CN1065339A - Method and apparatus for magnetic particle-based luminescence assays containing composite magnets - Google Patents
Method and apparatus for magnetic particle-based luminescence assays containing composite magnets Download PDFInfo
- Publication number
- CN1065339A CN1065339A CN 92101900 CN92101900A CN1065339A CN 1065339 A CN1065339 A CN 1065339A CN 92101900 CN92101900 CN 92101900 CN 92101900 A CN92101900 A CN 92101900A CN 1065339 A CN1065339 A CN 1065339A
- Authority
- CN
- China
- Prior art keywords
- particle
- electrode
- measuring
- electrogenerated chemiluminescence
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/66—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light electrically excited, e.g. electroluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
-
- 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
-
- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
-
- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2458/00—Labels used in chemical analysis of biological material
- G01N2458/30—Electrochemically active labels
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Plasma & Fusion (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
本申请公开并且要求保护通过电极测定电致化 学发光对样品中存在的有意义的分析物进行结合测 量的方法和装置。所述方法采用磁性感应颗粒。本 发明的方法和装置需要产生磁场的南北向复合电磁 体或永久磁体用于收集颗粒。
The present application discloses and claims a method and apparatus for the binding measurement of an analyte of interest present in a sample by electrochemiluminescence determined by an electrode. The method employs magnetically sensitive particles. The method and apparatus of the present invention require north-south compound electromagnets or permanent magnets to generate a magnetic field for particle collection.
Description
The present invention relates to by measuring electrogenerated chemiluminescence on the electrode in conjunction with the method for meaningful analyte in the measuring samples, the measurement that compound wherein comprises sample, contain the component that is connected with the tagged compound that can induce electrogenerated chemiluminescence is with the material and the formation Magnetic Induction suspended particle that can combine with analyte or material; Make the method that produces magnetic field on the particle by compound north-south permanent magnet or electromagnet, collect this compound at electrode surface; With the tagged compound induced luminescence; And measurement is penetrated luminous.The invention still further relates to and be used to implement the permanent magnet with compound north-south of this method or the device of electromagnet.
About detect and quantitative test biochemical substances and biological substance in analyte, research and development many methods and system.Method and the system that can measure trace microorganism, medicine, hormone, virus, antibody, nucleic acid and other albumen have great importance to researchist and clinical staff.
Adopt known association reaction method, can detect and analyze big quantity of material, antigen-antibody reaction for example, nucleic acid hybridization technique and albumen-ligand system.High degree of specificity in many biochemistry and the biological articulated system has produced research and has diagnosed significant many measuring methods and system.Specifically, on one or more bond materials, whether there is the label of observable, can illustrates whether there is significant analyte.Especially meaningfully, label is made luminescent material by photochemistry, chemistry and electrochemical method." photoluminescence " is a kind of method that can induced luminescence when the absorbed electromagnetic radiation.Fluorescent and phosphorescence then are the exemplary of photoluminescence." chemiluminescence " method then is that the chemical conversion by energy provides luminous method." electrogenerated chemiluminescence " is to provide luminous by electrochemical method.
The chemiluminescence measuring technique that has now grown up is to mix mutually with reactant with the chemiluminescent labels mark containing the sample of meaning analysis thing, and this reaction mixture is after inducing, and the labeling reaction of some part combines with analyte.After inducing the bound fraction of potpourri is separated with bound fraction not, the concentration of label can be measured by chemiluminescence method in these two parts or the part wherein.The chemiluminescent amount of being measured in these two parts or its part can illustrate the content of significant analyte in the biological sample.
Electrogenerated chemiluminescence (ECL) measuring technique is the improvement to chemiluminescence.This method provides sensitive existence and the concentration thereof of critically measuring meaningful analyte.When using these class methods, will pass through a volt-ampere working electrode stimulated luminescence through the sample of inducing.In specific chemical environment, this electrogenerated chemiluminescence is in the regular hour and adopts the voltage that is applied on the working electrode under the condition of certain method to excite.The light that measurement is produced by label, thus existence or its content of analyte can be described.About the detailed introduction of electrogenerated chemiluminescence, can consult the disclosed patent application document of PCT: US85/01253(WO86/02734), US87/00987 and US88/03947, above-mentioned document also is the list of references of this paper.
Carrying out electrogenerated chemiluminescence when measuring, being preferably in does not need separating step in the process of measurement, and the signal modulation of variable concentrations analyte is put at utmost, thereby can obtain the measurement of accurate sensitivity.In the existing method of no separating and measuring, microparticle is suspended in the measuring samples, combine in conjunction with component with one or more measurements.
United States Patent (USP) 4305925 relates to and uses nephelometry and turbidimetry detection and mensuration and clinical proteins associated and peptide, disclosed method to comprise antigen or antibody are combined with the latex particle with light scattering or optical absorption.
United States Patent (USP) 4480042 relates to the technology of the particle reagents that use is made up of the shell core particle, and its particle shell contains meaningful biologic artifact can covalently bound with it functional group; And the high index of Response of shell core has formed the susceptibility to light scattering measurement.This technology is based on agglutinating reaction, and it produces the agglutinator that can in all sorts of ways and detect and/or measure by bivalent antibody and polyvalent antigen reaction.
United States Patent (USP) 4419453 also relates to the application that can be used for detecting the band look latex agglutination assay method that whether has immuno-chemical substance (for example antibody and immunogene).According to this prior art, as if also microparticle can not be used to measure luminescence phenomenon.But be expected by luminously being absorbed of partly producing of free chemiluminescence or electrogenerated chemiluminescence, scattering even also can be subjected to the interference of microparticle.
United States Patent (USP) 539389(PCT application number U.S.89/04919 unexpectedly) specificity of sensitivity that discloses luminescence phenomenon is in conjunction with assay method, and wherein the inertia microparticle can carry out specificity with an association reaction thing in the mensuration system and combines.This measurement can be carried out in heterogeneous (one or more separating step), carries out then more favourable in homogeneous phase (no separating step).
United States Patent (USP) 89/04919 relates to the composition of the association reaction that is used to measure luminescence phenomenon, and said composition comprises the composite suspension particle with the surface that can combine with the component in measuring potpourri.On the other hand, also provide the system of detection or quantitative test analyte in sample, this system can realize measuring method with measurement composition of the present invention.This system is included in the measuring media the device of tagged compound induced luminescence and is used for the luminous measurement mechanism of test sample analyte.
Have now found that, the component that partly is connected with electrogenerated chemiluminescence in the measuring system combines with the microactuator suspension particle, can modulate the intensity of the luminous signal that partly produces by the electrogenerated chemiluminescence that is connected with this component greatly, thereby the device of specificity association reaction in the monitoring measuring system is provided.Find also that more surprisingly suspended particle is to the very little or not influence of influence of the luminous signal intensity that produced by the electrogenerated chemiluminescence part that is connected with component in this system, the component in the described system does not combine with the microactuator suspension particle yet.
Therefore, described in the United States Patent (USP) 89/04919 in the test sample method of analyte comprise the steps:
(1) form composition, said composition contains:
(a) contain the sample that might exist required analyte;
(b) be used to the material measured, be selected from: (ⅰ) need analyte or the similar analysis thing measured; (ⅱ) analyte of need measuring or similar analysis thing in conjunction with object; (ⅲ) can with (ⅰ) or the reactive component that (ⅱ) combines, a kind of material of the wherein said material that is used for measuring is connected with the tagged compound with chemical part that can induced luminescence; With
(c) can be with analyte and/or (b) (ⅰ), (ⅱ) or (ⅲ) described in the composite suspension particle that combines of material specificity;
(2) incubation composition forms the compound that contains particle and described tagged compound;
(3) with the tagged compound induced luminescence; And
(4) measure luminous by the composition emission, detect the analyte that is present in the sample.
By measuring the luminous contrast of composition of the luminous of composition and the analyte that contains known quantity, above-mentioned identical method all can be used for the quantitative test content of analyte in sample.
Similar analyte can be natural, also can synthesize, and they are the compounds with the binding specificity that can do to contrast with analyte, but also comprises the compound that binding ability is higher or lower.Be suitable for and of the present inventionly be in conjunction with object that the public is familiar with, antibody, enzyme, nucleic acid, lectin, co-factor and acceptor are for example arranged.Can be secondary antibodies or with analyte or its analog and/or the reactive component that combines the object combination with it such as the albumen of albumin A or Protein G, also can be avidin or biotin, or other components that can participate in association reaction known in the art.
It is luminous that luminous electrogenerated chemiluminescence (ECL) of preferably being induced through the effect of volt-ampere working electrode by tagged compound (no matter whether it has combined object combination or not combination with specificity) produces.The electrogenerated chemiluminescence reaction mixture applies voltage in the regular hour with certain method on working electrode, make it to produce light, thereby controlledly launches bright dipping.Though visible emitting is better, composition or system also can launch the electromagnetic radiation of other types, for example infrared light or ultraviolet light, X ray, microwave etc.Term used herein " electrogenerated chemiluminescence ", " electrogenerated chemiluminescence ", " luminous ", " luminous " and " emission light " all comprise emission light and other forms of electromagnetic radiation.
The method that United States Patent (USP) 89/04919 is introduced requires in the various measurements that research and clinicing aspect carry out detection and the very small amount of analyte of quantitative test.The needs of researchist and clinical staff make it to become and reduce all detection limit that these methods of employing are measured very urgently, improve the sensitivity of these measurements and the speed that they can measure.
Before various labels are measured, earlier they are concentrated, thereby improve the signal that produces by their, be known to the public about the whole bag of tricks of this respect.In United States Patent (USP) 4652333, can before measurement, concentrate by microfiltration with the particle of fluorescence, phosphorescence or atomic fluorescence label mark.
The method that concentrates the labelled immune chemical substance before measurement is also understood by the public of this area, for example the Magnetic Induction marking particle can be shifted near the surface of measuring vessel.In United States Patent (USP) 4731337,4777145 and 4115535, earlier particle is shifted near chamber wall exactly, then the fluorescence of launching excitation light.
In United States Patent (USP) 4945045, particle is concentrated on the magnetic pole.Electrochemical reaction occurs on the electrode of being simplified by the mark chemical mediator.Generation in conjunction with the time, the immunochemistry association reaction has changed the efficient of the amboceptor that produces modulation signal.
Existing method does not all relate to the method that surface selectivity of the present invention excites.Illustrated that owing to do not excite any mechanics of (for example electrogenerated chemiluminescence) to explain can think that so the label on the solid-phase complex is inevitable oxidized on electrode, this just needs electronics to be moved to electrode by label in conjunction with usable surface.Can also think that this " transition " of electronics is because known tunnel(l)ing produces, promptly electronics needn't " be crossed " potential barrier and just can pass through space (zone that potential energy is very high, for example solution).Electronics can need not other intake by potential barrier, promptly can be moved to another molecule by a molecule, or be moved to electrode by a molecule.But this tunnel(l)ing just can occur in the very short distance, and the probability of tunnel(l)ing is with the index decreased of distance between two kinds of increases.If distance is less than 25A(2.5nm), the probability that then occurs in tunnel(l)ing between the two is very high; If distance is very big, then probability is very low.The distance of 25A is the thumb rule that this area professional uses, and is not absolute limitations.
Therefore, those electrogenerated chemiluminescence labels that only have a 25A electrode surface just can be expected to participate in the process of electrogenerated chemiluminescence.In general, the particle area in 25A electrode surface scope is very little.
Therefore, can not expect to measure any significant amount by the electrogenerated chemiluminescence that particle surface produces.But must could arrive photomultiplier cell by particle by the light that the electrogenerated chemiluminescence method produces.Because particle is opaque (concentrated suspension liquid of particle is black) basically,, can not expect that luminous energy passes through particle and measures with photomultiplier cell even can produce a large amount of light by electrogenerated chemiluminescence.In a word, prior art does not provide or advises using compound north-south magnet, however this method and apparatus of the present invention just.
