on the different colours microballoon basis, this technology has solved the difficult problem of thousands of kinds of materials of quantitative measurement simultaneously, but has also had the following disadvantages with the different colours different mensuration projects of encoding:
The one, can only obtain 100 kinds of more stable different colours microballoons at most, this can't satisfy, and increasing clinical laboratory is detected and the requirement of medical research project, belongs to limited flux determination techniques.
The 2nd, may there be error in checkout equipment to the meticulous interpretation of color category, can not detect the countless versions test substance simultaneously,
The 3rd, the microballoon color is measured the light signal of microsphere surface, can produce interference.
The 4th, the different colours microballoon, because of the holding time long, may occur the decolouring.
The 5th, sensing equipment requirement condition height.Need high-sensitive color to judge and can eliminate the microsphere surface light signal quantitative measurement equipment of microballoon self color,, measure this liquid chip technology.This is difficult to exploitation and produces this sensing equipment in the not too flourishing country of technology, is unfavorable for the laboratory diagnosis equipment and the reagent production domesticization demand for development of national requirements.
Summary of the invention
Impedance variable liquid chip technology of the present invention is once brand-new improvement and the innovation on U.S.'s conventional liquid chip technology, is the once leap in the design.Microballoon impedance variable is different with color coding, and impedance microballoon coding can be accomplished unlimited many, has determined the present invention to have purposes widely at biomedical sector.
Proteomics and genomics need be measured the countless versions test substance.Medical diagnosis on disease and observation of curative effect also need more and more to detect index.Final purpose of the present invention is, impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis replaces all ELISA projects of present hospital laboratory or medical research universities and colleges, radio-immunity project and genetic test project etc. and is based upon antigen-antibody reaction and the technical all items of making nucleic acid molecular hybridization.Thoroughly change the project division of labor and the instrument configuration of hospital laboratory, following hospital and research institution only need an impedance variable liquid biochip technical measurement instrument, promptly can survey any antigen, antibody and unknown gene fragment, can also carry out cell count.The liquid biochip analyzer is a multi-functional laboratory diagnosis equipment.
In order to reach above purpose, the present invention divides following step.
1) according to method of the present invention, production impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis.Comprise that (1) makes different impedance magnetic microspheres, (2) are coated on antigen, antibody and known dna fragment on the different impedance magnetic microspheres, (3) other matched reagent.
2) according to method of the present invention and design concept, production impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis sensing equipment.Comprise that on behalf of the approval of different mensuration projects and standardization, (1) national departments concerned work out to different impedance magnetic microspheres.(2) a kind of while can be measured the design of magnetic microsphere impedance and magnetic microsphere surface light signalling arrangement, exploitation and production.
3) promote the use of to domestic and foreign hospitals and scientific research institution.Comprise that the unknown determined dna sequence of countless versions is identified in (1) genomics, a large amount of proteantigen substance-measurings of (2) proteomics countless versions, countless versions index determining in (3) medical diagnosis on disease, countless versions index determining in observation of (4) curative effect of disease and the judgement.
Technical scheme of the present invention is:
1. impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis overall technical architecture feasibility study of the present invention.
A lot of patents of invention are too high because of technical requirement, and are difficult to develop and promote.Color identifying technical requirement height surpasses 100 kinds of colors, and general technology is difficult to realize, develops a kind of equipment of differentiating the countless versions color, and is more impossible.
Resistance sizes can represent that the different test substance kind of unlimited a plurality of resistance sizes magnetic microsphere coding countless versions can be arranged in theory with continuous quantitative value.Patented technology of the present invention requires low relatively, and the result is more reliable.Every producer that can produce blood cell cell counter and flow cytometer all can develop the designed unlimited flux diagnose magnetic microsphere of the impedance variable quantified system analysis of the present invention.
1.1 the different impedance measuring technical feasibilities of magnetic microsphere countless versions are analyzed
The ultimate principle of blood counting instrument, the impedance of measuring each cell of blood is different and carry out differential count.Micropore resistance measurement principle can detect the subtle change of magnetic microsphere impedance, and proven technique can satisfy the detection to magnetic microsphere impedance variable fully at present.
In addition, different impedance magnetic microsphere (impedance variable magnetic microsphere) mature production technology, the method that adopts conductive materials (as metal powder, polyaniline etc.) and synthesizing magnetic microballoon the raw material styrene etc. of magnetisable material (as contain) to mix, but production countless versions impedance variable magnetic microsphere.Because conductive compositions can be mixed by arbitrary proportion with synthesizing magnetic microballoon raw material, can produce the different magnetic microsphere of countless versions resistance sizes theoretically, synthetic magnetic microsphere is after carboxylated or aminated processing, covalently bound with antigen, antibody, DNA or RNA produced the different impedances diagnosis of countless versions magnetic magnetic microsphere.
1.2 the unlimited flux diagnose magnetic microsphere development of impedance variable of the present invention technical feasibility analysis.
The different impedance magnetic microspheres of countless versions surface is by carboxylated and aminated processing, can firmly connect on different proteantigen, antibody or the DNA by covalent bond, make the unlimited flux diagnose magnetic microsphere of impedance variable of the present invention, this technology is a proven technique.
1.3 the unlimited flux diagnose magnetic microsphere of impedance variable of the present invention quantitative analysis tech feasibility analysis.
The magnetic microsphere resistance sizes changes and the instrument of magnetic microsphere surface fluorescence or luminous signal power as long as can detect simultaneously, can satisfy equipment requirements of the present invention.Being the combination of blood counting instrument and flow cytometer on principle, also is proven technique.
2. content of the present invention is divided two parts.The one, the development of the unlimited flux diagnose magnetic microsphere of impedance variable quantified system analysis, the 2nd, the unlimited flux diagnose magnetic microsphere of impedance variable quantified system analysis is in bio-medical applications.
2.1 the development of the unlimited flux diagnose magnetic microsphere of impedance variable quantified system analysis
The magnetic diagnosis magnetic microsphere that the volume of the different impedance magnitude of (1) production countless versions is identical
Resistance is a kind of attribute of material, and resistance sizes belongs to continuous variable on mathematics.The different chemical composition magnetic microsphere has different resistance.Innovation part of the present invention is that the raw material of conductive materials (as metal powder and polyaniline etc.) and the synthesizing magnetic microballoon styrene of ferromagnetic powder (as contain) is mixed by arbitrary proportion, can produce the identical magnetic microsphere of the volume of the different impedance characteristics of countless versions as shown in Figure 1.
With styrene is that raw material synthetic latex magnetic microsphere is an example, at first with conducting polymer composite polyaniline and the styrene that contains the magnetic oxide powder, pressing different proportion mixes, the control reaction conditions is produced satisfactory different impedance polystyrene latex magnetic microsphere by ripe latex production technology.Conducting polymer composite polyaniline content can change arbitrarily, can produce the different magnetic magnetic microsphere (table 2) of countless versions impedance magnitude.
Table 2: the factory formula of different impedance polystyrene magnetic microspheres
Figure A20071030697500071
Annotate: this table only plays the signal effect.Concrete prescription is not limited to this table data and raw material.
In order to guarantee to mix the activity that metal ingredient does not influence latex magnetic microsphere surface, the present invention's the 2nd innovation part is to make " diplocardia latex " as shown in Figure 2.At first produce the magnetic microsphere core that contains the different metal composition, the outside is wrapped up the not latex shell of containing metal composition of one deck more then, forms " diplocardia latex " magnetic microsphere.This " diplocardia latex " magnetic microsphere does not influence the surfactivity of latex magnetic microsphere, can not influence the effect that known antigens, antibody and DNA are coated on the latex surface.
The unequal magnetic microsphere of volume of the different impedance magnitude of (2) production countless versions
Same resistivity (or conductivity) magnetism of material microballoon, the magnetic microsphere volume is big more, and resistance is big more.For from distinguishing the magnetic microsphere of different impedances in appearance, same magnetic microsphere raw material according to the strict controlled condition of proven technique, can be made into the magnetic microsphere of different volumes size, and the magnetic microsphere volume is big more, and resistance is just big more.
(3) the different impedance diagnosis of preparation countless versions magnetic microsphere
The different impedance magnetic microspheres of the countless versions of purchase or oneself development, according to proven technique and document chemical treatment is carried out on the magnetic microsphere surface, make the carboxylation or the amination polystyrene latex magnetic microsphere of the different impedance series of countless versions, but covalency firmly connects antigen, antibody and DNA chemical molecular, and the different impedance series diagnosis of preparation countless versions magnetic microsphere as shown in Figure 3.
(4) different impedance magnitude magnetic microsphere coding countless versions test substances of countless versions or unknown materials project kind.
I determines that different magnetic microsphere resistance (or scope) are corresponding one by one with the test substance composition, formulates different impedances or the impedance ranges magnetic microsphere different test substance corresponding tables of encoding.Owing to magnetic microsphere resistance variations unlimitedness, also determining the unlimitedness of project to be measured.Different resistance magnetic microspheres comprise the equal volume magnetic microsphere of heterogeneity and the different volumes magnetic microsphere of identical component.A1, a2, certain field project of the equivalent hypergene thing of a3 medical science (as the medical diagnosis on disease project), b1, b2, the equivalent hypergene thing of b3 medical science another one field (measuring project as genomics or proteomics) sees Table 3.
