CN1107863C - Method and apparatus for magnetic microparticulate based luminescrnce assay including plurality of magnets - Google Patents

Method and apparatus for magnetic microparticulate based luminescrnce assay including plurality of magnets Download PDF

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Publication number
CN1107863C
CN1107863C CN 92101900 CN92101900A CN1107863C CN 1107863 C CN1107863 C CN 1107863C CN 92101900 CN92101900 CN 92101900 CN 92101900 A CN92101900 A CN 92101900A CN 1107863 C CN1107863 C CN 1107863C
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particle
compound
electrogenerated chemiluminescence
electrode
measuring
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CN1065339A (en
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J·K·利兰
H·P·沙
J·H·肯滕
J·E·古德曼
G·E·洛克
难波勇三郎
G·F·布莱克本
R·J·梅西
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Eisai Co Ltd
IGEN Inc
IGEN International Inc
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Eisai Co Ltd
IGEN Inc
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    • 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
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    • 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
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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    • 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
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    • 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
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    • 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
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

Disclosed and claimed are methods and apparatus for performing a binding assay for an analyte of interest present in a sample based upon measurement of electrochemiluminescence at an electrode. The method uses magnetically responsive particles. The method and apparatus call for a plurality of electromagnets or permanent magnets in north-south orientation for imposing a magnetic field so as to collect the particles.