The objective of the invention is to provides separation (heterogeneous) or does not have separation (homogeneous phase) specificity associated methods and device by measuring the electrogenerated chemiluminescence by the measurement composition emission that contains magnetic corpuscular material and the compound north-south of use magnet.
The method and apparatus that another object of the present invention is to provide, compared with the prior art, sensitivity, specificity and precision are all higher, and Measuring Time is rapid and detection limit is lower.
Used term is defined as among the application: " electrogenerated chemiluminescence part ", " metallic electrogenerated chemiluminescence part ", " label ", " tagged compound " and " mark substance " all commutative use." electrogenerated chemiluminescence part ", " containing metal electrogenerated chemiluminescence part ", " organometallics ", " metallo-chelate ", " transition metal chelate ", " rare-earth chelates ", " tagged compound ", " mark substance " all are connected with molecule with " label ", for example analyte or its analog, analyte or its analog in conjunction with object, and described in conjunction with object in conjunction with object, or can or combine the reactive component of object combination with described analyte or its analog, the above all belongs to scope of the present invention.Above-mentioned all kinds of can also being connected with one or more compositions in conjunction with object and/or one or more reactive components.In addition, above-mentioned all kinds of can also be connected with in conjunction with object, reaction combination or one or more analyte or its analogs in conjunction with the composition combination of object and/or one or more reactive components.Belonging to above-mentioned directly all kinds of or other molecules from what has been discussed above of also having of the scope of the invention combines with analyte or its analog.In brief, these ligands all can be used as measurement of species.
Term " detection " and " quantitative test " are all represented to measure, and can be understood as: quantitative test can require to prepare relevant composition and calibration.
Term " is collected and concentrated compound " and can be used for describing the collection concentrated and compound on electrode surface of measuring the composition compound mutually with exchanging.
Description of drawings:
Fig. 1 represents to implement measuring chamber and the built-up magnet that magnetic corpuscular of the present invention separates or do not have separating measuring method with Fig. 2; The magnetic field line that the built-up magnet of magnet system produces all parallels with electrode surface basically.
Fig. 3 represents to use the sedimentation measurement chamber of the electromagnet of Fig. 1 and Fig. 2, and compound is deposited on the electrode surface.
Fig. 4 is illustrated respectively in the relative speed of microparticle compound deposition under the influence of magnetic field (Fig. 1 and Fig. 2) and gravity, be the deposition that causes of magnetic field (Fig. 1 and Fig. 2) and the contrast of the microparticle sedimentation time between the gravity sedimentation, wherein deposition value in magnetic field is represented with soft dot, and the gravity sedimentation value is represented with the black round dot.
Fig. 5 represents to comprise the collecting chamber of the permanent magnet of Fig. 1 and Fig. 2.
Fig. 6 represents the increase and the time function relation of measuring with Fig. 5 measuring chamber of electrogenerated chemiluminescence intensity, and promptly acquisition time is to the influence of electrogenerated chemiluminescence intensity.
Fig. 7 is illustrated near the magnetic line of force the electrode surface of magnet (Fig. 1 and 2) under the electrode surface.
Fig. 8 represents to implement the measuring chamber that microparticle of the present invention does not have separation and separating and measuring, and this measuring chamber adopts working electrode and built-up magnet illustrated in figures 1 and 2.
Fig. 9 represents the sketch with the voltage-operated device of measuring chamber when measuring of Fig. 8.
In a embodiment the wideest of the present invention, be method in conjunction with analyte in the measuring samples, its measuring process comprises:
(a) form composition, it contains:
(ⅰ) described sample;
(ⅱ) measure and to use material, its contain the component that is connected with the tagged compound that can induce electrogenerated chemiluminescence and
(ⅲ) can carry out the composite magnetic induction suspending particle that specificity combines with material with analyte or described measurement;
(b) the described composition of incubation is formed the compound that contains particle and described tagged compound;
(c) described composition is imported measuring chamber;
(d) by magnetic field being applied on the described particle, collect described compound at electrode surface;
(e) by voltage being applied on the described electrode, with the tagged compound induced luminescence in the collected compound; And
(f) penetrated in the electrode surface measurement luminous, thereby the analyte in the measuring samples, wherein under the magnet in compound north-south, permanent magnet or the electromagnet arranged perpendicular electrode surface in the horizontal direction.
The present invention also according to measuring electrogenerated chemiluminescence at electrode surface, provides the device that carries out in conjunction with analyte in the measuring samples, and this device comprises:
(a) sample measurement chamber comprises container, doorway device, electrode and collect the device of equally distributed compound basically on described electrode surface;
(b) on described electrode, apply the device of voltage; And
(c) device of the electrogenerated chemiluminescence that measurement produces on described electrode, make the equally distributed device of compound, comprise that the magnetic field that makes generation is suitable for the device of electrode, make the magnetic line of force in magnetic field and the electrode surface almost parallel in described surface region, and the device that produces magnetic field, comprise the north-south and the built-up magnet of isolating at the nonmagnetic substance under the electrode by arranged perpendicular.
Adopt said method, collect and concentrate compound, can implement to various differences heterogeneous and homogeneous phase and form thing and carry out combination measurement at electrode surface.,, before measuring produce by label luminous, should earlier compound be separated with composition in conjunction with in measuring heterogeneous.In homogeneous phase measurement, to separating with unconjugated labelled reagent in conjunction with (solid phase).
In heterogeneous measurement, when compound was concentrated on working electrode surface, the measuring-signal that is produced by label was much larger than the signal of collecting step.In contrast, but and no change by the signal that produces without compound labelled reagent.Therefore, although exist not compound labelled reagent in measuring chamber, the signal that is produced by the compound of collecting far is better than the signal of not collecting compound when measuring.Collect the result of step, significantly improved in conjunction with the detection limit of measuring.
In the present invention, all combine to measure and to comprise separating step on the spot.Will measure that composition (be sample, measure with material and particle) pumps into measuring chamber and by working electrode intercepting and capturing compound after, another liquid is pumped in the measuring chamber that does not contain label or labelled reagent, thus wash on the spot or with compound with measure composition in do not separate in conjunction with component.This process of measurement says to be heterogeneous in conjunction with measuring from technical standpoint, and still the advantage of separating in measuring chamber is that it does not need additional tripping device, and in general, handles also than externally separating rapidly.
Employing the inventive method can be carried out heterogeneous combination measurement with the mutual mixing of each component of measurement composition and with their reaction preset times.To measure composition then and separate, comprising separating of solution and particle.Then measure the electrogenerated chemiluminescence in compound or the solution.With the electrogenerated chemiluminescence of measurement compound may make the Measurement and analysis thing have higher precision and lower detection limit concentrating afterwards without concentrated comparing.
From following introduction to some preferred embodiments, will be to the present invention and purpose and the clearer understanding all sidedly of character.
The present invention can be widely used in participating in the various analytes of association reaction.These reactions comprise: Ag-Ab, and the ligand acceptor, DNA and RNA react to each other and other known response.The present invention relates to the method and apparatus of this class analyte in qualitative and the detection by quantitative multicomponent sample, this method and apparatus comprises compound north-south magnet.
Sample
The sample that contains analyte can be solid, emulsion, suspending liquid, liquid or gas, can be derived by cell and cell derivative, water, food, blood, bloodstain, scared, sweat, urine, ight soil, tissue, saliva, oils, organic solvent or air.Sample can also be water, acetonitrile, dimethyl sulfoxide, dimethyl formamide, n-methyl pyrrolidone or alcohols, and their potpourri.
Analyte
Typical analyte is intact cell or surface antigen in the sample, the subcellular fraction particle, virus, Prion, viroid, antibody, antigen, haptens, fatty acid, nucleic acid, protein, lipoprotein, polysaccharide, lipopolysaccharides, glycoprotein, peptide, polypeptide, products of cellular metabolism, hormone, medicine, synthetic organic molecule, the organic metal molecule, sedative, barbiturates, alkaloid, steroid, vitamin, amino acid, sugar, lectin, recombinant or derived protein, biotin, avidin, streptavidin or inorganic molecule.In general, the concentration of analyte is 10
-3The mole or be lower than 10
-3Mole, for example 10
-12Mole or lower.
The measurement material
The measurement that combines with the sample that contains analyte contains a kind of following material that is selected from least with material: (ⅰ) add above-described analyte or its analog, (ⅱ) analyte or its analog in conjunction with object, (ⅲ) can with (ⅰ) or the above-mentioned reactive component that (ⅱ) combines, described measurement is connected with a kind of material in the material and compound or part the electrogenerated chemiluminescence part of induced luminescence (for example can).Material through mark can be intact cell or surface antigen, the subcellular fraction particle, virus, Prion, viroid, antibody, antigen, haptens, lipid, fatty acid, nucleic acid, protein, lipoprotein, polysaccharide, lipopolysaccharides, glycoprotein, peptide, polypeptide, products of cellular metabolism, hormone, medicine, synthetic organic molecule, the organic metal molecule, sedative, barbiturates, alkaloid, steroid, vitamin, amino acid, sugar, non-biological polymer (preferred solubility), lectin, recombinant or derived protein, biotin, avidin, streptavidin or inorganic molecule.In one embodiment, reagent is a kind of electrogenerated chemiluminescence part, itself and antibody, antigen, nucleic acid, haptens, short nucleotide sequence, oligomer, ligand, enzyme or biotin, avidin, streptavidin, a-protein, protein G, or their compound, or react to each other by protein can be secondary to object with nascent other of object combination of combining.
The analog of analyte can be natural, also can synthesize.In general, the bonding properties of this compounds should be suitable with analyte, but also can be to have compound higher or low binding ability.The reactive component of the electrogenerated chemiluminescence part that can be connected with analyte in conjunction with object and by them with analyte or its analog and/or its is secondary antibodies or albumen preferably, for example albumin A or Protein G, or avidin or biotin, or other components that can participate in association reaction known in the art.
Label
Electrogenerated chemiluminescence partly is a metallo-chelate, and the metal of this chelate is suitable for using any metal, for example promptly is applied under the electrical conditions on the reactive system of being discussed metallo-chelate that can be luminous under electrochemical conditions.The metal of this metalloid chelate for example has transition metal or rare earth metal.Described metal preferentially uses ruthenium, osmium, rhenium, iridium, rhodium, platinum, indium, palladium, molybdenum, technetium, copper, chromium or tungsten, preferably uses ruthenium and osmium.
The ligand that is connected with metal in the chelate is generally natural heterocycle or organic compound, and whether they are water-soluble at the decision metallo-chelate, play an important role in organic solvent or other non-aqueous solvents.Ligand can be a polydentate compound, and can be substituted.There is tooth compound ligand to comprise aromatics and aliphatic ligand.The aromatics polydentate compound ligand that is fit to comprises the aromatic heterocycle ligand.Preferable aromatic heterocycle ligand all is nitrogenous, and for example dipyridine joins pyrazoles, terpyridyl and phenanthrol.Suitable substituting group comprises alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, carboxylate, formaldehyde, formamide, cyano group, amino, hydroxyl, imido grpup, hydroxyl carbonyl, amino carbonyl, amidine, Guanidinium salt, uride, contains sulfenyl, phosphorous-containigroups groups and N-hydroxy-succinamide carboxylate.Chelate can contain one or more monodentate compound ligands, and wherein a large amount of ligands all is known.Suitable monodentate compound ligand comprises phosphine, amine, stilbene and the arsine of carbon monoxide, prussiate, isocyanide, fontanelle compound and aliphatic series, aromatics and heterocycle.