Table 3: different impedance magnetic microspheres and composition corresponding tables to be measured
Figure A20071030697500081
Annotate: this table only plays the signal effect.Concrete impedance and project corresponding data are not limited to this table data
Ii is the different test substance kind of information of different impedance-encoded, the unlimited flux diagnose magnetic of input impedance variable quantitative analysis device software processing system.Software processing system is analyzed different impedances (project) magnetic microsphere surface light signal automatically.
(5) the unlimited flux diagnose magnetic microsphere of production impedance variable.
Adopt ripe chemical covalent labeling technology, to be used to measure specific antibody, specific antigen or the known dna of disparity items) chemical crosslinking is on corresponding different resistance magnetic microspheres surface, produces the unlimited flux diagnose magnetic microsphere of a series of impedance variablees.Concrete operation method can be consulted in related data and teaching material, is not described in detail here.
(6) the required matched reagent of preparation impedance variable unlimited flux diagnose magnetic microsphere quantitative test.
According to the different in kind of test substance or unknown materials, design different matched reagents.
1. composition to be measured is protein, polysaccharide, lipid antigen material.
This class material comprises, a) surperficial specific antigen (belonging to the property made a definite diagnosis diagnosis), kinds of tumors label, body internal protein peptide materials (as c reactive protein, hormone, rheumatoid factor etc.) such as the known or unknown bacterium in medical diagnosis on disease and curative effect of disease observation field, mycoplasma, Chlamydia, virus.B) at biomedical scientific research field (as genomics, proteomics) countless versions protein, polysaccharide, lipid antigen material etc.
Detect this class material composition, need its specific antibody of magnetic microsphere pan coating, measure.With latex magnetic microsphere surface is the reaction platform, adopts the double antibodies sandwich method to be illustrated, and the matched reagent that this class is measured project comprises following several composition.
(1) free corresponding multiple specific antibody solution
(2) second antibody (antiantibody) solution of fluorescence or shiner mark
(3) antigen-antibody binding reaction damping fluid
(4) latex magnetic microsphere lavation buffer solution
(5) fluorescence or luminescence-producing reaction damping fluid
Above damping fluid all can find prescription on disclosed data document or teaching material, no longer describe in detail here.Specific monoclonal antibody and mark second antibody can obtain or production voluntarily by buying.
2. test substance is a protein antibody.This class material mainly comprises a) in medical diagnosis on disease and curative effect of disease observation field and since the patient infection bacterium, mycoplasma, Chlamydia, virus, and the specific antibody that produces.In addition, some disease also can be diagnosed the pathologic antibody that self material produces.B), need the countless versions protein antibody material of mensuration etc. in biomedical scientific research field (as genomics, proteomics).
This class test item needs the corresponding specific antigen composition of latex magnetic microsphere surface indicia, measures.The matched reagent that this class is measured project comprises following several composition.
(1) free corresponding fluorescence or the multiple specific antigen solution of shiner mark
(2) antigen-antibody binding reaction damping fluid
(3) latex magnetic microsphere lavation buffer solution
(4) fluorescence or luminescence-producing reaction damping fluid
Above damping fluid all can find prescription on disclosed data document or teaching material, no longer describe in detail here.Specific monoclonal antibody and antigen (characteristic of bacteria albumen, virion), the mark second antibody can obtain or production voluntarily by buying.
3. test substance is DNA or RNA.This class material mainly comprises, a) in medical diagnosis on disease and curative effect of disease observation field, internal microorganism especially virus specific DNA or RNA fragment (property made a definite diagnosis diagnosis), b) at biomedical scientific research field (as genomics, proteomics), countless versions DNA that need measure or RNA etc.
The detection of this intermediate item needs corresponding DNA of latex magnetic microsphere pan coating or RNA fragment, analyzes.The matched reagent of this intermediate item comprises following several composition.
(1) free dna fragmentation (gene probe) solution with fluorescence or shiner mark
(2) hybridization reaction damping fluid
(3) latex magnetic microsphere lavation buffer solution
(4) fluorescence or luminescence-producing reaction damping fluid
Above reagent source is convenient, the manufacturing technology maturation.Here no longer describe in detail.
(7) assembling impedance variable liquid biochip kit.
To wrap by the countless versions magnetic microsphere of countless versions antigen, antibody and DNA (RNA) (the unlimited flux diagnose magnetic microsphere of impedance variable) and matched reagent thereof, assembly packaging becomes the unlimited flux diagnose magnetic microsphere of impedance variable quantitative test kit.The diagnosis magnetic microsphere that the different resistance of how many kinds of are arranged in the kit, just representative can be measured the how many kinds of project simultaneously.
(8) impedance variable unlimited flux diagnose magnetic microsphere detection method and device.
The unlimited flux diagnose magnetic microsphere of impedance variable detection method is that at first, while or priority are measured the resistance sizes and the magnetic microsphere surface light signal intensity of the unlimited flux diagnose magnetic microsphere of impedance variable.Then, according to the disparity items kind of different impedance magnetic microsphere correspondences, judge those test substance feminine genders in the sample, those test substance positive and content.At last, print result report.The unlimited flux diagnose magnetic microsphere of impedance variable detection method principle is seen Fig. 4.
Impedance variable unlimited flux diagnosis magnetic microsphere assay method of the present invention is undertaken by matched reagent box and device.The matched reagent box is as aforementioned.The unlimited flux diagnose magnetic microsphere of impedance variable determinator comprises 2 determinators and 1 sets of data process software as shown in Figure 5.
First determinator is exactly the micropore impedance measuring instrument.This device can accurately be measured the resistance sizes by the magnetic microsphere of micropore, and the principle of work of its principle and cell counter is basic identical.According to the project kind of different impedance-encoded, the micropore impedance measuring instrument is the mensuration project category of interpretation magnetic microsphere accurately.
Second determinator is to the fluorescence or the luminous signal on the magnetic microsphere surface of passing through micropore, carries out the detection by quantitative device.Its principle and flow cytometer principle of work are basic identical.
Magnetic microsphere liquid biochip determination techniques of the present invention, from being exactly the combination of full-automatic blood count technology and fluidic cell determination techniques in essence, this device technique maturation, the blood counting instrument that some is high-grade, possessed this function, here be not described in detail.
The 3rd software analysis system, be exactly magnetic microsphere resistance signal and this magnetic microsphere surface light signal to priority or reception simultaneously, according to the impedance-encoded repertory, accurately analyze in countless versions mensuration project, those material feminine genders, those material positives, and under the standard reference condition, carry out quantitatively as shown in Figure 6.
The mensuration project (table 3) of at first default magnetic microsphere resistance value (or scope) representative of software analysis system, then, analyze the fluorescence or the light signal strength (content of material) on different impedance magnetic microspheres (mensuration project) surface, do reference with reference material, calculate test substance content, transfer data to printer at last, the print result report.
Table 3: the different mensuration projects of different latex magnetic microsphere impedance-encoded
Figure A20071030697500101
What deserves to be explained is, be human body blood leucocyte, red blood cell and hematoblastic resistance characteristic value if we set certain impedance, and this device can possess the function of blood counting instrument simultaneously.If we replace magnetic microsphere to measure with cells such as marrow, this device can possess the function of flow cytometer simultaneously by changing parameter.
Impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis is in the purposes of biomedical sector
1. the disease association Antigens is measured
Medical diagnosis on disease and observe the curative effect need be measured multiple antigenic substance in the body.Along with the research to the disease genesis mechanism is goed deep into, increasing disease association antigen is found, conventional method comprises that color-coded diagnosis magnetic microsphere quantitative determination system once also can only finish 100 kinds of left and right sides antigenic substances and measure, and can not satisfy the needs of hospital's laboratory diagnosis future development far away.The inventor adopts impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis once can measure the countless versions determined antigen simultaneously.
The inventor is an example to measure n kind (n is greater than 1 and less than just infinite) disease association Antigens simultaneously, is illustrated.
1) oneself produces or buys the latex magnetic microsphere that can be used to chemical covalent bond mark of the different impedances of n kind.
1) the latex magnetic microsphere is classified from small to large by impedance and encode n kind various disease related antigen.
2) formulate the different impedance magnetic microsphere coding of n kind determined antigen corresponding tables.
4) oneself buy or own production at the monoclonal antibody of n kind antigen.
5) according to different impedance magnetic microsphere coding determined antigen corresponding tables, with n kind monoclonal antibody, covalent bonds is on the magnetic microsphere surface of the different impedances of n kind respectively, make the diagnosis magnetic microsphere, centrifugal 10 minutes of 1500g, or be placed on the magnetic sheet and made the magnetic microsphere precipitation in 2-12 hour, discard free unlabelled antibody.N kind diagnosis magnetic microsphere mixes by equal proportion after washing with phosphate buffer, and is resuspended with phosphate buffer, makes every liter and contains 10 5The n unit impedance diagnosis magnetic microsphere mixed liquor of individual magnetic microsphere concentration.