Description

The method and apparatus that contains the magnetic particle base luminescence assays of a plurality of magnets
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 a plurality of north-souths 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 a plurality of north-souths 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 are 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.
Unexpectedly United States Patent (USP) 539389 (PCT application number U.S.89/04919) luminescence phenomenon is disclosed the specificity of sensitivity in conjunction with assay method, 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 a plurality of suspended particles 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) need the analyte measured 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 a plurality of suspended particles of combining of the specificity of material (iii);
(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 can only occur in the very short distance.When the distance between described first molecule and second molecule (or electrode) increased, the probability of tunnel(l)ing was with index decreased.If distance is less than 25 (2.5nm), the probability that then occurs in the tunnel(l)ing between described first molecule and second molecule (or electrode) is very high; If distance is very big, then probability is very low.The distance of 25 is the thumb rule that this area professional uses, and is not absolute limitations.
Therefore, those electrogenerated chemiluminescence labels that only have 25 electrode surfaces just can be expected to participate in the process of electrogenerated chemiluminescence.In general, the particle area in 25 electrode surface scopes 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 a plurality of north-souths 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 a plurality of north-souths 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 a plurality of magnet that magnetic corpuscular of the present invention separates or do not have separating measuring method with Fig. 2; The magnetic field line that a plurality of magnets of magnet system produce 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 a plurality of 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 use material, it contains with the tagged compound that can induce electrogenerated chemiluminescence and connects
The component that connects and
(iii) can carry out a plurality of magnetic that specificity combines with material with analyte or described measurement
Sexuality is answered suspended particle;
(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 a plurality of north-souths, 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 a plurality of magnets 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 a plurality of north-souths 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 -/2That 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
Particle has the microparticle material that diameter is 0.001-200 μ m (for example 0.05 μ-200 μ m), 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
As described in disclosed PCT application US89/04859 (denomination of invention: the reductive agent that utilizes amine to derive carries out the electrogenerated chemiluminescence reaction), 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: Following formula: compound (X represents 40 in the formula) with commodity Triton N-401 (NPE-40) by name: 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 p H 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 a plurality of north-souths 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.
Device 10 comprises electrochemical chamber 12, and light detects measurement mechanism 14 (can be a photomultiplier, photodiode, charge-coupled device (CCD), analogs such as photosensitive sheet or emulsion) and pump 16 (can be a peristaltic pump), and it passes through and carries liquid by chamber 12.In addition, also can use positive-dispacement pump.One of the configuration mechanism of opening and closing 18 between chamber 12 and photomultiplier 14, this switchings machine-processed 18 only when electrogenerated chemiluminescence is measured photomultiplier 14 be exposed under the situation of chamber 12 and control open operation.Switching mechanism can be closed (when for example turning round).Device 10 also can comprise chamber against sunshine (not marking among Fig. 8), and it is made up of various assemblies, when carrying out the electrogenerated chemiluminescence measurement, makes photomultiplier 14 not be subjected to the influence of any ambient light.
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 42, 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 48 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.
Feed pipe 22 intersects with sample chamber 30 at an end 50 of the sample chamber 30 of contiguous annular space 38, and drainage conduit 24 intersects with sample chamber 30 at the other end 52 of the sample chamber 30 of contiguous annular space 38.Therefore, the combination of feed pipe 22, sample chamber 30 and drainage conduit 24 has constituted the narrow continuous stream that is essentially laminar flow of solution, and flow of solution is to, flow path chamber 12 and can be flowed out by chamber 12.Arrow A and B represent the inflow and the outflow of feed pipe 22 and drainage conduit 24 respectively.
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.A plurality of north-souths magnet 27/37 arranged perpendicular, 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 operates 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 running 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. Reverse electrode 72,74 and working electrode 56,58 joint face is provided, and they are applied on the solution in the sample chamber 30 voltage, energy providing chemical reaction, 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 running 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 running of two electrodes, promptly with working electrode 56 and as oppositely/electrode 58 of reference electrode also can make voltage controller 66 operate.During with these two electrode runnings, 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 operates 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.
The flow direction structure of device 10 makes working electrode can accept variable voltage or keeps original running voltage, makes one or more solution obtain continuously handling simultaneously, does not make working electrode 56,58 (or reverse and reference electrode 72,74,70) be subjected to air influence again.Influenced by air and then can make circuit be subjected to the interference of reference electrode 70, thereby make voltage that unknown variation arbitrarily, the repeatability of destruction work electrode 56,58 surface conditions take place.Flow direction structure makes the initial step that purifies and monitor electrode system 54 and measures between the measuring process of ripple of one or more forms and obtains rapidly changing, and maybe can quicken to excite electrogenerated chemiluminescence.
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 the particle of mean diameter as 0.001-200 μ m (as 0.05-200 μ m), and 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 (being the application's list of references) have been 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 a plurality of north-souths 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 little place of Hamamatsu R374 PMT (low gain red-sensitive cell)
The luminometer of reason machine control; The variable voltage of PMT is the 0-1400 volt
(2) material
(a) electrogenerated chemiluminescence label: Ru (bpy) 3 2+
(b) electrogenerated chemiluminescence damping fluid: 112mM KH 2PO 4, 88mM K 2HPO 43H 2O,
50μM?NaCl，6.5mM?NaN 3，0.8μM
Triton X-100，0.4mM?Tween?20，100mM
The aqueous solution of 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 3，0.43mM
The aqueous solution of bovine serum albumin
(d) Ru (bpy) 3 2+-NHS:Ru (2,2 '-bipyridyl) 2(4-(3-(1,3-dioxy penta
Propyl group)-4 ring-2-yl) '-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 is provided by Dynal (NY 11021 for 45 North Station Plaza, Great Neck)
(ii) Dynal M-280 Dynabeads, the super paramagnetic particle of 2.8 μ M diameters, 10mg/mL is provided by Dynal (NY 11021 for 45 North Station Plaza, Great Neck)
(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
A plurality of north-souths 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 a plurality of electromagnets (as illustrated in fig. 1 and 2).
The plane along continuous straight runs orientation of settling chamber, a plurality of 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 a plurality of north-souths 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 running 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, a plurality of as illustrated in fig. 1 and 2 permanent magnets of 37 expressions.
The plane along continuous straight runs orientation of collecting chamber, a plurality of 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 a plurality of magnets 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
By magnetic separation method, (Norway), the each washing used 2ml solution to the Magnetic Induction polystyrene M-450 DYNABEADS that the 4.5 μ m of washing 30mg (1ml) do not coat for DYNAL, Oslo with 150mM phosphate buffer (pH7.5).With 150 μ g Ru (bpy) 3 2+Phosphate-buffered saline (the PBS of mark mouse IgG (Jackson Immunochemicals), 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, lml3%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.Table I has provided the light emission measure of the electrogenerated chemiluminescence that the Magnetic Induction particle that coated by labelled protein obtains.
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 SiO2 1150
Gold 1.0-2.0 19.3 Au 1100
Embodiment 6: the Dynal particle that the goat-anti thyrotropic hormone (TSH) of physisorption coats
Preparation (reagent I)
Have on the surface that does not coat by magnetic separation washing lml4.5 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 lml 3% BSA/PBSW/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 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), 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 that ouabain (4.6ml) is added drop-wise to 108mg bovine serum albumin (BSA) (Miles Fraction V), and (pH7.8) in the solution, ouabain is 40: 1 with the ratio of BSA for 5ml, 0.15M.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 of the Dynal particle that the ouabain of physisorption-BSA coats
(reagent III)
Have-the magnetic polystyrene particle (DYNAL of OH residue by the surface that magnetic separation washing 5mg4.5 μ m does 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 3% (weight).
Embodiment 9:Ru (bpy) 3 2+The preparation of the mouse-anti digoxin of mark (reagent IV)
With Ru (bpy) 3 2+Mark 1mg mouse-anti digoxin (Cambridge MedicalTechnologies 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) 3 2+Can use behind-the NHS.With 0.18mg Ru (bpy) 3 2+-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 3 2+: 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, 0.15MNaCl and 0.05% heavy Sodium azide, pH7.2) purifying.Collect and precipitation Ru (bpy) 3 2+The mouse-anti digoxin part of mark, labelled protein (reagent IV) after measured, every protein molecular has 12 labels.
Embodiment 10:Ru (bpy) 3 2+The preparation (reagent V) of the mouse-anti thyrotropic hormone (TSH) of mark
With Ru (bpy) 3 2+Mark 0.5mg mouse-anti TSH (CIBA) uses Centricon 30 little concentrators (Amicon) will resist TSH antibody buffering to exchange among 0.15M kaliumphosphate buffer, the 0.15M NaCl (pH7.8) again, and final volume is 0.35ml.With 0.5mg Ru (bpy) 3 2+-NHS can use after being dissolved into 75 μ l anhydrous dimethyl sulfoxides (Aldrich).With 0.176mg Ru (bpy) 3 2+-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 3 2+Label: 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.(collect and precipitation Ru (bpy) for 1 * 20cm, 0.15M potassium phosphate, 0.15M NaCl and 0.05% heavy Sodium azide by pH7.2) purifying through Sephadex G-25 post for reaction mixture 3 2+The 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) 3 2+After the ECL buffer solution of the ECL buffer solution of the mouse-anti TSH of mark (reagent V) 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.Table II has provided the value of measuring curve.
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 Lumi TAG Kit), 25 μ l Ru (bpy) 3 2+After the ECL buffer solution of the ECL buffer solution of the mouse-anti TSH of mark (reagent V) 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.Table III has provided the value of measuring curve.
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, and (Chicago is IL) with 25 μ lRu (bpy) for TDx Assay, Abbott Labs 3 2+After the ECL buffer solution of the mouse-anti digoxin of mark (reagent IV) merges, at room temperature incubation mixed in 20 minutes, the slow middle liquid of ECL 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.Table IV has provided the value of measuring curve.
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, and (Chicago is IL) with 25 μ lRu (bpy) for TDx Assay, Abbott Labs 3 2+After 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).Table V has provided the value of measuring curve.
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 by read period
Step is not separated comparative determination
50 μ l are demarcated serum, and (Chicago is IL) with 25 μ lRu (bpy) for TDx Assay, Abbott Labs 3 2+After 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.Table VI has provided the value of measuring curve.
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 warm the region between the heart and the diaphragm 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 3 2+Labeled 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 the DynalM450 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 DynalM-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, and (Clontech, San Diego CA, dimethyl sulfoxide 5002-1) are added among the 15mg BSA (in 2-3ml PBS), 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 in the washed Dynabead of sodium carbonate/bicarbonate damping fluid (Dynal 14002), and 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, these particles of ECL thinning agent once washing with 10ml are suspended in the ECL dilution and 100 μ l tRNA (10mg/ml) of 10ml then again.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 in the ECL of 29.7ml dilution and 300 μ l tRNA (10mg/ml), 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) 3 2+Mark 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 lECL 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 Table VII.
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.
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Claims (8)