Suitable chelate can comprise: two ((4,4 '-carbomethoxy)-2,2 '-dipyridine 2-(3-(4-methyl-2,2 '-dipyridine-4-yl) propyl group)-1,3-dioxolanes ruthenium (II); Two (2,2 '-dipyridine) (4-(fourth-1-aldehyde)-4 '-methyl-2,2 '-dipyridine) ruthenium (II); Two (2,2 '-dipyridine) (4-(4 '-methyl-2,2 '-dipyridine-4 '-yl)-and butyric acid) ruthenium (II); Three (2,2 '-dipyridine) ruthenium (II); (2,2 '-dipyridine) (two-two (1, the 2-diphenylphosphino) ethene) 2-(3-(4-methyl-2,2 '-dipyridine-4 '-yl) propyl group)-1,3-dioxolanes osmium (II); Two (2,2 '-dipyridine) (4-(4 '-methyl-2,2 '-dipyridine)-butylamine) ruthenium (II); Two (2,2 '-dipyridine) (1-bromo-4(4 '-methyl-2,2 '-dipyridine-4-yl) butane) ruthenium (II); Two (2,2 '-dipyridine) the maleimide guanidine-acetic acid, 4-methyl-2,2 '-dipyridine-4 '-butyramide ruthenium (II).Other electricity causes the optics luminous component can consult PCT application US87/00987 and 88/0394, and these documents also are the application's list of references.
The effect of electrogenerated chemiluminescence part is to launch electromagnetic radiation, thereby it is imported the electrochemical energy reactive system.For this reason, they must be stimulated to become to be excited the energy state and can also to launch the electromagnetic radiation that is transformed into from excited state, for example the photon in the light.The constraint of not wishing to be subjected to the mechanism theory of electrogenerated chemiluminescence subparticipation electrogenerated chemiluminescence reaction to analyze, we think by electrochemical energy is imported reactive system and oxidation, then by with reactive system in the reactant phase reaction be converted into excited state.This state is also unstable comparatively speaking, and metallo-chelate is converted into comparatively stable status rapidly.So chelate sends the detected electromagnetic radiation of energy, for example photon in the light.
The amount of the used metallo-chelate of the present invention or other containing metal electrogenerated chemiluminescences part changes along with different system.In general, the consumption of this part can make effectively by the emission that produces the electromagnetic energy that can detect (can carry out quantitative measurment when needing) in above-mentioned composition or the system.In general, the detection of analyte and/or quantitative measurment are that luminous and luminous the comparing that partly demarcate with the analyte and the electrogenerated chemiluminescence of known quantity that will contain the sample of analyte and electrogenerated chemiluminescence part obtains.This is to carry out homogeneous phase measurement.As for heterogeneous measurement, then, measure again after before carrying out Electrochemiluminescprocess process, separating earlier by as previously mentioned.
One of skill in the art is appreciated that the homogeneity and the consumption of metal electrogenerated chemiluminescence part change with actual conditions in different system.One of skill in the art need not undue experimentation according to information provided herein, can rule of thumb determine to obtain required result's suitable containing metal electrogenerated chemiluminescence part and enough consumptions thereof.
Particle
It is for example 0.05 μ-200 μ m of 0.001-200 μ m(that particle has diameter) the microparticle material, preferred diameter is 0.1 μ m-100 μ m, especially be the best with 0.5 μ m-10 μ m, its surface component can combine with analyte and/or above-mentioned one or more other materials.For example the microparticle material can be crosslinked starch, glucosan, cellulose, protein, organic polymer, styrol copolymer, for example styrene/butadiene copolymers, acrylonitrile/butadiene/styrene multipolymer, acrylic acid vinyl acetate copolymer or vinyl chloride/acrylic copolymer, the inert inorganic particle, chromium dioxide, the oxide of iron, silicon and silicon potpourri, and protein matter, or their potpourri.Preferably with particle suspending in the electrogenerated chemiluminescence system.But particle must be maybe must comprise the Magnetic Induction particle.Also must be noted that: sample, analyte, measurement comprise above-described any combination and component with material, label, measuring media, reagent and other measurement component and particle, undoubtedly all belong to scope of the present invention.
Measuring media
For the system by electrode importing electrochemical energy can be operated, need provide the dielectric that can immerse electrode and contain the electrogenerated chemiluminescence part.Dielectric carries electric charge by ion.In general, dielectric is liquid phase, the solution of being made up of one or more salt or other class materials and water, organic liquid or liquid organic mixture or water and one or more liquid organic mixtures.But, in some embodiment of the present invention, also can use other forms of dielectric.For example dielectric can be the dispersions of one or more materials in fluid (as liquid, steam or supercritical fluid), also can be the solution of one or more materials at solid, steam or supercritical fluid.
Suitable dielectric is the aqueous solution of salt.Salt can preferably use sodium salt or sylvite, also suits but add other kations in some embodiment, as long as this kation does not influence the order of electrogenerated chemiluminescence reaction.Anion salt can be a phosphate, but also can use other negative ion in some embodiment of the present invention, as long as selected negative ion does not influence the order of electrogenerated chemiluminescence reaction.
Composition is non-aqueous composition also.Yet in some embodiment, can use supercritical fluid, more particularly, can use the dielectric of the non-aqueous composition that contains organic liquid.The same with the water power medium, nonaqueous dielectric also is to carry electric charge by ion.In general, this means that salt is dissolved in the organic liquid medium.Suitable organic liquid has acetonitrile, dimethyl sulfoxide (DMSO) (DMSO), dimethyl formamide (DMF), methyl alcohol, ethanol, or above-mentioned two or more potpourri.Need to prove and use the tetraalkylammonium salt (for example tetrafluoro boric acid tetrabutylammonium) that dissolves in organic solution, it forms nonaqueous dielectric with the above.
In some embodiment of the present invention, dielectric is a buffer system, and phosphate buffer commonly used then meets desirable.Sodium phosphate-sodium-chloride water solution and sodium phosphate-sodium fluoride aqueous solution then are the examples of this respect.
Other measurement component
Apply for the US89/04859(denomination of invention as disclosed PCT: the reductive agent that utilizes amine to derive carries out the electrogenerated chemiluminescence reaction) as described in, desirable reductive agent, be (more macromolecular) amine or contain amine moiety in general, can be oxidized and after natural decomposition, be converted into high reductive agent.This article is also as the list of references of this paper.Can think, amine or contain amine moiety also can be oxidized by electrochemical energy is imported reactive system.Amine or contain amine moiety and lose an electronics, deprotonation or rearrange is converted into strong reductant then.This reductive agent with through the containing metal electrogenerated chemiluminescence partial reaction of peroxidating, make it become aforesaid excited state.For this reason, amine or contain the residue that amine moiety preferably has the carbon atom center, in the deprotonation process that forms reductive agent, it contains an electronics of being supplied with by this carbon atom and the alpha-carbon atom that can play the proton donor effect.The reductive agent that amine is derived provides the containing metal electrogenerated chemiluminescence partly has been converted into the required stimulus of its excited state, and launching thus can detected electromagnetic radiation.
Many amine or contain amine moiety accordingly and all can be used for implementing the present invention.In general, amine or the selection that contains amine moiety should be suitable for carrying out the pH value of the system of Electrochemiluminescprocess process.Another relevant factor is that amine or the environmental facies that contain the effect that amine moiety should must produce when analyzing adapt to, and promptly adapts with possible water or non-water environment.Another factor of need considering then is the reductive agent that amine that amine or the selection that contains amine moiety should form under concrete condition is derived, and it is enough in this system reduction through the containing metal electrogenerated chemiluminescence part of peroxidating.
Being used for amine of the present invention (and by its appropriate section of deriving) is the aliphatic amine of aliphatic amine and replacement, for example primary alkyl amine, secondary alkyl amine and tertiary alkyl alkane, and wherein alkyl respectively contains 1-3 carbon atom.Comparatively speaking, tripropyl amine (TPA) is particularly preferred amine, because of it can launch high-intensity especially electromagnetic radiation, as shown in some embodiment, improves the sensitivity and the degree of accuracy of detection and quantitative measurment.Diamines (as hydrazine) and the polyamine (as polyethyleneimine) in addition that are suitable for.The example that is suitable for implementing other amine of the present invention has: triethanolamine, triethylamine, 1,4-diaza-bicyclo (2.2.2)-octane, 1-piperidines ethanol, 1,4-piperazine-two (ethane sulfonic acid), tri-isopropyl amine and poly-triolefin imines.
In general, the component that to be used for containing metal electrogenerated chemiluminescence of the present invention partly be limited reactions.Therefore, more particularly, the amine that provides or contain the chemistry amount that amine moiety should be excessive.Use the concentration of amine or amine moiety to be 50-150mM, in order to use when the pH7 left and right sides, concentration is that 100mM usually is favourable.In some embodiment, amine or the upper limit concentration that contains amine moiety are by amine or contain amine moiety maxima solubility decision of (for example in water) in environment for use.In general, amine should be enough to make the containing metal electrogenerated chemiluminescence through peroxidating partly to be converted into its excited state with the consumption that contains amine moiety, thereby produces luminous.One of skill in the art according to information provided herein, need not undue experimentation according to the experience of oneself, just can determine in concrete analysis system amine or contain the consumption of amine moiety.
As be entitled as and increase (content of this article is as the application's list of references) as described in the PCT of the electrogenerated chemiluminescence reaction application US89/04915, measurement of the present invention is preferably in promoter to be carried out under existing, and specifically is the compound of following formula:
R represents hydrogen or C in the formula
nH
2n+1, R ' expression C
nH
2n, X represents 0-70, and n represents 1-20, and wherein n is preferably 1-4.For instance, can be called the following formula: compound (X represents 9-10 in the formula) of Triton X-100 by the commodity that commercial sources obtains:
With commodity Triton N-401(NPE-40 by name) following formula: compound (X represents 40 in the formula):
In general, the consumption of promoter should be enough to make the electromagnetic radiation of emission to be increased to cater to the need.Specifically, consumption is 0.01%-5.0%, more specifically is 0.1%-1.0%(V/V).
Be used for electrogenerated chemiluminescence part of the present invention and launch electromagnetic radiation by its stimulation is induced for excited state, this finishes by acting on this system, and the electrogenerated chemiluminescence part combines with electrochemical energy in this system.The possibility of oxidation that electrogenerated chemiluminescence part can take place and form the compound of strong reductant depends on their exact chemical structures and such as the pH value of system be used to import the factors such as character of the electrode of electrochemical energy.One of skill in the art knows how to determine the emission wavelength of best possibility and electrogenerated chemiluminescence system.In disclosed PCT patented claim US89/01814, the some preferred approach that stimulate the electrogenerated chemiluminescence system are disclosed, its content is as the reference of this paper.
Measure the device of electrogenerated chemiluminescence
The device of implementing the present invention's measurement is shown in Fig. 1,2,3,5,7,8 and 9.Fig. 8 discloses preferred electrogenerated chemiluminescence device.The inventive method also can be used other types electrogenerated chemiluminescence device, and this device should comprise that compound north-south magnet and working electrode or other trigger the surface, make the electrochemical energy that provides that electrogenerated chemiluminescence is partly triggered and are electrogenerated chemiluminescence.When method of the present invention was implemented with the static mode or the type of flow, device 10 comprised circulation chamber, and it provides significant advantage for many kinds of samples (comprising in conjunction with measuring samples).It is open in the US89/04854 of PCT application and US90/01370 to implement device details that electrogenerated chemiluminescence of the present invention measures.