6) 250 microlitre n unit impedance diagnosis magnetic microsphere mixed liquor is put into 1 milliliter centrifuge tube, add 250 microlitre serum or other mensuration samples again, abundant mixing, 37 ℃ of concussions are incubated 1 minute～2 hours, make the abundant combination of determined antigen in magnetic microsphere surface antibody and the sample.
7) centrifugal 10 minutes of centrifuge tube 1500g or be placed on the magnetic sheet static 2-12 hour, supernatant discarded, resuspended with PBS, centrifugal again or be placed on the magnetic sheet static 2-12 hour, abandoning supernatant again, n unit impedance diagnosis magnetic microsphere is washed, thoroughly free test substance in the flush away centrifuge tube.
8) add 500 milliliters of n kind monoclonal antibody phosphate buffers containing anti-n kind antigen, 37 ℃ of concussions are incubated 30 minutes～1 hour, and they are reacted with the test substance that is combined on the magnetic microsphere respectively, repeat the 7th) step, magnetic microsphere is washed abandoning supernatant.
9) add fluorescently-labeled anti-human immunoglobulin(HIg) antibody (antiantibody) 500 milliliters, 37 ℃ of concussions are incubated 45 minutes, with the antibodies that is combined in the magnetic microsphere surface, and repeating step 7), the magnetic microsphere precipitation is stayed in thoroughly washing.
10) with the resuspended magnetic microsphere precipitation of magnetic microsphere fluorometric assay damping fluid, use can be measured the equipment of magnetic microsphere impedance and magnetic microsphere surface fluorescence intensity simultaneously, carries out magnetic microsphere impedance (mensuration project) and magnetic microsphere surface fluorescence intensity (content of material) and measures.
11) interpretation as a result: if the n1 impedance magnetic microsphere surface fluorescence positive illustrates serum n1 antigen positive, fluorescence intensity and n1 antigen concentration are proportional.Adopt n1 antigen normal concentration to do reference and measure, can carry out accurately quantitatively n1.If n kind impedance magnetic microsphere has only n1, n2, n3, n4, five kinds of impedance magnetic microspheres such as n5 surface fluorescence are positive simultaneously, illustrate to have n1 in the blood simultaneously, and n2, n3, n4, five kinds of antigenic substances of n5, other n-5 kind antigens are all negative.If n kind impedance magnetic microsphere surface fluorescence signal is all positive, illustrate to have n kind antigenic substance in the blood simultaneously.And the like.
Technical solution of the present invention also is applicable to the mensuration of countless versions determined antigen in the biomedical proteomics.
If survey n kind antigenic substance, need the magnetic microsphere of the different resistance of n kind, operate the same.Technique scheme only plays an exemplary role, and specifically sees the relevant technologies data for details.
2. the disease association antibody class is measured
Measure the antibody of some material in the body, can infect some material in the side light body, medical diagnosis on disease and observe the curative effect need be measured multiple antibody materials in the body.Along with the research to the disease genesis mechanism is goed deep into, increasing disease association antibody is found, conventional method comprises that color-coded diagnosis magnetic microsphere quantitative determination system once also can only finish 100 kinds of left and right sides antibody materials and measure, and can not satisfy the needs of hospital's laboratory diagnosis future development far away.The inventor adopts impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis once can measure countless versions antibody to be measured simultaneously,
The inventor is an example to measure n kind (n is greater than 1 and less than just infinite) disease association antibody class simultaneously, is illustrated.
1) oneself produces or buys the latex magnetic microsphere that can be used to chemical covalent bond mark of the different impedances of n kind.
2) the latex magnetic microsphere is classified from small to large by impedance and encode n kind various disease associated antibodies.
3) formulate the different impedance magnetic microspheres of the n kind antibody corresponding tables to be measured of encoding.
4) oneself buy or own production at the antiantibody or the corresponding antigen of n kind antibody.
5) according to the different impedance magnetic microspheres antibody corresponding tables to be measured of encoding, with the n kind can with the n kind antiantibody or the corresponding antigen of n kind antibodies to be measured, covalent bonds is on the magnetic microsphere surface of the different impedances of n kind respectively, make the diagnosis magnetic microsphere, centrifugal 10 minutes of 1500g or be placed on the magnetic sheet static 2-12 hour discards free unlabelled antiantibody or corresponding antigen.N kind diagnosis magnetic microsphere mixes by equal proportion after washing with phosphate buffer, and is resuspended with phosphate buffer, makes every liter and contains 10 5The n unit impedance diagnosis magnetic microsphere mixed liquor of individual magnetic microsphere concentration.
6) 250 microlitre n unit impedance diagnosis magnetic microsphere mixed liquor is put into 1 milliliter centrifuge tube, add 250 microlitre serum or other mensuration samples again, abundant mixing, 37 ℃ of concussions are incubated 1 minute～2 hours, make the abundant combination of antibody to be measured in magnetic microsphere surface and the sample.
7) centrifugal 10 minutes of centrifuge tube 1500g or be placed on the magnetic sheet static 2-12 hour, supernatant discarded, resuspended with PBS, centrifugal again or be placed on the magnetic sheet static 2-12 hour, remove supernatant again, n unit impedance diagnosis magnetic microsphere is washed, thoroughly free test substance in the flush away centrifuge tube.
8) add 500 milliliters of the n kind antiantibody contain anti-n kind antibody or corresponding antigen phosphate buffers, 37 ℃ of concussions are incubated 30 minutes～1 hour, and they are reacted with the test substance that is combined on the magnetic microsphere respectively, repeat the 7th) step, magnetic microsphere is washed abandoning supernatant.
9) antibody of fluorescently-labeled anti-n kind antiantibody of adding or corresponding antigen is 500 milliliters, and 37 ℃ of concussions are incubated 45 minutes, with the antibodies that is combined in the magnetic microsphere surface, and repeating step 7), thoroughly washing stays magnetic microsphere to precipitate.
10) with the resuspended magnetic microsphere precipitation of magnetic microsphere fluorometric assay damping fluid, use can be measured the equipment of magnetic microsphere impedance and magnetic microsphere surface fluorescence intensity simultaneously, carries out magnetic microsphere impedance (mensuration project) and magnetic microsphere surface fluorescence intensity (content of material) and measures.
11) interpretation as a result: if the n1 impedance magnetic microsphere surface fluorescence positive illustrates serum n1 antibody positive, fluorescence intensity and n1 antibody concentration are proportional.Adopt n1 antibody normal concentration to do reference and measure, can carry out accurately quantitatively n1.If n kind impedance magnetic microsphere has only n1, n2, n3, n4, five kinds of impedance magnetic microspheres such as n5 surface fluorescence are positive simultaneously, illustrate to have n1 in the blood simultaneously, and n2, n3, n4, five kinds of antibody materials of n5, other n-5 kind antibody are all negative.If n kind impedance magnetic microsphere surface fluorescence signal is all positive, illustrate to have n kind antibody materials in the blood simultaneously.And the like.
If survey n kind antibody materials, need the magnetic microsphere of the different resistance of n kind, operate the same.Technique scheme only plays an exemplary role, and specifically sees the relevant technologies data for details.
3. the disease association nucleic acid material is measured
Measure the specific nucleic acid fragment of certain microorganism, existence that can this microorganism of specific diagnostic.Conventional method comprises that color-coded diagnosis magnetic microsphere quantitative determination system once also can only finish 100 kinds of left and right sides nucleic acid fragments and measure, and can not satisfy the needs of hospital's laboratory diagnosis future development far away.The inventor adopts impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis once can measure countless versions specific nucleic acid fragment simultaneously.
The inventor is an example to measure n kind (n is greater than 1 and less than just infinite) disease association DNA or RNA fragment simultaneously, is illustrated.
1) oneself produces or buys the latex magnetic microsphere that can be used to chemical covalent bond mark of the different impedances of n kind.
2) the latex magnetic microsphere is classified from small to large by impedance and relevant DNA of the n kind various disease of encoding or RNA fragment.
3) formulate the different impedance magnetic microspheres of n kind encode DNA to be measured or RNA fragment corresponding tables.
4) oneself buy or own production at the complementary DNA fragment of n kind DNA to be measured or RNA fragment.
5) according to relevant DNA of the different impedance magnetic microsphere coding of n kind various disease or RNA fragment corresponding tables, with the n kind can with the different complementary DNA fragments of n kind of n kind DNA to be measured or RNA fragment specific bond, covalent bonds is on the magnetic microsphere surface of the different impedances of n kind respectively, make the diagnosis magnetic microsphere, centrifugal 10 minutes of 1500g or be placed on the magnetic sheet static 2-12 hour discards free unlabelled complementary DNA fragment.After n kind diagnosis magnetic microsphere fully washs with phosphate buffer, mix by equal proportion, resuspended with phosphate buffer, make every liter and contain 10 5The n unit impedance magnetic microsphere dna probe mixed liquor of individual magnetic microsphere concentration.