Chamber 12 itself also comprises first assembled block 20, and feed pipe 22 and drainage conduit 24 run through this assembled block, preferably are made of stainless steel.Assembled block 20 has first outside surface 26 and second inside surface 28, keeps a side of the volume 30 of sample with delimit chamber 12.When device 10 turned round, chamber 12 can purify and/or check and/or measure solution.Feed pipe and drainage conduit 22,24 to inside surface 28 assembled block 20 of flowing through, and are led to sample chamber 30 by outside surface 26.Second assembled block of being made by stainless steel 32 also has first outside surface 34 and second inside surface 36.The annular space 38 and first assembled block 20 that second assembled block 32 is made by a teflon or other non-materials that pollutes separate.Therefore, the outside surface 34 of assembled block 32 limits the part of sample chamber 30 opposite sides.Annular space 38 has periphery 40 and center pit 40, and its inner rim 44 limits the sidewall of sample chamber 30.Periphery 40 separates the inside surface 28 of first assembled block 20 and the outside surface 34 of second assembled block 32, to prevent that solution is by flowing out in the sample chamber 30 between two surfaces 28 and 34.Assembled block 32 also has center pit 46, and wherein observation window 40 is sealed, to limit the remainder (being the continuation part of outside surface 34) of sample chamber 30 opposite sides.The material that constitutes observation window 48 should be transparence substantially in the light wave strong point of the electrogenerated chemiluminescence of partly being launched by electrogenerated chemiluminescence.Therefore, observation window 48 should be made by materials such as glass, plastics, quartz.
In one embodiment, working electrode system 54 is configured in the inside surface of first assembled block 20, and it comprises first and second working electrodes 56 and 58.In another embodiment, can dispose a working electrode, or only with electrode 56 as working electrode.On working electrode 56,58, carry out the reaction of galvanochemistry and electrogenerated chemiluminescence.Working electrode 56,58th, therefore the volt-ampere electrode of solid can be made by platinum, gold, carbon or satisfactory other materials.Wire barrel 60,62 with working electrode 56,58 connections passes first assembled block 20 respectively.Built-up magnet 27/37 arranged perpendicular in north-south, will go through to electrode 56 or electrode 56,58 following (being shown in Fig. 1 and Fig. 2) below in level.And see also Fig. 3,5 and 7 and following discussion.
Wire barrel 60,62 is connected (being shown in Fig. 9) with first " working electrode " terminal 64 of voltage controller 66.Therefore, voltage controller 66 moves by the mode of Voltagre regulator, and voltage signal is supplied with working electrode 56,58, and can measure the electric current by its outflow as required when measuring electrogenerated chemiluminescence.In addition, wire barrel 60,62 can be connected with the end of voltage controller 66 separately, carries out running separately.
The action of the Voltagre regulator of voltage controller 66 also can produce useful effect by reverse electrode 68 and reference electrode 70.In one embodiment, assembled block 32 is made by stainless steel, and reverse electrode 68 is in the exposure 72,74 of assembled block 32.The joint face that reverse electrode 72,74 and working electrode 56,58 constitutes is applied to voltage on the solution in the sample chamber 30, the reaction of energy providing chemical, and the electrogenerated chemiluminescence in the excited sample and/or provide energy to be used to purify surface with inspection chamber 12. Reverse electrode 72,74 is connected with another " reverse electrode " terminal 78 of voltage controller 66 by wire barrel 76.
Reference electrode 70 provides reference voltage, and the voltage that is applied by working electrode 56,58 is by its generation, for example+and 1.2V is to reference voltage.Reference electrode 70 is configured in drainage conduit 24 on the position 80 of chamber 12, and is connected with the 3rd " reference electrode " terminal 84 of voltage controller 66 by wire barrel 82.Under the situation of three electrodes, electric current can not flow by reference electrode 70.Reference electrode 70 can be used for the action of three electrodes, and so that balanced, known and stable voltage to be provided, so it is made or a saturated calomel electrode by silver/silver chloride (Ag/AgCl).Only with the action of two electrodes, promptly with working electrode 56 and as oppositely/electrode 58 of reference electrode also can make voltage controller 66 move.During with these two electrode actions, the voltage controller end 78,84 on reverse/reference electrode 58 and the voltage controller 66 is electrically connected.In this case, voltage controller 66 is being worked as a battery basically.Voltage controller 66 is supplied with working electrode 56 and reverse electrode 58 with voltage signal, and can measure the electric current that flows by electrode separately as required.Reference electrode 70 also can be a what is called of being made by platinum, gold, stainless steel or other materials " accurate reference " electrode, and it provides the voltage of less stable, but can measure with regard to solution.When using two and three electrodes, reference electrode 70 or 58 provides reference in the time of can putting on voltage on the working electrode 56 for measurement.It is rather favourable that stable Voltage Reference now is considered to.When voltage controller 66 moves with its Voltagre regulator, by the known voltage that on working electrode 56,58, provides according to reference electrode 70, control various electrode, simultaneously the electric current between surveying work electrode 56,58 and the reverse electrode 72,74.The Voltagre regulator that is used for this purpose all is known, and what the inner structure of voltage controller 66 can be equivalent to any routine passes through the available Voltagre regulator with above-mentioned functions of commercial sources, so itself does not constitute a part of the present invention.Voltage controller 66 in the structure of device 10 also can not comprise, and can be suitable for being connected with the external voltage voltage stabilizer, thereby can control respectively, desired voltage signal is offered electrode 56,58,72,74 and 70.These voltage signals (see as described below, use in a certain way) provide repeatably entry condition as the surface of working electrode 56,58 and the surface of chamber 12, and this feature can significantly improve the precision that electrogenerated chemiluminescence is measured as a whole.
Pump 16 is configured on the position of drainage conduit 24, and sample solution is drawn in the feed pipe 22 along arrow direction A.Solution flow path feed pipe 22, sample chamber 30 and drainage conduit 24 again through reference electrode 70, are discharged along arrow direction B.In addition, pump 16 also can be configured on the feed pipe 22, makes flow of solution through installing 10.All solution of chamber 12 of flowing through all use identical stream with fluid, promptly flow through feed pipe 22, sample chamber 30 and drainage conduit 24, thereby make the fluid that flowed out by chamber 12 staticize processing through fluid dynamic.Pump 16 can be controlled running, can both keep solution in chamber 12 in making at any time.
When implementing the inventive method and using apparatus of the present invention, need to use reagent composition.The ingredient of this agent combination system measuring system of the present invention, i.e. (a) dielectric, (b) contain electrogenerated chemiluminescence tagged compound partly, (c) particle comprises the Magnetic Induction particle, (d) analyte or its analog, (e) analyte or its analog in conjunction with object, (f) can with (d) or (e) reactive component of reaction, (g) reductive agent, or (h) promoter of electrogenerated chemiluminescence reaction.Reagent can combine with another kind of common agents, can prepare the potpourri of two kinds of components, three kinds of components and more kinds of components, if these components duration of storage not with other component phase reactions, so that their effect of infringement in required measurement.Reagent preferably contains the potpourri of the two kinds of components or the various ingredients of particle and one or more other components.
The introduction of the preferred embodiment of the present invention
When carrying out particle sizing of the present invention, can use various particles, particle is the magnetic perception or comprises that Magnetic Induction particle, its density are 1.0-5.0g/mL in general, preferred density is 1.1-2g/mL.The selection of optimum density belongs to this area professional's working range, and gravimetric settling rate can and need suitably be selected between the compound that obtains conforming layer on the electrode surface at measuring speed.
The particle of various mean grain sizes all can use, and for example can use mean diameter to be 0.001-200 μ m(such as 0.05-200 μ m) particle, preferred average diameter is the particle of 0.01-10 μ m.
In measuring composition, also can use the particle of various concentration, for example can use the concentration range of 1-10000 μ g/mL, the concentration range of preferred 5-1000 μ g/mL.The selection of the density of particle, particle diameter and concentration preferably can make the speed of solids precipitation reach 0.5mm/ minute at least, preferred faster rate.
Prior art by the agency of a large amount of magnetic-particles, these particles can be used for measurement of the present invention.For example, United States Patent (USP) 4628037,4695392,4695393,4698302,4554088, BrP 2005019A and European patent 0180384(are the application's list of references) introduced the various magnetic-particles that can successfully use.Particle can be a paramagnetic particle, also can be ferromagnetic particle, and can coat the various materials that can combine with binding compounds, makes magnetic-particle can be used in immunizing dose.Be used for magnetic-particle of the present invention and preferably have the magnetic susceptibility of 0.001cgs unit at least, preferred magnetic susceptibility is at least 0.01cgs unit.The density of magnetic-particle can have wide range, is lower than the density of water basically, i.e. 0.01-5g/mL, and preferred density is 0.5-2g/mL.Particle diameter can be 0.001-200, for example 0.001-100 or 0.05-200 μ m, and preferable range is 0.01-10 μ m.The scope of granule density is also very wide, can be 1-10000 μ g/mL, and preferable range is 5-1000 μ g/mL.
Described as European patent 0180384, the used magnetic particle is preferably low magnetic-particle, make remove magnetic field from electrode surface after, with the particle degaussing, and can purify measuring chamber.The selection of the density of magnetic-particle, concentration and particle diameter should make the settling time reach 0.5mm/ minute at least, is preferably in this more than speed.When operating,, before inducing electrogenerated chemiluminescence, remove magnet apparatus from pole surface earlier in order not disturb the running of photomultiplier in the magnetic chamber.
Measure
Adopt the inventive method can carry out various measurements, these measurements will detailed introduction in following each embodiment.
Below be non-limiting embodiment, these embodiment are for the present invention is described, rather than restriction the present invention.Under the situation that does not break away from the spirit and scope of the present invention, obviously can make various changes.
Embodiment: instrument, material and method
(1) instrument
As Fig. 1,2,3,5,7, flow to device shown in 8 and 9, use three electrodes (seeing shown in Fig. 8 and 9), especially used north-south built-up magnet as illustrated in fig. 1 and 2.
Working electrode: Au sheet, diameter 3mm
Reverse electrode: Au sheet, diameter 3mm
Reference electrode: Ag/AgCl
The teflon pad: 0.15 is " thick
The organic glass panel
Feed pipe: 0.042 ", polypropylene
The speed of exhaust: 0.01-5mL/ minute (variable)
Voltagre regulator: with Hamamatsu R374 PMT(low gain red-sensitive cell) microprocessor-controlIed luminometer; The variable voltage of PMT is the 0-1400 volt
(2) material
(a) electrogenerated chemiluminescence label: Ru(bpy)
2+ 3
(b) electrogenerated chemiluminescence damping fluid: 112mM KH
2PO
4, 88mM K
2HPO
43H
2O, 50 μ M NaCl, 6.5mM NaN
3, 0.8 μ M Triton X-100,0.4mM Tween 20, the aqueous solution of 100mM tripropyl amine (TPA)
(c) electrogenerated chemiluminescence thinning agent: 37.5mM KH
2PO
4, 109.2mM K
2HPO
43H
2O, 151.7mM NaCl, 0.65mM NaN
3, the aqueous solution of 0.43mM bovine serum albumin
(d) Ru(bpy)
2+ 3-NHS:Ru(2,2 '-bipyridyl)
2(4-(3-(1,3-dioxolanes-2-yl) propyl group)-4 '-methyl-2,2 '-dipyridine)
2+
(e) Dynal particle
(ⅰ) Dynal M-450 Dynabeads, the super paramagnetic particle of 4.5 μ m diameters, 30mg/mL be by Dynal(45 North Station Plaza, Great Neck, NY 11021) provide
(ⅱ) Dynal M-280 Dynabeads, the super paramagnetic particle of 2.8 μ M diameters, 10mg/mL be by Dynal(45 North Station Plaza, Great Neck, NY 11021) provide
(3) the electrogenerated chemiluminescence cycle (running of three electrode chambers)
The electrogenerated chemiluminescence cycle is made up of three steps: (1) pre-service, (2) are measured and (3) purify.Pre-treatment step comprises the speed of voltage triangular wave with per second 2.0V is become-1.0V-+0.6V by 0.0V-+2.2V.Measuring process comprises with per second 1.0V triangular wave is become+2.0V by+0.6V-+2.8V.Purifying step comprises voltage square wave is become-0.5V-0.0V by+0.0V-+3.0V.All voltages all relate to the Ag/AgCl reference electrode.