6) 250 microlitre n unit impedance magnetic microsphere dna probe mixed liquor is put into 1 milliliter centrifuge tube, add 250 microlitre serum or other mensuration samples again, abundant mixing, 37 ℃ of concussions are incubated 1 minute～12 hours, make DNA to be measured or the abundant combination of RNA in magnetic microsphere surface and the sample.
7) centrifugal 10 minutes of centrifuge tube 1500g or be placed on the magnetic sheet static 2-12 hour, supernatant discarded, resuspended with phosphate buffer, centrifugal again or be placed on the magnetic sheet static 2-12 hour, remove supernatant again, n unit impedance magnetic microsphere dna probe mixed liquor is washed, thoroughly free test substance in the flush away centrifuge tube.
8) add 500 milliliters of dna fragmentation phosphate buffers that can combine containing fluorescence or luminescent marking with DNA to be measured or RNA complementary series, 37 ℃ of concussions are incubated 30 minutes～12 hours, make they respectively be combined in magnetic microsphere on DNA to be measured or RNA complementary DNA fragment fully combine, repeat the 7th) step, magnetic microsphere is washed abandoning supernatant.
9) with the resuspended magnetic microsphere precipitation of magnetic microsphere fluorometric assay damping fluid, use can be measured the equipment of magnetic microsphere impedance and magnetic microsphere surface fluorescence intensity simultaneously, carries out magnetic microsphere impedance (mensuration project) and magnetic microsphere surface fluorescence intensity (content of material) and measures.
10) interpretation as a result: if the n1 impedance magnetic microsphere surface fluorescence positive illustrates the serum n1 DNA to be measured or the RNA fragment positive, fluorescence intensity and n1 DNA to be measured or RNA fragment concentrations are proportional.Adopt n1 DNA to be measured or RNA fragment normal concentration to do reference and measure, can carry out accurately quantitatively n1 DNA to be measured or RNA fragment.If n kind impedance magnetic microsphere has only n1, n2, n3, n4, five kinds of impedance magnetic microspheres such as n5 surface fluorescence are positive simultaneously, illustrate to have n1 in the blood simultaneously, n2, n3, n4, five kinds of DNA to be measured of n5 or RNA fragment, other n-5 kinds DNA to be measured or RNA fragment are all negative.If n kind impedance magnetic microsphere surface fluorescence signal is all positive, illustrate to have n kind DNA to be measured or RNA fragment in the blood simultaneously.And the like.
Technical solution of the present invention also is applicable to countless versions DNA to be measured or RNA mensuration in the biomedical proteomics.
If survey n kind DNA to be measured or RNA fragment, need the magnetic microsphere of the different resistance of n kind, operate the same.Technique scheme only plays an exemplary role, and specifically sees the relevant technologies data for details.
4. biomedical scientific research field is used
Impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis has measures countless versions test substance or unknown materials simultaneously.In the gene age behind the mankind, the DNA of a large amount of thousands of kinds in the human body, RNA fragment and countless versions known antigens or antibody need to measure.DNA in the human body, RNA and antigenic substance and antibody materials complexity are various, and conventional method comprises that color-coded diagnosis magnetic microsphere quantitative determination system once also can only finish 100 kinds of left and right sides substance-measurings, can not satisfy the needs of scientific research development far away.
The single job of impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis can be measured the countless versions test substance simultaneously, by huge computer processing system, will reduce the research work personnel labor intensity greatly, improves research work efficient.
7.4.1 existing whole known mRNA kinds are example in human certain cell to measure, and the application of impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis in genomics is described.
1) suppose that this cellular genome has n kind (n can be arbitrary integer) gene expression, and base sequence is clear and definite.
2) buy or the different impedances of the own n of production kind can be for the latex magnetic microsphere of nucleic acid marking, and set n kind difference impedance magnetic microspheres and encode respectively and represent n kind mRNA to be measured.
3) formulate the different mRNA corresponding tables to be measured of the different impedance magnetic microsphere coding of n kind n kind.
4) the complementary base sequence DNA of purchase or oneself synthetic n kind mRNA to be measured.
5) according to formulating the different mRNA corresponding tables to be measured of the different impedance magnetic microsphere coding of n kind n kind, the complementary base sequence dna fragmentation with purchase or oneself synthetic n kind mRNA to be measured covalently bind in corresponding n kind impedance magnetic microsphere surface respectively.Centrifugal 10 minutes of 1500g or be placed on the magnetic sheet static 2-12 hour, discard free unlabelled dna fragmentation, resuspended with phosphate buffer, centrifugal again 10 minutes of 1500g or be placed on the magnetic sheet static 2-12 hour, supernatant discarded keeps the diagnosis magnetic microsphere in the precipitation.The different impedances diagnosis of n kind magnetic microsphere mixes by equal proportion respectively with the phosphate buffer washing, carries out resuspendedly with containing hybridization buffer, makes every liter and contains 10 5The n kind magnetic microsphere dna probe of individual magnetic microsphere concentration.
6) get the different impedance magnetic microsphere of n kind equal-volume and mix, make the different impedance magnetic microsphere of n kind dna probe.
7) get the different impedance diagnosis of n kind magnetic microsphere 250 microlitres and put into 1 milliliter centrifuge tube, add 250 microlitre cell total rna extracts again, abundant mixing, 37 ℃ of concussion insulations 1 minute～20 hours make in magnetic microsphere surface known dna and the sample RNA to be measured fully hybridize combination.
8) centrifugal 10 minutes of 1500g or be placed on the magnetic sheet static 2-12 hour, supernatant discarded, resuspended with phosphate buffer, centrifugal again or be placed on the magnetic sheet static 2-12 hour, remove supernatant again, n kind impedance magnetic microsphere is washed, thoroughly the RNA fragment free to be measured that does not combine in the flush away centrifuge tube with magnetic microsphere surface DNA.
9) add contain fluorescence or luminescent marking at 500 milliliters of the hybridization buffers of DNA to be measured or RNA complementary DNA fragment, 37 ℃ of concussions are incubated 30 minutes～1 hour, make they respectively be combined in magnetic microsphere on RNA to be measured hybridization combine, repeat the 8th) step, magnetic microsphere is thoroughly washed, free fluorescence that abandoning supernatant is not hybridized or luminescent substance dna fragmentation keep the magnetic microsphere precipitation.
10) with magnetic microsphere fluorescence or luminescence assays damping fluid, resuspended magnetic microsphere precipitation, use can be measured the equipment of magnetic microsphere impedance and magnetic microsphere surface fluorescence or luminous intensity simultaneously, carries out magnetic microsphere impedance (the n kind is measured project) and magnetic microsphere surface fluorescence intensity (rna content to be measured) and measures.
11) interpretation as a result: if the n1 impedance magnetic microsphere surface light signal positive illustrates the expression of this cell a1 gene masculine.The n2 impedance magnetic microsphere surface light signal positive shows the expression of this cell a2 gene masculine, and the like.If, n1, n2, n3, n4, impedance magnetic microsphere surface light signal total positiveses such as n5 show a1 in the liver cell, a2, a3, a4, the a5 gene is positive expression simultaneously, and other n-5 kind gene expressions are all negative.And the like.
If survey more kinds of mRNA materials, need the magnetic microsphere of more kinds of different resistance, operate the same.
More than the operation example only plays an exemplary role, and specifically sees relevant technologies data and document for details.
Above scheme also is used to measure biomedical research field countless versions specific DNA fragment, antigen or antibody fragment.
7.4.2 existing unknown mRNA is an example in the human normal cell to measure, and the application of impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis in genomics is described.
Discover recently, limited though human gene quantity is huge, and human protein's quantity almost is unlimited.Because effects such as processing after the genetic transcription and modifications, the mRNA of encoding proteins (mRNA) quantity also is far longer than gene dosage.Most of mRNA and gene order are human and unclear, how to find that unknown gene, unknown mRNA and agnoprotein matter are the technical barriers that faces the human genome times afterwards comprehensively.
Impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis can detect countless versions DNA or RNA fragment simultaneously, provides laboratory facilities for solving direct searching unknown gene.Unknown gene (mRNA) is an example to the inventor in human certain cell to seek, and the purposes of impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis in seeking unknown gene is described.
In cell cdna library, directly filter out unknown gene with countless versions random probe (the known dna fragment of mark), technology all is difficult to accomplish at present.The present invention adopts computer code countless versions testing gene with the different resistance magnetic microspheres of countless versions, and preparation countless versions random probe just can filter out all unknown genes theoretically.
Technology of the present invention has the feature of unlimited flux, just in time satisfies the technical requirement of screening unknown gene with numerous random probe in cell cdna library.The method of impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis screening unknown gene is as follows:
1) branch carries out many times, produces the different impedance magnetic microspheres of n kind (n is an arbitrary integer) at every turn,
2) computing machine automatically with magnetic microsphere from impedance value record from small to large.
3) the synthetic at every turn random oligonucleotide fragment of n kind more than 7.
4) computing machine sorts by impedance magnitude, and the artificial synthetic automatically pairing target gene fragment of random oligonucleotide fragment of the n kind of encoding respectively.