Embodiment 1: the electrogenerated chemiluminescence apparatus and method of precipitation microparticle
Carrying out magnetic with the settling chamber collects
Compound north-south magnet with Fig. 1 and 2 produces the measuring chamber (for example settling chamber) of magnetic force with microparticle deposition (as shown in Figure 3).Label 48 expression transparency windows among Fig. 3,122 expression pads, the charging aperture in the 22 expression settling chambers, 56,58 expression working electrodes, 24 expression sample discharge gates, 20 expression settling chambers itself, 27 expression composite electromagnets (as illustrated in fig. 1 and 2).
The plane along continuous straight runs orientation of settling chamber, built-up magnet 27 arranged perpendicular are below magnet.With peristaltic pump the mark microparticle (Dynal) in the electrogenerated chemiluminescence damping fluid is transported to the settling chamber, after microparticle is transported to the settling chamber, pump cuts out.Use the magnetic field that produces by compound north-south electromagnet (as illustrated in fig. 1 and 2, usually shown in label among Fig. 3 27) microparticle in the settling chamber to be transported to working electrode, available 12V of the action of built-up magnet and 1.2A.Owing to adopted electromagnet, the rate of sedimentation of microparticle is considerably beyond the speed (as shown in Figure 4) of only carrying out particle deposition with action of gravity.
Embodiment 2: the electrogenerated chemiluminescence apparatus and method of deposition microparticle
Carrying out magnetic with collecting chamber collects
Measurement is to carry out in the collecting chamber as described in Figure 5.Label 48 expression transparency windows among Fig. 5,132 expression pads, the charging aperture in the 22 expression collecting chambers, 56,58 expression working electrodes, 20 expression collecting chambers itself, 24 expression sample discharge gates, the compound as illustrated in fig. 1 and 2 permanent magnet of 37 expressions.
The plane along continuous straight runs orientation of collecting chamber, built-up magnet 37 arranged perpendicular are below magnet.With peristaltic pump the mark microparticle (Dynal) in the electrogenerated chemiluminescence damping fluid is transported to electrochemical chamber, before importing sample, earlier the interval of permanent magnet 37 with 0.035 inch directly is fixed under working electrode/solution interface.When sample delivery arrived electrochemical chamber, microparticle was deposited on the surface that is limited by the magnet area on the working electrode.After whole sample deposition, pump cuts out, remove demagnetizing field.Acquisition time is long more, and the particle of deposition is many more.Along with the raising of granule density on the working electrode, cause the intensity of electrogenerated chemiluminescence to increase (as shown in Figure 6).
Embodiment 3: use magnet deposition microparticle
Magnetic field orientating
As shown in Figure 7, it is consistent with the direction in magnetic field 98 to be attracted on built-up magnet 27/37 as illustrated in fig. 1 and 2 the microparticle 96 on (no matter it is permanent magnet or electromagnet), the particle alignment 96 that generates parallels with the surface of working electrode 56/58, and near this surface.
Fig. 1 and Fig. 2 represent magnet 27/37 shown in measuring chamber and Fig. 3,5,7,8 and 9, and this measuring chamber disposes the magnet system that can produce with the field line of electrode surface 56,58 plane parallel.This magnet system is made up of stacked and each permanent magnet or electromagnet that align, and the two poles of the earth, north and south of magnet 27/37 are staggered in stacked.Each magnet in the magnet system 27/37 is isolated by air or any non magnetic inductive material.Arrangement illustrated in figures 1 and 2 makes the magnetic line of force act on zone on the working electrode that almost becomes level with electrode surface, thereby causes aligning of Magnetic Induction particle.Wherein particle is positioned on the electrode surface, and is easy to use the electrochemical energy of being supplied with by electrode (consulting Fig. 7).
The magnetic field line that is also advantageous in that of magnet system 27/37 illustrated in figures 1 and 2 does not stretch out very long distance (consulting Fig. 7) from magnet structure.Therefore, the magnetic field that is produced by a kind of like this magnet system can not produce permanent magnetism near the ferromagnetic material the electrode assembly, and can not have a strong impact near the running of the photomultiplier of liquid stream chamber device.
Embodiment 4: with the mark nonspecific proteins coated particle of medium surface concentration
With 150mM phosphate buffer (pH7.5) by magnetic separation method, washing 30mg(1ml) the Magnetic Induction polystyrene M-450 DYNABEADS(DYNAL that do not coat of 4.5 μ m, Oslo, Norway), each washing 2ml solution.With 150 μ g Ru(bpy)
2+ 3Mark mouse IgG(Jackson Immunochemicals) phosphate-buffered saline (PBS, 1ml) be added in the particle with 0.05% thiomersalate, this potpourri can at room temperature rotate and be incubated overnight, and magnetic separation goes out solution and is removed from particle then.For the unreacted position of blockading, 1ml3% BSA/PBS and 0.05% sodium azide are added in the particle, the solution of generation is incubation 2 hours at room temperature, washing granule 5 times, each 2ml is suspended in the identical damping fluid of 6ml then, preserves standby.
Embodiment 5: with Magnetic Induction particle sizing electrogenerated chemiluminescence
Coat uniformly and uneven, polymerization and non-polymeric Magnetic Induction particle (Dynal, Oslo, Norway with embodiment 4 described labelled proteins; Polysciences, Warrington, PA 18976; Cortex Biochem, San Leandro, CA 94577; Aldrich, Milwaukee, WI 53201).Wash 3 times with the electrogenerated chemiluminescence damping fluid through coated pellet, prepare the 300 μ g/ml suspending liquid of 2ml then.With peristaltic pump the particle suspension liquid of 500 μ l is transported to liquid stream chamber (embodiment 2).When grain flow during to working electrode, because the magnet effect is attracted and concentrates on the working electrode surface.Electrogenerated chemiluminescence with the Hamamatsu R374 photomultiplier measurement magnetic-particle that is configured in liquid stream chamber (particle concentrates on working electrode surface) central upper portion.The table I has provided the light emission measure of the electrogenerated chemiluminescence that the Magnetic Induction particle that coated by labelled protein obtains.
The table I: the electrogenerated chemiluminescence of different Magnetic Induction particles is measured
Particle type diameter (μ m) density (g/ml) raw material electrogenerated chemiluminescence amount
Glass 8.0 2.4 soda-lime glasss 2200
2.0 2.4 soda-lime glasss 8500
Quartzy 0.3-3.5 2.5 SiO
21150
Gold 1.0-2.0 19.3 Au 1100
Embodiment 6: the preparation (reagent I) of the Dynal particle that the goat-anti thyrotropic hormone (TSH) of physisorption coats
Have on the surface that does not coat by magnetic separation washing 1ml 4.5 μ m with 150mM sodium carbonate/bicarbonate solution (pH9.6)-the magnetic polystyrene particle (DYNAL of OH residue, DYNABEADS M-450, DYNAL A.S. Oslo Norway), washs with 2ml at every turn.With the goat-anti TSH of 0.5mg affinity purification, the sodium carbonate/bicarbonate solution (1ml) of the antibody that HCG is pure (CIBA) is added in the particle.This potpourri is incubated overnight at room temperature, and then solution is separated by magnetic force with particle, and removes solution.Add the heavy Sodium azide of 1ml 3% BSA/PBS W/0.05%, at room temperature stirred incubation 2 hours, the unreacted position of blockading.With 5 times (using 2ml) of particle washing at every turn, be suspended in then in the identical damping fluid of 1ml, preserve standby.The ultimate density of this reagent I is a 3%(weight).
Embodiment 7: the preparation of ouabain-BSA bond (reagent II)
The activation of ouabain
With 60.4mg ouabain eight hydrates (Aldrich Cat# 14, deionized water 193-3) (6ml) (blister-pack) solution mixes with 87mg sodium metaperiodate (Mallinckrodt Cat#1139), and this potpourri at room temperature rotated incubation 2 hours.Reaction mixture is by being equipped with Dowex 1 * 8-50 ion exchange resin (Aldrich Cat#21,740-9) cessation reaction of deionized water.Add 200 μ l 1M sodium phosphates (pH7.2), the pH of solution is transferred to 7.0.
The ouabain of activation combines with BSA
50mg is activated the PBS(5ml that ouabain (4.6ml) is added drop-wise to 108mg bovine serum albumin (BSA) (Miles Fraction V), 0.15M, pH7.8) in the solution, ouabain is 40: 1 with the ratio of BSA.Reactant is incubation 2 hours at room temperature.When mixing, add the 30mg sodium cyanoborohydride rapidly.Free ouabain and excessive sodium cyanoborohydride are by removing dialysing in the heavy Sodium azide (0.15M PBS W/0.05%) of pH7.8 under 4 ℃.Ouabain-BSA binding reagents II is housed in 4 ℃.
Embodiment 8: the preparation (reagent III) of the Dynal particle that the ouabain of physisorption-BSA coats
Have-the magnetic polystyrene particle (DYNAL of OH residue by the surface that magnetic separation washing 5mg 4.5 μ m do not coat with 150mM sodium carbonate/bicarbonate solution (pH9.6), DYNABEADS M-450, DYNAL A.S. Oslo, Norway), each washing 2ml.The sodium carbonate/bicarbonate solution (5ml) of 3mg crow echugin-BSA bond (binding reagents II) is added in the particle.This potpourri at room temperature rotates and is incubated overnight, and solution is removed after magnetic force and particle separation.Add the heavy Sodium azide of 5ml 3% BSA/PBS W/0.05%, at room temperature incubation is 2 hours, rotates to the non-reacted parts of blockading.5 times (at every turn using 2ml) of particle washing is suspended in the identical damping fluid of 1ml at last, preserves standby.The ultimate density of reagent III is a 3%(weight).
Embodiment 9:Ru(bpy)
2+ 3The preparation of the mouse-anti digoxin of mark (reagent IV)
Use Ru(bpy)
2+ 3Mark 1mg mouse-anti digoxin (Cambridge Medical Technologies Cat#200-014 Lot A3575), with Centricon 30 little concentrators (Amicon) monoclonal antibody (anti digoxin antibody) buffering is exchanged to (0.15M NaCl in the 0.15M kaliumphosphate buffer, pH7.8), final volume is 0.5ml.With 125 μ l anhydrous dimethyl sulfoxides (Aldrich) dissolving 0.5mg Ru(bpy)
2+ 3Can use behind-the NHS.With 0.18mg Ru(bpy)
2+ 3-NHS(45 μ l) vibration joins protein solution, can obtain being respectively in molecular weight the Ru(bpy of 25: 1 mol ratios of 1057 and 150000)
2+ 3: albumen.Under room temperature and dark, reaction tube vibration incubation 30 minutes.Add 25 μ l 1M glycocoll and make reaction terminating, incubation is 10 minutes again.Reaction mixture is by Sephadex G-25 post (1 * 20cm, 0.15M potassium phosphate, 0.15M NaCl and 0.05% heavy Sodium azide, pH7.2) purifying.Collect and precipitation Ru(bpy)
2+ 3The mouse-anti digoxin part of mark, labelled protein (reagent IV) after measured, every protein molecular has 12 labels.
Embodiment 10:Ru(bpy)
2+ 3The preparation (reagent V) of the mouse-anti thyrotropic hormone (TSH) of mark
Use Ru(bpy)
2+ 3Mark 0.5mg mouse-anti TSH(CIBA), use Centricon 30 little concentrators (Amicon) will resist TSH antibody buffering to exchange to 0.15M kaliumphosphate buffer, 0.15M NaCl(pH7.8 again) in, final volume is 0.35ml.With 0.5mg Ru(bpy)
2+ 3-NHS can use after being dissolved into 75 μ l anhydrous dimethyl sulfoxides (Aldrich).With 0.176mg Ru(bpy)
2+ 3-NHS(26.4 μ l) vibration is added in the protein solution, can obtain being respectively in molecular weight the Ru(bpy of 50: 1 mol ratios of 1057 and 150000)
2+ 3Label: albumen.Under room temperature and dark, reaction tube vibration incubation 30 minutes.Add 25 μ l 1M glycocoll and make reaction terminating, incubation is 10 minutes again.Reaction mixture is through Sephadex G-25 post (pH7.2) purifying is collected and precipitation Ru(bpy for 1 * 20cm, 0.15M potassium phosphate, 0.15M NaCl and 0.05% heavy Sodium azide)
2+ 3The mouse-anti TSH part of mark.Measure the albumen (reagent V) of mark, every protein molecular has 14 labels.