5), the synthetic at random oligonucleotide fragment of n kind more than 7 is marked at the magnetic microsphere surface of the different impedances of n kind respectively according to the impedance-encoded table of comparisons.
6) by impedance variable unlimited flux diagnosis magnetic microsphere quantitative analysis device, analyzing magnetic microsphere surface positive signal, and, determine unknown cDNA fragment according to magnetic microsphere impedance value and magnetic microsphere impedance-encoded cDNA sequence corresponding tables thereof.
7) repeating step 1) to step 6), up to filtering out the unknown gene fragment.
Produce different impedance magnetic microspheres, magnetic microsphere surface indicia oligonucleotide fragment, and operations such as magnetic microsphere surface positive signal and impedance value judgement all can be finished automatically.Impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis can be used for screening unknown gene.
The present invention is fit to present and following biomedical sector and comprises that hospital, scientific research institution etc. are based upon the mensuration of antigen-antibody reaction and the technical all items of making nucleic acid molecular hybridization (as the ELISA project, radiation or electrochemiluminescent immunoassay project and genetic test project etc.).The body self that application relates to proteomics, genomics and various diseases laboratory diagnosis and the required mensuration of observation of curative effect produces or external range protein, polypeptide and lipid and glucide mensuration.
Impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis of the present invention (impedance variable liquid biochip) is the revolution that is based upon on the magnetic microsphere surface platform detection technique, it is infinitely many that on behalf of different mensuration projects, different impedance magnetic microspheres make the present invention measure project, content of material is measured in the representative of magnetic microsphere light signal strength, makes quantitatively accurately to be easy to quality control.Magnetic microsphere impedance measuring and magnetic microsphere surface light signal measuring principle are simple, diagnostic kit development and laboratory diagnosis equipment development technology maturation, and being one is worth the new invention applied at biomedical sector.The ever-increasing test substance type requirements of biomedical sector has been satisfied in the present invention and application thereof.Have quick and precisely, quantitatively, be easy to advantages such as quality control.
The present invention makes the microballoon feature more stable with impedance variable magnetic microsphere replacement color variable microballoon.Magnetic microsphere can directly precipitate rapidly under strong magnetic field action not by manually centrifugal, is easy to the whole-course automation operation that microballoon is handled and detected.Because microballoon resistance variations quantity of information is unlimited theoretically, thereby improving the unlimitedness of chip detection project, is biochip truly.The technology of measuring microballoon resistance is than interpretation microballoon color technology, want ripe and simple, present internal reagent and instrument development industry, can satisfy the measuring principle of liquid chip technology of the present invention fully, can produce fully and measure kit and relevant detection equipment,, break developed country's technical monopoly for allowing the whole world can produce liquid chip high-tech technology, promote towards whole world medical and health industry, create conditions.
The present invention is a kind of huge innovation of practicality, and maximum characteristics are:
1) unlimited flux.Adopt different impedances to represent (coding) different mensuration projects, the impedance variable is infinitely many, and the project that decision is measured simultaneously is infinitely many, guarantees the unlimited flux determination techniques requirement of scientific research and clinical needs.Countless versions test substance or unknown materials single job are finished.
2) good stability.Magnetic microsphere resistance measurement technology maturation, experimental error is little, as a result the precision height.
3) be easy to preserve.The different impedance magnetic microspheres of producing are preserved more stable than different colours microballoon, manufacture craft is simpler, and it is more convenient to originate.
4) highly sensitive.Magnetic microsphere itself is not with color, can not disturb the quantitative measurement of magnetic microsphere surface light signal, and the denier fluorescence signal just can detect positive signal.
5) automaticity height.The magnetic microsphere that the present invention adopts can precipitate rapidly under strong magnetic field action, does not need manually centrifugally, can realize the substantial length of operationization from the application of sample to the printed report.
6 are easy to produce and promote.Magnetic microsphere resistance and surface fluorescence signal measuring technology maturation.See Table 1.
Table 1: impedance variable liquid chip of the present invention is the color variance liquid chip technology comparison of inventing with the U.S.
Technical indicator is estimated Impedance variable liquid chip The color variance liquid chip
The interpretation of mensuration project Different impedance latex magnetic microspheres are represented different measure material, the bigger and easy detections of resistance variations quantity of information. The different colours microballoon is represented the different materials of measuring, and quantity of information is relatively little, and the color interpretation is prone to error, requires the technology height.
Measure project kind Impedance can consumption limit formula, and the magnetic microsphere impedance variation is unlimited, decision project unlimited amount About 100 kinds of colors
Test substance is quantitative Magnetic microsphere surface light signal is not subjected to the microballoon impedance disturbances The microsphere surface light signal is subjected to the microballoon color interference.
Equipment needed thereby Magnetic microsphere impedance measuring and magnetic microsphere surface light signal measuring device, technical requirement is relatively low. Interpretation of microballoon color and microsphere surface light signal determinator, technical requirement is higher relatively.
The chip manufacturing difficulty Different impedance magnetic microspheres are convenient for production The different colours microballoon is produced difficulty
The interpretation of microballoon variable The micropore impedance measuring Color is judged mensuration
DEVELOPMENT PROSPECT Relatively easily, cost is lower, and market is good Difficult relatively, the cost height, market, backward areas is little
Advantage of the present invention and purposes are as follows:
Solid-state biochip technology, elisa technique and radio-immunity (RIA) technology of liquid biochip of the present invention and domestic maturation relatively has the fine quality (seeing Table 3) that none substitutes.Mainly show the following aspects:
1 different impedance latex magnetic microspheres, stable, the holding time is long.
2 automaticity height.Magnetic microsphere can precipitate rapidly under strong magnetic field action, can centrifugally be separable and the washing magnetic microsphere.
3 magnetic microsphere impedance magnitude interpretations are simple.Can measure with micropore impedance method blood counting instrument.Add a voltage at the micropore two ends, when magnetic microsphere enters micropore, will produce a current impulse, the current impulse difference that the magnetic microsphere of different impedances produces.
4 synthesizing magnetic microballoon raw materials are different with the conductive materials ratio, and magnetic microsphere resistance can have or not several variations theoretically, determining the present invention can measure the countless versions test substance simultaneously theoretically.
5 magnetic microsphere surface light signal detection technique maturations.The ordinary stream cell instrument just can be finished the quantitative measurement to magnetic microsphere surface light signal.
6 magnetic microsphere impedance variable liquid biochips of the present invention are measured devices needed, on Machine Design and principle of work, are the combinations of cell counter and flow cytometer, and this equipment can be finished exploitation at home and produce.
Table 3: technology of the present invention and domestic proven technique are relatively
Methodology relatively Impedance variable liquid chip The solid-state chip technology Elisa technique The RIA technology
Reaction principle The antigen-antibody reaction making nucleic acid molecular hybridization The antigen-antibody reaction making nucleic acid molecular hybridization Antigen-antibody reaction Antigen-antibody reaction
Reaction system Liquid phase Solid phase and liquid phase Liquid phase Liquid phase
The reaction platform The liquid phase magnetic microsphere surface of dissociating Support surface The liquid phase free molecule The liquid phase free molecule
The mensuration project Infinitely Limited Single Single
Project indicates Different resistance magnetic microspheres are represented different mensuration projects Different coordinate positions are represented different mensuration projects A kind of material of a kind of kit measurement A kind of material of a kind of kit measurement
Can be quantitative Energy Can not Energy Energy
Quality control Easily Difficult Difficult Easily
Detection method Measure the light signal on different impedance magnetic microspheres surface Measure the light signal on the diverse location Observe colour developing Radioactive intensity is measured
Required instrument Magnetic microsphere impedance and microsphere surface light signal determinator Fluorescent microscope Microplate reader Full-automatic radioimmunoanalyzer
The development technique requirement Low High Low Low
Promotion prospect Good Better Good Good
The production domesticization prospect Good Good Good Good
The inventive method is simple to operate, the equal mature and reliable of required technology.Patent cooperation or transfer object are
1. diagnostic reagent is researched and developed personnel both at home and abroad.
2. domestic and international laboratory diagnosis equipment research and developer.
3. blood counting instrument manufacturer.
4. stream type cell analyzer manufacturer.
5. magnetic microsphere synthesizes and manufacturer.
6. latex diagnostic reagent manufacturer.
7. hospital laboratory.
8. R﹠D institution of medical colleges and schools.
9. computer software research and development personnel.
10. comprehensively possess top all professional entity and individual's entities.
Description of drawings
Fig. 1 is the magnetic microsphere synoptic diagram that contains different conductive materials concentration.Conductive materials content is high more, and resistance is more little, and vice versa.
Fig. 2 is a diplocardia magnetic microsphere synoptic diagram.The electrically conductive core volume is big more in the same volume magnetic microsphere, and impedance is more little, and vice versa.
Fig. 3 is different impedance series diagnosis magnetic microspheres.Different specific antibodies, antigen, DNA or RNA material are carried in different resistance magnetic microspheres surface.Represent different antibody, antigen, DNA or RNA material.
Fig. 4 is an impedance variable unlimited flux diagnosis magnetic microsphere detection method principle schematic.