Embodiment 11: a step of thyrotropic hormone (TSH) is separated sandwich assay
100 μ l are demarcated serum (London Diagnostics TSH Lumi TAG Kit), 25 μ l Ru(bpy)
2+ 3After the ECL buffer solution of the ECL buffer solution mouse-anti TSH(reagent V of mark) and 25 μ l goat-anti TSH-DYNAL particles (reagent I) merges, at room temperature place the polypropylene tube incubation to mix in 15 minutes, by the magnetic separation washing granule, again with particle suspending in the ECL damping fluid of 500 μ l, this step repeats 2 times again and washs.At last with particle suspending in the ECL of 1ml damping fluid, by embodiment 2 described methods read the electrogenerated chemiluminescence of a plurality of samples.Electrogenerated chemiluminescence amount be directly proportional with the concentration of analyte in sample (electrogenerated chemiluminescence amount be directly proportional increase) with the concentration of analyte.The table II has provided the value of measuring curve.
The table II: a step separates interlayer and measures: detect TSH
TSH concentration electrogenerated chemiluminescence amount
(μ IU/ml) double sample
0.00 1918 1885
0.05 2584 2530
0.10 3365 3288
0.50 8733 8652
2.50 35688 35347
10.0 125316 136994
25.0 300248 288272
50.0 549034 564948
Embodiment 12: a step of thyrotropic hormone (TSH) is not separated sandwich assay
100 μ l are demarcated serum (London Diagnostics TSH LumiTAG Kit), 25 μ l Ru(bpy)
2+ 3After the ECL buffer solution of the ECL buffer solution mouse-anti TSH(reagent V of mark) and 25 μ l goat-anti TSH-DYNAL particles (reagent I) merges, at room temperature place the polypropylene tube incubation to mix in 15 minutes, before obtaining a result, add 1ml ECL damping fluid earlier, it is described to press embodiment 2 then, read the electrogenerated chemiluminescence number of a plurality of samples.Electrogenerated chemiluminescence amount be directly proportional with the concentration of analyte in sample (electrogenerated chemiluminescence amount be directly proportional increase) with the concentration of analyte.The table III has provided the value of measuring curve.
The table III: a step does not separate interlayer and measures: detect TSH
TSH concentration electrogenerated chemiluminescence amount
(μ IU/ml) (double sample)
0.00 2610 2769
0.05 2870 2894
0.10 2970 2950
0.50 3473 3403
2.50 5588 5495
10.0 13051 13139
25.0 26468 27306
50.0 47104 48664
Embodiment 13: two steps of digoxin are separated comparative determination
50 μ l are demarcated serum (Chicago is IL) with 25 μ l Ru(bpy for TDx Assay, Abbott Labs)
2+ 3After the ECL buffer solution of the mouse-anti digoxin of mark (reagent IV) merges, at room temperature incubation mixed in 20 minutes, the ECL damping fluid that adds ouabain-BSA-DYNAL particle (reagent III), at room temperature incubation mixed in 20 minutes again, by the magnetic separation washing granule, again with particle suspending in the ECL damping fluid of 500 μ l, this washing step repeats 2 times again.At last with particle suspending in the ECL of 1ml damping fluid, by embodiment 2 described methods read the electrogenerated chemiluminescence number of a plurality of samples.The concentration of electrogenerated chemiluminescence amount and analyte in sample be inversely proportional to (electrogenerated chemiluminescence amount increase and reduce) along with the concentration of analyte.The table IV has provided the value of measuring curve.
The table IV: two steps were separated measurement of comparison: detect digoxin
Digoxin concentration electrogenerated chemiluminescence amount
(ng/ml) (double sample)
0.0 22031 21154
0.5 13367 13638
1.0 9506 9607
2.0 5244 5129
3.0 2959 2994
5.0 1581 1631
Embodiment 14: two steps of digoxin are not separated comparative determination
50 μ l are demarcated serum (Chicago is IL) with 25 μ l Ru(bpy for TDx Assay, Abbott Labs)
2+ 3After the ECL buffer solution of the mouse-anti digoxin of mark (reagent IV) merges, at room temperature incubation mixed in 20 minutes, the ECL damping fluid that adds 25 μ l ouabain-BSA-DYNAL particles (reagent III), at room temperature incubation mixed in 20 minutes again, before reading with particle suspending in the ECL of 1ml damping fluid, by embodiment 2 described methods read the electrogenerated chemiluminescence number of a plurality of samples.The concentration of electrogenerated chemiluminescence amount and analyte in sample be inversely proportional to (the electrogenerated chemiluminescence amount reduces along with the increase of the concentration of analyte).The table V has provided the value of measuring curve.
The table V: two steps were not separated measurement of comparison: detect digoxin
Digoxin concentration electrogenerated chemiluminescence amount
(ng/ml) (double sample)
0.0 42051 39643
0.5 28721 28074
1.0 22190 21364
2.0 14660 14542
3.0 11315 11893
5.0 9161 8945
Embodiment 15: add on electrode and wash the end reaction sample and carry out digoxin two step by read period and do not separate comparative determination
50 μ l are demarcated serum (Chicago is IL) with 25 μ l Ru(bpy for TDx Assay, Abbott Labs)
2+ 3After the ECL buffer solution of the mouse-anti digoxin of mark (reagent IV) merges, at room temperature incubation mixed in 20 minutes, the ECL damping fluid that adds 25 μ l ouabain-BSA-DYNAL particles (reagent III), at room temperature incubation mixed in 20 minutes again, before reading earlier with particle suspending in the ECL of 1ml damping fluid, by embodiment 2 described methods read the electrogenerated chemiluminescence number of a plurality of samples.The concentration of electrogenerated chemiluminescence amount and analyte in sample be inversely proportional to (electrogenerated chemiluminescence amount increase and reduce) along with the concentration of analyte.The table VI has provided the value of measuring curve.
The table VI: two steps were separated measurement of comparison: detect digoxin
Digoxin concentration electrogenerated chemiluminescence amount
(ng/ml) (double sample)
0.0 42613 35309
0.5 24211 24168
1.0 17561 17206
2.0 10491 9909
3.0 6712 7145
5.0 4680 4603
Embodiment 16: the preparation of nucleic acid magnetic-particle and the application of electrogenerated chemiluminescence
(6) according to a conventional method activate the DynalM450 particle with toluene-4-sulfonic acid 2-fluoro-1-picoline, and these particles that will activate then and oligonucleotides JK8 and JK8C reaction are with 650 μ l 0.1M NaHCO of 33nmole oligonucleotides
3Solution is added in the Dynal particle that 100mg activates, and incubation mixed in 3 hours, adds 4ml monoethanolamine (0.1M) particle of blockading again.The particle of combination is mixed with the ECL damping fluid of 0.5mg/ml salmon sperm single stranded DNA, and washing is 4-5 time in the ECL damping fluid, is resuspended in the ECL damping fluid that 10mg/ml contains 100 μ g/ml salmon sperm single stranded DNAs.
By particle and Ru(bpy) in conjunction with JK8 and JK8C
2+ 3Labeled oligonucleotide JK7 hybridization, can detect with particle hybridization after electrogenerated chemiluminescence, JK7 and the complementation of JK8 order, but not with the complementation of JK8C order.In the ECL damping fluid a large amount of particles (300g) with contain respectively 12.5,6.3,3.01 and the 50 μ l ECL damping fluids of 1.5fmole mark JK7 mix, these potpourris are after hybridizing 4 hours under 52 ℃, with the washing of 1ml ECL damping fluid, be suspended in then in the 830 μ l ECL damping fluids.Press embodiment 2 described methods analyst samples, when 12.5fmole, the electrogenerated chemiluminescence amount of JK8 particle is higher more than 1000 times than JK8C particle; When 6.3fmole, the electrogenerated chemiluminescence amount of JK8 particle is higher about 1000 times than JK8C; 3.02 with during 1.5fmole, the electrogenerated chemiluminescence amount of JK8 particle than the high about 5-3 of JK8C particle doubly.This explanation can be detected by specific hybrid by electrogenerated chemiluminescence and directly be fixed on specificity order on the particle surface.
Embodiment 17: the preparation of streptavidin magnetic-particle I
(6) according to a conventional method activate Dynal M450 particle with toluene-4-sulfonic acid 2-fluoro-1-picoline, then with the particle and streptavidin (Sigma Ltd) reaction that activate.Use 0.1M NaHCO
3Washing jihuokeli (50mg) adds the 1.5mg streptavidin again, and reaction is spent the night.Add 4ml monoethanolamine (0.1M) particle of blockading.In conjunction with particle mix with the ECL damping fluid of 0.5mg/ml salmon sperm single stranded DNA, in the ECL damping fluid washing 4-5 time, be suspended in the 10mg/ml ECL damping fluid that contains 100 μ l/ml salmon sperm single stranded DNAs.The streptavidin that is obtained by Dynal M-280 also proves significant, but then signal is lower with present measuring sequence.In order to carry out immunizing dose, behind the damping fluid conjugated antigen or antibody of available passive coating usefulness, it is particles used to blockade with BSA again.
Embodiment 18: the preparation of streptavidin magnetic-particle II
105 μ l are contained 50mg/ml biotin-x-NHS(Clontech, and San Diego CA, dimethyl sulfoxide 5002-1) are added to 15mg BSA(in 2-3ml PBS) in, incubation 30 minutes at room temperature after the mixing.Add 30 μ l 1M glycocoll and make reaction terminating, again incubation 10 minutes at room temperature.This reaction mixture is through gel permeation chromatography (Biorad, Bio-Gel P6) purifying.Biotin-BSA is with 0.2 μ m syringe filtering.The 10ml 0.2M sodium carbonate/bicarbonate damping fluid (pH9.6) of 5mg biotin is added to 300mg through the washed Dynabead(Dynal 14002 of sodium carbonate/bicarbonate damping fluid) in, potpourri is incubated overnight at room temperature mixing after whirling motion.These particles are after magnetic separation, the ECL dilution and the 100 μ l tRNA(10mg/ml that add 10ml), mixed at room temperature incubation 3-4 hour again, with these particles of ECL thinning agent once washing of 10ml, be suspended in ECL dilution and the 100 μ l tRNA(10mg/ml of 10ml then) in.Mix the back and under 2-6 ℃, be incubated overnight, proteinaceous solid is fixed on the particle.Particle is suspended among the 10ml PBS that contains 15mg streptavidin (Scripps S1214) after magnetic separation, mixes 1 hour, and particle is the ECL of 10ml dilution washing 4 times, and each washing all mixed through 5 minutes.At last with particle suspending at the ECL of 29.7ml dilution and 300 μ l tRNA(10mg/ml) in, make ultimate density reach 10mg/ml particle+100 μ g/ml tRNA.
Embodiment 19: the mensuration of specific gene group DNA sequence
This routine described mensuration has been used 2 oligonucleotides, and all with close mutually identical DNA chain hybridization, one of them is captured, another labeled complex (interlayer hybridization).This mensuration has been used has specific e. coli dna and probe to trp E/D gene regions.E. coli dna is (1) preparation according to a conventional method, and salmon sperm contrast DNA is provided by Sigma company.With 14 μ l hybridization buffers (10 * PBS, 10mM EDTA and 0.7% SDS), the biotin labeled TRP.CO4 of 2ng and 5ng Ru(bpy)
2+ 3Mark TRP.CO3 is added in the DNA sample, and water becomes 10 μ l with specimen preparation, is heated to 97 ℃, 97 ℃ of following incubations 10 minutes, is cooled to 50 ℃ then again, hybridizes 2 hours.The magnetic-particle II that 20 μ l streptavidins are coated is added in the sample, at room temperature mixes 2 hours, and particle washs 4 times in the ECL damping fluid, is resuspended in the 500 μ l ECL damping fluids, analyzes by embodiment 2 described methods.Positive DNA is Escherichia coli, and negative DNA is a salmon sperm, the results are shown in the table VII.
The table VII
DNA content electrogenerated chemiluminescence average magnitude
Positive 10 184
25 257
50 266.5
Negative 10 87
25 70
50 75
The The above results explanation uses interlayer hybridization measuring method on the DNA of not amplification, and the electrogenerated chemiluminescence measuring system can be used in the genomic gene that detects in the Escherichia coli.In this embodiment, the magnetic-particle II that coats with streptavidin is the same, the magnetic-particle I that also can use streptavidin to coat.
Above-mentioned preferred embodiment describes the present invention in detail, but can not therefore limit the present invention, under the situation that does not break away from essence of the present invention, according to the introduction of instructions, obviously can make various changes, but this also should belong to scope of the present invention undoubtedly.
List of references
1.Beaucage SL，Caruthers MH.Deoxynucleoside phosphoramidites，a new class of key intermediates for deoxypolynucleotide synthesis.Tetrahedron Lett 1982;22：1859-62.
2.Shibata DK，Arnheim N，Martin JW.Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction.J Exp Med 1988;167：225-30.
3.Yanofsky，C.et al（1981）Nucleic Acids Res.24，6647-6668.
4.Updyke TV，Nicolson GL.Immunoaffinity isolation of membrane antigens with biotinylated monoclonal antibodies and streptavidin-agarose.Methods Enzymol 1986;121：717-25.
5.Cardullo RA，Agrawal S，Flores C，Zamecnik DC，Wolf DE.Detection of nucleic acid hybridization by monradiative fluorescence resonance energy transfer.Proc Natl Acad Sci 1988;85：8790-4.
6.Ngo TT.Procedure for activating polymers with primary and or secondary hydroxyl groups.Makromol Chem Macromol Symp 1988;17：224-39.
7.Coutlee F，Bobo L，Mayur K，Yolken RH，Viscidi RP.
59
Immunodetection of DNA with biotinylated RNA probes：A study of reactivity of a monoclonal antibody to DNA-RNA hybrids.Anal Biochem 1989;181：96-105.
8.Casadei J，Powell MJ，Kenten JH.Expression and secretion of aequorin as a chimeric antibody using a mammalian expression vector.Proc Natl Acad Sci 1990;87：2047-51.
9.Molecular cloning，a laboratory manual 2nd Ed Sambrook，J.Cold Spring Harbor Laboratory New York
10.Heney，G.and Orr，G.A.（1981）Anal Biochem.114，92-96.
11.Mullis KB，Faloona FA.Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction.Methods Enzymol 1987;155：335-50.
12.Lyons J，Janssen JWG，Bartram C，Layton M，Mufti GJ.Mutation of Ki-ras and.N-ras oncogenes，in myelodysplastic syndromes.Blood 1988;71：1707-12.
13.Saiki RK，Gelfand DH，Stoffel S，Scharf SJ，Higuchi R.Horn GT，Mullis KB，Erlich HA.Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.Science 1988;239：487-91.
14.Yee C，Krishnan-Hewlett I，Baker CC，Schlegel R，Howley PM.Presence and Expression of Human
60
Papillomavirus sequences in human cervical carcinoma cell lines.Am J Pathol 1985;119：361-6.
15.Reddy EP，Reynolds RK，Santo E，Barbacid M.A point mutation is responsible for the acquisition of the transforming properties by the T24 humanbladder carcinoma oncogene.Nature 1982;300：149-52.
16.Marmur，J.（1961）J.Mol.Biol 3，208.
Claims (8)
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US65242791A | 1991-02-06 | 1991-02-06 | |
| US652,427 | 1991-02-06 | ||
| US82726992A | 1992-02-03 | 1992-02-03 | |
| US827,269 | 1992-02-03 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1065339A true CN1065339A (en) | 1992-10-14 |
| CN1107863C CN1107863C (en) | 2003-05-07 |
Family
ID=27096296
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 92101900 Expired - Lifetime CN1107863C (en) | 1991-02-06 | 1992-02-07 | Method and apparatus for magnetic microparticulate based luminescrnce assay including plurality of magnets |
Country Status (7)
| Country | Link |
|---|---|
| JP (2) | JP3013937B2 (en) |
| CN (1) | CN1107863C (en) |
| AU (1) | AU1530492A (en) |
| IE (1) | IE69371B1 (en) |
| IL (1) | IL100866A (en) |
| NZ (1) | NZ241538A (en) |
| WO (1) | WO1992014138A1 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1300598C (en) * | 2001-02-16 | 2007-02-14 | 量子设计有限公司 | Method and apparatus for detection and measurement of accumulations of magnetic particles |
| CN100501403C (en) * | 2001-11-20 | 2009-06-17 | 斯达高诊断公司 | method for detecting analytes using magnetic colloidal particles |
| CN102089661A (en) * | 2008-07-10 | 2011-06-08 | 英国政府创新、大学与技能部 | Sample carrier for effecting chemical assays |
| CN103954664A (en) * | 2014-04-10 | 2014-07-30 | 深圳普门科技有限公司 | Electrode disk, preparation method thereof and electrochemical flow cell based on same |
| CN104471380A (en) * | 2012-07-06 | 2015-03-25 | 株式会社日立高新技术 | Analysis device and analysis method |
| CN111148572A (en) * | 2017-06-06 | 2020-05-12 | 西北大学 | Cross-interface magnetic separation |
| CN116106297A (en) * | 2013-06-19 | 2023-05-12 | 豪夫迈·罗氏有限公司 | Electrochemiluminescence method and analysis system for detecting analytes in liquid samples |
Families Citing this family (61)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5466416A (en) * | 1993-05-14 | 1995-11-14 | Ghaed; Ali | Apparatus and methods for carrying out electrochemiluminescence test measurements |
| US7036019B1 (en) | 1994-04-01 | 2006-04-25 | Intarsia Software Llc | Method for controlling database copyrights |
| JPH07271865A (en) | 1994-04-01 | 1995-10-20 | Mitsubishi Corp | Database copyright management method |
| US6744894B1 (en) | 1994-04-01 | 2004-06-01 | Mitsubishi Corporation | Data management system |
| US7302415B1 (en) | 1994-09-30 | 2007-11-27 | Intarsia Llc | Data copyright management system |
| EP0715241B1 (en) | 1994-10-27 | 2004-01-14 | Mitsubishi Corporation | Apparatus for data copyright management system |
| US6424715B1 (en) | 1994-10-27 | 2002-07-23 | Mitsubishi Corporation | Digital content management system and apparatus |
| DE69535013T2 (en) | 1994-10-27 | 2006-12-28 | Intarsia Software LLC, Las Vegas | Copyright data management system |
| US5744367A (en) * | 1994-11-10 | 1998-04-28 | Igen International, Inc. | Magnetic particle based electrochemiluminescent detection apparatus and method |
| US5538687A (en) * | 1994-12-16 | 1996-07-23 | Behringer Mannheim Gmbh | Apparatus for generating optically detectable signals by applying electrical potentials to sample liquids |
| US6319670B1 (en) | 1995-05-09 | 2001-11-20 | Meso Scale Technology Llp | Methods and apparatus for improved luminescence assays using microparticles |
| WO1996036870A1 (en) * | 1995-05-18 | 1996-11-21 | Igen, Inc. | Method for derivitizing electrodes and assay methods using such derivitized electrodes |
| US8595502B2 (en) | 1995-09-29 | 2013-11-26 | Intarsia Software Llc | Data management system |
| US7801817B2 (en) | 1995-10-27 | 2010-09-21 | Makoto Saito | Digital content management system and apparatus |
| EP0859229A1 (en) * | 1997-02-10 | 1998-08-19 | Gist-Brocades B.V. | Detection of analytes using electrochemistry |
| AU5301598A (en) * | 1997-02-10 | 1998-08-13 | Gist-Brocades B.V. | Detection of analytes using electrochemistry |
| EP0859230A1 (en) * | 1997-02-10 | 1998-08-19 | Cranfield University | Detection of analytes using electrochemistry |
| US6146838A (en) * | 1997-03-18 | 2000-11-14 | Igen International, Inc. | Detecting water-borne parasites using electrochemiluminescence |
| DE19803528A1 (en) * | 1998-01-30 | 1999-08-05 | Roche Diagnostics Gmbh | Method for analyzing a sample using an electrochemiluminescence binding reaction test |
| GB9815042D0 (en) * | 1998-07-10 | 1998-09-09 | Imperial College | Detector |
| JP4763866B2 (en) | 1998-10-15 | 2011-08-31 | インターシア ソフトウェア エルエルシー | Method and apparatus for protecting digital data by double re-encryption |
| WO2001006257A1 (en) * | 1999-07-16 | 2001-01-25 | Wm. Marsh Rice University | Metal nanoshells for biosensing applications |
| US6136268A (en) * | 1999-08-17 | 2000-10-24 | Orion Diagnostica | Method for luminescence measurements |
| EP1325333A4 (en) * | 2000-10-10 | 2006-03-22 | Aviva Biosciences Corp | Individually addressable micro-electromagnetic unit array chips in horizontal configurations |
| EP1512009A2 (en) * | 2002-05-08 | 2005-03-09 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Magneto-controlled method and system for determining an analyte in a liquid medium |
| AU2003277611A1 (en) | 2002-11-07 | 2004-06-07 | Mitsubishi Kagaku Iatron, Inc. | Magnetic particle collecting magnetic force body and its use |
| US7920906B2 (en) | 2005-03-10 | 2011-04-05 | Dexcom, Inc. | System and methods for processing analyte sensor data for sensor calibration |
| US9247900B2 (en) | 2004-07-13 | 2016-02-02 | Dexcom, Inc. | Analyte sensor |
| AT413153B (en) * | 2004-03-11 | 2005-11-15 | Christian Doppler Labor Fuer S | METHOD AND DEVICE FOR DETECTING MARKED MICROPARTICLES |
| WO2006127694A2 (en) | 2004-07-13 | 2006-11-30 | Dexcom, Inc. | Analyte sensor |
| US7713574B2 (en) | 2004-07-13 | 2010-05-11 | Dexcom, Inc. | Transcutaneous analyte sensor |
| US20090061466A1 (en) | 2006-03-09 | 2009-03-05 | Wolfgang Hoesel | Anti-drug antibody assay |
| GB2436616A (en) * | 2006-03-29 | 2007-10-03 | Inverness Medical Switzerland | Assay device and method |
| CA2662416C (en) | 2006-09-12 | 2014-12-16 | F. Hoffmann-La Roche Ag | Anti-drug antibody assay |
| KR101503510B1 (en) | 2007-02-09 | 2015-03-18 | 어드밴스드 리퀴드 로직, 아이엔씨. | Droplet actuator devices and methods employing magnetic beads |