Fig. 5 is an impedance variable unlimited flux diagnosis magnetic microsphere pick-up unit principle of work synoptic diagram.
Fig. 6 is an impedance variable unlimited flux diagnosis magnetic microsphere software analysis system works principle schematic.Ω represents the magnetic microsphere impedance magnitude, the different test substances of different impedance-encoded.(light signal strength is represented test substance content to candela for cd, candela) expression magnetic microsphere surface light signal intensity.
Specific embodiment
Embodiment 1, disease association Antigens are measured
Medical diagnosis on disease and observe the curative effect need be measured multiple antigenic substance in the body.Along with the research to the disease genesis mechanism is goed deep into, increasing disease association antigen is found, conventional method comprises that color-coded diagnosis magnetic microsphere quantitative determination system once also can only finish 100 kinds of left and right sides antigenic substances and measure, and can not satisfy the needs of hospital's laboratory diagnosis future development far away.The inventor adopts impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis once can measure the countless versions determined antigen simultaneously.(HBsAg, HBeAg HbcAg) are example to the inventor, add an explanation to measure three antigenic substances of hepatitis type B virus simultaneously.
1) oneself produces or buys the latex magnetic microsphere that can be used to chemical covalent bond mark of different impedances.
2) the latex magnetic microsphere is divided into Low ESR, three kinds of middle impedance and high impedances.
3) oneself buy or own production anti-HBsAg three kinds of monoclonal antibodies of HBeAg and HbcAg.
4) with anti-HBsAg, three kinds of monoclonal antibodies of HBeAg and HbcAg, covalent bonds is made the diagnosis magnetic microsphere on the magnetic microsphere surface of basic, normal, high three kinds of different impedances respectively, centrifugal 10 minutes of 1500g or be placed on the magnetic sheet static 2-12 hour discards free unlabelled antibody.Three kinds of diagnosis magnetic microspheres mix by equal proportion with phosphate buffer (PBS) washing, carry out resuspendedly with containing antigen-antibody reaction PBS damping fluid, make every liter and contain 10 5The ternary magnetic microsphere liquid chip of individual magnetic microsphere concentration.
5) 250 microlitre ternary magnetic microsphere chips are put into 1 milliliter centrifuge tube, add 250 microlitre serum or other mensuration samples again, abundant mixing, 37 ℃ of concussions are incubated 1 minute～2 hours, make the abundant combination of determined antigen in magnetic microsphere surface antibody and the sample.
6) centrifugal 10 minutes of centrifuge tube 1500g or be placed on the magnetic sheet static 2-12 hour, supernatant discarded, resuspended with PBS, centrifugal again or be placed on the magnetic sheet static 2-12 hour, remove supernatant again, ternary impedance magnetic microsphere chip is washed, thoroughly free test substance in the flush away centrifuge tube.
7) adding contains anti-HBsAg, 500 milliliters of HBeAg and three kinds of monoclonal antibody PBS of HbcAg solution, and 37 ℃ of concussions are incubated 30 minutes～1 hour, they are reacted with the test substance that is combined on the magnetic microsphere respectively, repeat the 6th) step, magnetic microsphere is washed abandoning supernatant.
8) add fluorescently-labeled anti-human immunoglobulin(HIg) antibody (antiantibody) 500 milliliters, 37 ℃ of concussions are incubated 45 minutes, with the antibodies that is combined in the magnetic microsphere surface, and repeating step 6), the magnetic microsphere precipitation is stayed in thoroughly washing.
9) with the resuspended magnetic microsphere precipitation of magnetic microsphere fluorometric assay damping fluid, use can be measured the equipment of magnetic microsphere impedance and magnetic microsphere surface fluorescence intensity simultaneously, carries out magnetic microsphere impedance (mensuration project) and magnetic microsphere surface fluorescence intensity (content of material) and measures.
10) interpretation as a result: if the Low ESR magnetic microsphere surface fluorescence positive illustrates the serum HBsAg positive, fluorescence intensity and HBsAg, concentration is proportional.Employing standard HBsAg concentration is done reference and is measured, and can HBsAg be carried out accurately quantitatively.If Low ESR and high impedance surface fluorescence are positive simultaneously, illustrate to have HBsAg and HbcAg in the blood simultaneously, and the like.
More than the operation example only plays an exemplary role, and specifically sees the relevant technologies data for details.If survey n kind antigenic substance, need the magnetic microsphere of the different resistance of n kind, operate the same.
Embodiment 2, disease association antibody class are measured
Measure the antibody of some material in the body, can infect some material in the side light body, medical diagnosis on disease and observe the curative effect need be measured multiple antibody materials in the body.Along with the research to the disease genesis mechanism is goed deep into, increasing disease association antibody is found, conventional method comprises that color-coded diagnosis magnetic microsphere quantitative determination system once also can only finish 100 kinds of left and right sides antibody materials and measure, and can not satisfy the needs of hospital's laboratory diagnosis future development far away.The inventor adopts impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis once can measure countless versions antibody to be measured simultaneously, we describe to measure hepatitis A virus (HAV) antibody, hepatitis type B virus (HBV) antibody and hepatitis C virus (HCV) antibody simultaneously and HIV (human immunodeficiency virus) (HIV) antibody is example.
1) purchase or oneself production 4 kinds of different impedances (n1, n2, n3, n4) latex magnetic microspheres of Gong the covalent cross-linking of feature antigen protein.
2) purchase or own producer gene engineering product HAV, HBV, HCV, HIV specific antigen fragment or inactivation of viruses protein body.
3) default n1, n2, n3, n4 impedance latex magnetic microsphere represent respectively and measure HAV, HBV, four kinds of antibody of HCV, HIV.
4) respectively with HAV, HBV, HCV, HIV antigen fragment chemistry covalent labeling at n1, n2, n3, four kinds of impedance latexes of n4 magnetic microsphere surface.Make quaternary diagnosis magnetic microsphere, centrifugal 10 minutes of 1500g or be placed on the magnetic sheet static 2-12 hour discards free unlabelled antigen.Four kinds of diagnosis magnetic microspheres mix by equal proportion with phosphate buffer (PBS) washing, carry out resuspendedly with containing antigen-antibody reaction PBS damping fluid, make every liter and contain 10 5The quaternary magnetic microsphere liquid chip of individual magnetic microsphere concentration.
5) 250 microlitre quaternary magnetic microsphere chips are put into 1 milliliter centrifuge tube, add 250 microlitre serum or other mensuration samples again, abundant mixing, 37 ℃ of concussions are incubated 1 minute～2 hours, make the abundant combination of antibody to be measured in magnetic microsphere surface antigen and the sample.
6) centrifugal 10 minutes of 1500g or be placed on the magnetic sheet static 2-12 hour, supernatant discarded, resuspended with PBS, centrifugal again or be placed on the magnetic sheet static 2-12 hour, supernatant discarded is washed quaternary impedance magnetic microsphere chip again, thoroughly free antibody to be measured in the flush away centrifuge tube.
7) add 500 milliliters of the PBS solution of HAV, the HBV contain fluorescence or luminescent substance specific mark, HCV, HIV four species specific antigens, 37 ℃ of concussions are incubated 30 minutes～1 hour, make they respectively with the antibody response to be measured that is combined on the magnetic microsphere, repeat the 6th) step, magnetic microsphere is thoroughly washed, abandoning supernatant keeps the magnetic microsphere precipitation.
8) with magnetic microsphere fluorescence or luminescence assays damping fluid, resuspended magnetic microsphere precipitation, use can be measured the equipment of magnetic microsphere impedance and magnetic microsphere surface fluorescence or luminous intensity simultaneously, carries out magnetic microsphere impedance (mensuration project) and magnetic microsphere surface fluorescence intensity (content of material) and measures.
9) interpretation as a result: if the n1 impedance magnetic microsphere surface fluorescence positive illustrates serum HAV antibody positive, fluorescence intensity and HAV antibody concentration are proportional.Adopt HAV antibody normal concentration to do reference and measure, can carry out accurately quantitatively HAV antibody.If n2, n3 and n3 impedance magnetic microsphere surface fluorescence are positive simultaneously, illustrate to have HAV antibody, HBV antibody, HCV antibody and HIV antibody in the blood simultaneously, and the like.
More than the operation example only plays an exemplary role, and specifically sees relevant technologies data and document for details.If survey n kind antibody materials, need the magnetic microsphere of the different resistance of n kind, operate the same.
Embodiment 3, disease association nucleic acid material are measured
Measure the specific nucleic acid fragment of certain microorganism, existence that can this microorganism of specific diagnostic.Conventional method comprises that color-coded diagnosis magnetic microsphere quantitative determination system once also can only finish 100 kinds of left and right sides nucleic acid fragments and measure, and can not satisfy the needs of hospital's laboratory diagnosis future development far away.The inventor adopts impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis once can measure countless versions specific nucleic acid fragment simultaneously.The inventor is an example to measure nipple warty virus, Chlamydia, mycoplasma specific DNA fragment simultaneously, describes.