| CA2677901A1 (en) | 2007-02-27 | 2008-09-04 | F. Hoffmann-La Roche Ag | Method for the assessment of the inhibitory activity of antibodies against insulin-like growth factor i receptor |
| RU2399041C2 (en) * | 2007-07-31 | 2010-09-10 | ЗАО "Нанотехнология-МДТ" | Electrochemical cell with magnetic system |
| MX2010005891A (en) | 2007-12-15 | 2010-06-09 | Hoffmann La Roche | Distinguishing assay. |
| US8614297B2 (en) | 2008-12-22 | 2013-12-24 | Hoffmann-La Roche Inc. | Anti-idiotype antibody against an antibody against the amyloid β peptide |
| EP2606362B1 (en) | 2010-08-19 | 2015-09-30 | Roche Diagniostics GmbH | An assay for measurement of antibodies binding to a therapeutic monoclonal antibody |
| JP5481326B2 (en) * | 2010-09-08 | 2014-04-23 | 株式会社日立製作所 | Measuring apparatus and measuring method using biomaterial detection element |
| WO2012143380A1 (en) | 2011-04-20 | 2012-10-26 | F. Hoffmann-La Roche Ag | Microwaves for immunohistochemical analysis |
| AR086982A1 (en) | 2011-06-22 | 2014-02-05 | Hoffmann La Roche | ELIMINATION OF DIANA CELLS BY SPECIFIC CYTOTOXIC T-CELLS OF VIRUSES USING COMPLEXES THAT INCLUDE MHC CLASS I |
| EP2795335B1 (en) | 2011-12-19 | 2017-09-06 | F. Hoffmann-La Roche AG | Method for the detection of free binding partner of a multispecific binder |
| CA2859268A1 (en) | 2012-02-01 | 2013-08-08 | F. Hoffmann-La Roche Ag | Method for the detection of a binding partner of a multispecific binder |
| EP2634579B1 (en) * | 2012-03-03 | 2014-12-17 | Astrium GmbH | Method for performing immuno-assays in zero gravity |
| MX352118B (en) | 2012-03-08 | 2017-11-09 | Hoffmann La Roche | Multiplexed chromatography-immunoassay method for the characterization of circulating immune complexes. |
| EP2872893B1 (en) | 2012-07-13 | 2017-03-29 | F. Hoffmann-La Roche AG | Method for the detection of a multispecific antibody |
| KR20150097688A (en) | 2012-12-21 | 2015-08-26 | 에프. 호프만-라 로슈 아게 | Disulfide-linked multivalent mhc class i comprising multi-function proteins |
| KR102248581B1 (en) | 2013-07-04 | 2021-05-06 | 에프. 호프만-라 로슈 아게 | Interference-suppressed immunoassay to detect anti-drug antibodies in serum samples |
| EP3368568B1 (en) | 2015-10-29 | 2022-04-06 | F. Hoffmann-La Roche AG | Anti-variant fc-region antibodies and methods of use |
| CN108351361A (en) | 2015-11-30 | 2018-07-31 | 豪夫迈·罗氏有限公司 | Immunoassays for Determining Fc Region Modified Antibodies |
| CA3081801C (en) | 2017-11-29 | 2022-12-20 | F. Hoffman-La Roche Ag | Target interference suppressed anti-drug antibody assay |
| WO2020141145A1 (en) | 2018-12-30 | 2020-07-09 | F. Hoffmann-La Roche Ag | Anti-rabbit cd19 antibodies and methods of use |
| CN113811770B (en) | 2019-05-13 | 2024-06-28 | 豪夫迈·罗氏有限公司 | Suppression of interference in pharmacokinetic immunoassays |
| US11913945B2 (en) | 2020-01-02 | 2024-02-27 | Hoffmann-La Roche Inc. | Method for determining the amount of a therapeutic antibody in the brain |
| KR102836238B1 (en) | 2020-06-16 | 2025-07-18 | 에프. 호프만-라 로슈 아게 | Method for measuring free antigen of antibodies in a sample |
| EP4320444A1 (en) | 2021-04-09 | 2024-02-14 | F. Hoffmann-La Roche AG | Process for selecting cell clones expressing a heterologous polypeptide |
| WO2023021055A1 (en) | 2021-08-19 | 2023-02-23 | F. Hoffmann-La Roche Ag | Multivalent anti-variant fc-region antibodies and methods of use |
| JP2025500334A (en) | 2021-12-21 | 2025-01-09 | エフ． ホフマン－ラ ロシュ アーゲー | Methods for determining hydrolytic activity |
| CN119790065A (en) | 2022-08-24 | 2025-04-08 | 美国安进公司 | Anti-drug antibody assay |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0030087A1 (en) * | 1979-11-13 | 1981-06-10 | Technicon Instruments Company Limited | Immunoassay method and apparatus and kit for carrying out the method |
| DE3013765A1 (en) * | 1980-04-10 | 1981-10-15 | Philips Patentverwaltung Gmbh, 2000 Hamburg | DETECTOR ARRANGEMENT, ESPECIALLY FOR LIQUID CHROMATOGRAPHY |
| US4652533A (en) * | 1983-04-28 | 1987-03-24 | Pandex Laboratories, Inc. | Method of solid phase immunoassay incorporating a luminescent label |
| US4628037A (en) * | 1983-05-12 | 1986-12-09 | Advanced Magnetics, Inc. | Binding assays employing magnetic particles |
| US5238808A (en) * | 1984-10-31 | 1993-08-24 | Igen, Inc. | Luminescent metal chelate labels and means for detection |
| GB8507706D0 (en) * | 1985-03-25 | 1985-05-01 | Genetics Int Inc | Magnetic nucleic acid sequences |
| WO1987006706A1 (en) * | 1986-04-30 | 1987-11-05 | Igen, Inc. | Electrochemiluminescent assays |
| US5093268A (en) * | 1988-04-28 | 1992-03-03 | Igen, Inc. | Apparatus for conducting a plurality of simultaneous measurements of electrochemiluminescent phenomena |
| US5061445A (en) * | 1988-11-03 | 1991-10-29 | Igen, Inc. | Apparatus for conducting measurements of electrochemiluminescent phenomena |
| US5068088A (en) * | 1988-11-03 | 1991-11-26 | Igen, Inc. | Method and apparatus for conducting electrochemiluminescent measurements |
-
1992
- 1992-02-04 IL IL10086692A patent/IL100866A/en not_active IP Right Cessation
- 1992-02-05 AU AU15304/92A patent/AU1530492A/en not_active Abandoned
- 1992-02-05 JP JP4507261A patent/JP3013937B2/en not_active Expired - Lifetime
- 1992-02-05 IE IE920381A patent/IE69371B1/en not_active IP Right Cessation
- 1992-02-05 NZ NZ24153892A patent/NZ241538A/en not_active IP Right Cessation
- 1992-02-05 WO PCT/US1992/000982 patent/WO1992014138A1/en not_active Ceased
- 1992-02-07 CN CN 92101900 patent/CN1107863C/en not_active Expired - Lifetime
-
1998
- 1998-07-15 JP JP10233430A patent/JP3128541B2/en not_active Expired - Lifetime
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1300598C (en) * | 2001-02-16 | 2007-02-14 | 量子设计有限公司 | Method and apparatus for detection and measurement of accumulations of magnetic particles |
| CN100501403C (en) * | 2001-11-20 | 2009-06-17 | 斯达高诊断公司 | method for detecting analytes using magnetic colloidal particles |
| CN102089661A (en) * | 2008-07-10 | 2011-06-08 | 英国政府创新、大学与技能部 | Sample carrier for effecting chemical assays |
| CN102089661B (en) * | 2008-07-10 | 2014-09-24 | 英国政府创新、大学与技能部 | Sample carrier for effecting chemical assays |
| CN104471380A (en) * | 2012-07-06 | 2015-03-25 | 株式会社日立高新技术 | Analysis device and analysis method |
| CN104471380B (en) * | 2012-07-06 | 2017-11-17 | 株式会社日立高新技术 | Analytical equipment and analysis method |
| CN116106297A (en) * | 2013-06-19 | 2023-05-12 | 豪夫迈·罗氏有限公司 | Electrochemiluminescence method and analysis system for detecting analytes in liquid samples |
| CN103954664A (en) * | 2014-04-10 | 2014-07-30 | 深圳普门科技有限公司 | Electrode disk, preparation method thereof and electrochemical flow cell based on same |
| CN111148572A (en) * | 2017-06-06 | 2020-05-12 | 西北大学 | Cross-interface magnetic separation |
Also Published As
| Publication number | Publication date |
|---|---|
| IL100866A (en) | 1995-10-31 |
| NZ241538A (en) | 1993-02-25 |
| JP3013937B2 (en) | 2000-02-28 |
| JPH11125601A (en) | 1999-05-11 |
| IE69371B1 (en) | 1996-09-04 |
| JP3128541B2 (en) | 2001-01-29 |
| IE920381A1 (en) | 1992-08-12 |
| AU1530492A (en) | 1992-09-07 |
| WO1992014138A1 (en) | 1992-08-20 |
| CN1107863C (en) | 2003-05-07 |
| JPH06508203A (en) | 1994-09-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1094593C (en) | Improved method and device for luminescence assay | |
| CN1065339A (en) | Method and apparatus for magnetic particle-based luminescence assays containing composite magnets | |
| CN100419414C (en) | Device for Luminescence Measurement | |
| AU2020232954B2 (en) | Electrochemiluminescent labeled probes for use in immunoassay methods, methods using such and kits comprising same | |
| US5935779A (en) | Methods for improved particle electrochemiluminescence assay | |
| CN1526024A (en) | Photoelectron detection system | |
| Kenten et al. | Rapid electrochemiluminescence assays of polymerase chain reaction products | |
| CN87107800A (en) | Catch the method for target and background and the device that compatibility is measured | |
| US6828109B2 (en) | Methods for detecting an analyte of interest using catalyzed reporter deposition of tyramide | |
| CN1582394A (en) | Rapid and sensitive detection of molecules | |
| CN100343668C (en) | Novel fluorescent label compounds | |
| CN1784600A (en) | Chromatographic analysis system | |
| Fan et al. | Chemiluminescence platforms in immunoassay and DNA analyses | |
| CN1521274A (en) | Systems and methods for detecting biological and chemical materials | |
| CN1553188A (en) | Microarray signal amplifying method | |
| US5770459A (en) | Methods and apparatus for improved luminescence assays using particle concentration, electrochemical generation of chemiluminescence detection | |
| CN1313564C (en) | Irradiant material with surface modification and activation | |
| CN1882839A (en) | Devices for the separation and/or analysis of different molecular targets in complex mixtures | |
| CN100346163C (en) | Method and apparatus for detecting substance | |
| CN1255552A (en) | Universal microarray | |
| CN1203188C (en) | Nano-class amplifying detection method for biochip | |
| CN1721852A (en) | Antigen and ligand PCR pipe detecting reagent case, its manufacturing method and application | |
| KR102925377B1 (en) | Electrochemiluminescently labeled probe for use in immunoassay methods, method of use thereof, and kit comprising same | |
| HK1066059B (en) | Apparatus for luminescence assays | |
| HK1015878B (en) | Method and apparatus for luminescence assays |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: IGEN INT INC Free format text: FORMER OWNER: EAGEN INTERNATIONAL INC. |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| C56 | Change in the name or address of the patentee |
Owner name: EAGEN INTERNATIONAL INC. Free format text: FORMER NAME OR ADDRESS: IGEN, INC. |
|
| CP01 | Change in the name or title of a patent holder |
Address after: Marley Lanzhou USA Co-patentee after: Eisai Co.,Ltd. Patentee after: Igen, Inc. Address before: Marley Lanzhou USA Co-patentee before: Eisai Co.,Ltd. Patentee before: Igen, Inc. |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20050513 Address after: American Maryland Co-patentee after: Eisai Co.,Ltd. Patentee after: IGEN INTERNATIONAL, Inc. Address before: Marley Lanzhou USA Co-patentee before: Eisai Co.,Ltd. Patentee before: Igen, Inc. |
|
| C17 | Cessation of patent right | ||
| CX01 | Expiry of patent term |
Expiration termination date: 20120207 Granted publication date: 20030507 |