1) purchase or the different impedances of oneself production can supply the latex magnetic microsphere of nucleic acid (DNA) mark.
On behalf of nipple warty virus, Chlamydia, mycoplasma and hepatitis B specific DNA fragment, 2) set different impedance microballoons (n1, n2, n3 and n4) measure project respectively.
3) purchase or oneself synthetic nipple warty virus, Chlamydia, mycoplasma and hepatitis B specific DNA fragment complementary base sequence DNA.
4) with nipple warty virus, Chlamydia, mycoplasma and hepatitis B specific DNA fragment complementary base sequence dna fragmentation, covalently bind in respective impedance magnetic microsphere surface, make the gene diagnosis magnetic microsphere.
5) centrifugal 10 minutes of 1500g or be placed on the magnetic sheet static 2-12 hour discards free unlabelled antigen.Four kinds of diagnosis magnetic microspheres mix by equal proportion with phosphate buffer (PBS) washing, carry out resuspendedly with containing antigen-antibody reaction PBS damping fluid, make every liter and contain 10 5The quaternary magnetic microsphere liquid chip of individual magnetic microsphere concentration.
6) 250 microlitre quaternary magnetic microsphere genetic chips are put into 1 milliliter centrifuge tube, add 250 microlitre serum or other mensuration samples (mixing at 1: 1) again as vaginal fluid and physiological saline, abundant mixing, 37 ℃ of concussion insulations 1 minute～2 hours make in magnetic microsphere surface known dna and the sample DNA to be measured fully hybridize combination.
7) centrifugal 10 minutes of 1500g or be placed on the magnetic sheet static 2-12 hour, supernatant discarded, resuspended with PBS, centrifugal again or be placed on the magnetic sheet static 2-12 hour, supernatant discarded again, quaternary impedance magnetic microsphere genetic chip is washed, thoroughly in the flush away centrifuge tube not with the DNA hybridization of latex surface on dna fragmentation free to be measured.
8) add 500 milliliters of the hybridization buffers of nipple warty virus, Chlamydia, mycoplasma and the hepatitis B specific DNA fragment (polynary probe) contain fluorescence or luminescent substance specific mark, 37 ℃ of concussions are incubated 30 minutes～1 hour, they are hybridized with the DNA to be measured that is combined on the magnetic microsphere respectively, repeat the 7th) step, magnetic microsphere is thoroughly washed, the free probe that abandoning supernatant is not hybridized keeps the magnetic microsphere precipitation.
9) with magnetic microsphere fluorescence or luminescence assays damping fluid, resuspended magnetic microsphere precipitation, use can be measured the equipment of magnetic microsphere impedance and magnetic microsphere surface fluorescence or luminous intensity simultaneously, carries out magnetic microsphere impedance (mensuration project) and magnetic microsphere surface fluorescence intensity (dna content) and measures.
10) interpretation as a result: if the n1 impedance magnetic microsphere surface fluorescence positive illustrates the human nipple warty of the serum viral DNA positive, fluorescence intensity and DNA concentration are proportional.Adopt human nipple warty viral dna fragment normal concentration to do reference and measure, can carry out accurately quantitatively human nipple warty viral DNA.If n1, n2, n3 and n4 impedance magnetic microsphere surface fluorescence are positive simultaneously, illustrate to have human nipple warty virus, Chlamydia, mycoplasma and hepatitis B specific DNA fragment in the blood simultaneously, and the like.
More than the operation example only plays an exemplary role, and specifically sees relevant technologies data and document for details.If survey n kind genetic stew, need the magnetic microsphere of the different resistance of n kind, operate the same.Above scheme also can be measured special RNA fragment.
Embodiment 4, biomedical scientific research field are used
Impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis has measures countless versions test substance or unknown materials simultaneously.In the gene age behind the mankind, the DNA of a large amount of thousands of kinds in the human body, RNA fragment and countless versions known antigens or antibody need to measure.DNA in the human body, RNA and antigenic substance and antibody materials complexity are various, and conventional method comprises that color-coded diagnosis magnetic microsphere quantitative determination system once also can only finish 100 kinds of left and right sides substance-measurings, can not satisfy the needs of scientific research development far away.
The single job of impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis can be measured the countless versions test substance simultaneously, by huge computer processing system, will reduce the research work personnel labor intensity greatly, improves research work efficient.
Now whole known mRNA kinds are example in the human normal liver cell to measure, and the application of impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis in the hepatic gene group is described.
1) suppose that the liver cell genome has 1,000,000 gene expressions, and base sequence is clear and definite.
2) buy or ownly produce 1,000,000 kinds of different impedances and can supply the latex magnetic microsphere of nucleic acid marking, and set 1,000,000 kinds of different impedances (n1, n2, n3...) magnetic microsphere represent respectively 1,000,000 kinds of mRNA to be measured (a1, a2, a3......).
3) the complementary base sequence DNA of purchase or oneself synthetic 1,000,000 kinds of mRNA to be measured.
4) will buy or the complementary base sequence dna fragmentation of oneself synthetic 1,000,000 kinds of mRNA to be measured, covalently bind in corresponding 1,000,000 kinds of impedance magnetic microsphere surfaces.Centrifugal 10 minutes of 1500g or be placed on the magnetic sheet static 2-12 hour, discard free unlabelled dna fragmentation, resuspended with phosphate buffer, centrifugal again 10 minutes of 1500g or be placed on the magnetic sheet static 2-12 hour, abandon supernatant, keep the diagnosis magnetic microsphere in the precipitation.1,000,000 kinds of different impedances diagnosis magnetic microspheres are used phosphate buffer (PBS) washing respectively, mix by equal proportion, carry out resuspendedly with containing antigen-antibody reaction PBS damping fluid, make every liter and contain 10 51,000,000 kinds of magnetic microsphere diagnostic reagents of individual magnetic microsphere concentration.
5) get 1,000,000 kinds every liter and contain 10 5The different impedance magnetic microsphere of individual magnetic microsphere concentration equal-volume mixes, and makes impedance variable 1,000,000 flux diagnose magnetic microspheres.
6) (every liter contains 10 to get impedance variable 1,000,000 flux diagnose magnetic microspheres 5Individual magnetic microsphere concentration) 250 microlitres are put into 1 milliliter centrifuge tube, add the total RNA extract of 250 microlitre liver cells again, abundant mixing, and 37 ℃ of concussion insulations 1 minute～2 hours make in magnetic microsphere surface known dna and the sample RNA to be measured fully hybridize combination.
7) centrifugal 10 minutes of 1500g or be placed on the magnetic sheet static 2-12 hour, supernatant discarded, resuspended with phosphate buffer, centrifugal again or be placed on the magnetic sheet static 2-12 hour, remove supernatant again, impedance variable 1,000,000 flux diagnose magnetic microspheres are washed, thoroughly in the flush away centrifuge tube not with the DNA hybridization of latex surface on RNA fragment free to be measured.
8) add 500 milliliters of the hybridization buffers of the complementary base sequence dna fragmentation of the mRNA to be measured contain 1,000,000 kinds of fluorescence or luminescent substance specific mark, 37 ℃ of concussions are incubated 30 minutes～1 hour, they are hybridized with the RNA to be measured that is combined on the magnetic microsphere respectively, repeat the 7th) step, magnetic microsphere is thoroughly washed, the complementary base sequence dna fragmentation of the free fluorescence that abandoning supernatant is not hybridized or the mRNA to be measured of luminescent substance specific mark keeps the magnetic microsphere precipitation.
9) with magnetic microsphere fluorescence or luminescence assays damping fluid, resuspended magnetic microsphere precipitation, use can be measured the equipment of magnetic microsphere impedance and magnetic microsphere surface fluorescence or luminous intensity simultaneously, carries out magnetic microsphere impedance (1,000,000 kinds of mensuration projects) and magnetic microsphere surface fluorescence intensity (rna content) and measures.
10) interpretation as a result: if the n1 impedance magnetic microsphere surface light signal positive illustrates the expression of liver cell a1 gene masculine.The n2 impedance magnetic microsphere surface light signal positive shows the expression of liver cell a2 gene masculine, and the like.If, n1, n2 impedance magnetic microsphere surface light signal total positives shows a1 in the liver cell, the a2 gene is positive expression simultaneously.If n1, n2, ten thousand kinds of magnetic microsphere surface light of n3....100 signal are all positive, show a1 in the liver cell, a2, a3 ... wait 1,000,000 kinds of equal positive expressions of gene.
More than the operation example only plays an exemplary role, and specifically sees relevant technologies data and document for details.If survey more kinds of mRNA materials, need the magnetic microsphere of more kinds of different resistance, operate the same.
Above scheme also is used to measure countless versions specific DNA fragment, antigen or antibody fragment.
Embodiment 5, existing unknown mRNA is an example in the human normal liver cell to measure, and illustrate that impedance variable unlimited flux diagnoses the application of magnetic microsphere quantified system analysis in the hepatic gene group.
Discover recently, limited though human gene quantity is huge, and human protein's quantity almost is unlimited.Because effects such as processing after the genetic transcription and modifications, the mRNA of encoding proteins (mRNA) quantity also is far longer than gene dosage.Most of mRNA and gene order are human and unclear, how to find that unknown gene, unknown mRNA and agnoprotein matter are the technical barriers that faces the human genome times afterwards comprehensively.
Impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis can detect countless versions DNA or RNA fragment simultaneously, provides laboratory facilities for solving direct searching unknown gene.Unknown gene (mRNA) is an example to the inventor in the liver cell to seek, and the purposes of impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis in seeking unknown gene is described.
General technology is difficult to accomplish, in cell cdna library, filters out unknown gene with numerous random probe (the known dna fragment of mark).The probability that filters out unknown gene for a kind of random probe is undoubtedly " looking for a needle in a haystack ", but uses the countless versions random probe, just can filter out all unknown genes theoretically.
Technology of the present invention has the feature of unlimited flux, just in time satisfies the technical requirement of screening unknown gene with numerous random probe in cell cdna library.The method of impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis screening unknown gene is as follows:
1) produces the magnetic microsphere of 1,000,000 different impedances at every turn, divide and carry out many times.
2) computing machine automatically with magnetic microsphere from impedance value record from small to large.
3) each synthetic at random 1,000,000 kinds of oligonucleotide fragments more than 7.
4) computing machine is by the impedance magnitude ordering and the 1000000 kinds of artificial target gene fragment of synthetic different oligonucleotide fragment representatives automatically of encoding respectively.
5), 1,000,000 kinds of synthetic at random oligonucleotide fragments more than 7 are marked at the magnetic microsphere surface of 1,000,000 different impedances respectively by impedance-encoded.
6) by impedance variable unlimited flux diagnosis magnetic microsphere quantitative analysis device, analyzing magnetic microsphere surface positive signal, and determine unknown cDNA fragment according to impedance value.
7) repeating step 1) to step 6), up to filtering out the unknown gene fragment.
Produce different impedance magnetic microspheres, magnetic microsphere surface indicia oligonucleotide fragment, and operations such as magnetic microsphere surface positive signal and impedance value judgement all can be finished automatically.Impedance variable unlimited flux diagnosis magnetic microsphere quantified system analysis can be used for screening unknown gene.

Claims (10)

1, a kind of impedance variable unlimited flux diagnosis magnetic microsphere, comprise the latex magnetic microsphere, it is characterized in that: the diagnosis magnetic microsphere has magnetic and contains the conductive materials of variable concentrations, comprise metal and carbon dust, conductive materials is to mix by arbitrary proportion with the latex raw material that contains magnetisable material.
2, impedance variable unlimited flux is diagnosed magnetic microsphere according to claim 1, it is characterized in that: 1) the different impedance series diagnosis of countless versions microballoon all has magnetic and precipitates under the action of a magnetic field, 2) the different specific antigens of different impedance series diagnosis magnetic microsphere surface covalent bond, antibody or specific DNA fragment, 3) the identical specific antigen of same impedance series diagnosis magnetic microsphere surface covalent bond, antibody or specific DNA fragment, 4) the covalently bound specific antigen in diagnosis magnetic microsphere surface, antibody or specific DNA or RNA fragment, be at having diagnosis in normal person or the patient's body, judge the corresponding antibodies to be measured that curative effect and scientific research are worth, antigen, DNA or RNA fragment.
3, the preparation method of impedance variable unlimited flux diagnosis magnetic microsphere as claimed in claim 1 or 2, it is characterized in that: 1) conductive materials and magnetic microsphere synthesis material are mixed by arbitrary proportion, produce the latex magnetic microsphere of different resistance characteristic, 2) represent different test substances by the magnetic microsphere of different impedance value or scope, work out different impedances or the scope magnetic microsphere different test substance corresponding tables of encoding, 3) represent different test substance corresponding tables according to different impedances or scope magnetic microsphere, with different impedances or scope magnetic microsphere surface, the specific antigen that mark is different, antibody, specific DNA or RNA fragment.
4, represent different test substances as the magnetic microsphere of claims 3 described different impedance value or scope, it is characterized in that: 1) test substance is a biomedical sector, comprise diagnosing the illness, observe test substance or unknown materials that curative effect of medication and scientific research need; 2) the different impedance magnetic microspheres of the preparation countless versions different test substances of countless versions of can representing or encode; 3) magnetic microsphere impedance different range series can be represented different series test substance kind.
5, a kind of detection method of impedance variable unlimited flux diagnosis magnetic microsphere as claimed in claim 1 or 2, it is characterized in that: 1) different impedance series diagnosis magnetic microspheres are mixed with testing sample, carry out antigen-antibody specific bond and making nucleic acid molecular hybridization, wash magnetic microsphere with phosphate buffer then; 2) will wash specific antibody, antigen or the dna fragmentation of back magnetic microsphere and fluorescence or other shiner mark, carry out specific bond, make different impedance diagnosis magnetic microspheres surface fluorescence or luminous signal occur, fluorescence or luminous magnetic microsphere suspend with the phosphate buffer washing more again through centrifugal or be placed on the magnetic sheet and attract the magnetic microsphere post precipitation; 3) different impedances series fluorescence in washing back or luminous diagnosis magnetic microsphere by the magnetic microsphere impedance analysis, according to different impedances or the scope magnetic microsphere different test substance corresponding tables of encoding, are judged the detection material kind; 4) different impedance series immunofluorescences in washing back or luminous magnetic microsphere by magnetic microsphere surface light signal strength analysis, according to known standard product concentration, calculate test substance content.
6, detection method as claimed in claim 5, it is characterized in that: with the different impedance series diagnosis of the countless versions magnetic microsphere of 10% concentration, put into centrifuge tube, add capacity testing sample or standard items, 35-39 ℃ of incubation, the centrifugal 5--8 of 2000g minute or be placed on the magnetic sheet and magnetic microsphere precipitated fully in 2-12 hour, with phosphate buffer washing 2 times, abandoning supernatant, resuspended with phosphate buffer; The antibody, antigen or the dna fragmentation that add fluorescence or luminous plain mark again, 35-39 ℃ incubation 30--60 minute, the centrifugal 5--8 of 2000g minute, or be placed on the magnetic sheet and magnetic microsphere precipitated fully in static 2-12 hour, abandoning supernatant is left and taken magnetic microsphere, carries out resuspended with phosphate buffer, centrifugal again or be placed on the magnetic sheet and magnetic microsphere precipitated fully in static 2-12 hour, use phosphate buffer resuspended again.
7, detection method as claimed in claim 5, it is characterized in that: to the different impedance series diagnosis magnetic microspheres of combined with fluorescent or luminescent substance, after the phosphate buffer washing is resuspended, add fluorescence or luminescence assays reagent, measure its impedance by the magnetic microsphere impedance measuring instrument, detect fluorescence or luminous intensity by magnetic microsphere surface fluorescence or luminous intensity pick-up unit; Judge the test substance kind by the magnetic microsphere impedance measuring, judge test substance content by fluorescence or luminous signal strength detection.
8, detection method as claimed in claim 7, it is characterized in that: utilize a kind of device that detects the different impedance series diagnosis magnetic microsphere impedance magnitude of countless versions and magnetic microsphere surface fluorescence or luminous signal intensity simultaneously, this device comprises three parts, first is a magnetic microsphere impedance quantitative measurement device, can measure the single magnetic microsphere resistance sizes by micropore; Second portion is magnetic microsphere surface fluorescence or luminous signal detection by quantitative device, can measure the single magnetic microsphere surface light signal intensity of the different impedance series of countless versions; Third part is the software analysis system, can receive single magnetic microsphere impedance signal and this magnetic microsphere surface fluorescence or luminous signal, according to the different impedance magnetic microspheres different test substance corresponding relations of encoding, judge the test substance kind, according to the magnetic microsphere light signal strength of known standard product content, calculate test substance content.
9, as claims 8 described a kind of devices that detect the different impedance series diagnosis magnetic microsphere impedance magnitude of countless versions and magnetic microsphere surface fluorescence or luminous signal intensity simultaneously, it is characterized in that: 1) magnetic microsphere impedance quantitative measurement device, magnetic microsphere surface fluorescence or luminous signal detection by quantitative device, and the software analysis system, be system and device unified rather than that separate; 2) described software analysis system is that at first default different series magnetic microsphere impedance ranges coding different series is measured project, the different magnetic microsphere impedance value different test substance kinds of encoding, judge test substance according to the magnetic microsphere resistance value then, according to magnetic microsphere surface fluorescence or luminous signal, do reference with reference material, calculate test substance content, and transfer data to printer, print the report of countless versions test substance content results automatically.
10, the purposes of impedance variable unlimited flux diagnosis magnetic microsphere as claimed in claim 7 in biomedicine, it is characterized in that: quantitative measurement simultaneously is used to diagnose the illness with countless versions in a sample, observe the Antigens of curative effect of medication and scientific research, material to be measured or unknown materials such as antibody class or nucleic acid class.
CNA2007103069757A 2007-12-29 2007-12-29 Unlimited flux magnetic microsphere quantitative determination system and uses in biomedicine thereof Pending CN101349690A (en)

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