1. in conjunction with the method for meaningful analyte in the measuring samples, the step of this method comprises:
(a) form composition, said composition contains:
(i) described sample,
The measurement material that (ii) contains the component that is connected with the tagged compound that can induce electrogenerated chemiluminescence;
(iii) a plurality of Magnetic Induction suspended particles that can combine with the material specificity with analyte and/or described measurement;
(b) the described composition of incubation forms the compound that contains particle and described tagged compound;
(c) described composition is imported in the measuring cell;
(d) by on described particle, producing magnetic field, on electrode surface, collect this compound;
(e) by on described electrode, applying voltage, with the tagged compound induced luminescence in the collected compound;
(f) the potential electrode surface penetrated luminous, with existing of meaningful analyte in the measuring samples;
Wherein a plurality of north-souths magnet is vertically fixed on level below electrode surface.
2. by the process of claim 1 wherein that described measurement is that homogeneity is measured.
3. by the process of claim 1 wherein that described measurement is a heterogeneity test.
4. by the method for claim 3, wherein said heterogeneity measure also be included in incubation step (b) afterwards and measuring process (f) before, from compound, isolate unconjugated composition component.
5. by the method for claim 4, wherein said separating step is to induce step (e) to carry out before.
6. by the method for claim 4, wherein separating step is to carry out before in importing step (c).
7. by the method for claim 5, wherein separating step comprises: collecting step (d) afterwards, liquid is pumped in the measuring chamber, described liquid is substantially free of label or labelled reagent, thus any unconjugated composition component in the flush away compound.
8. by the device of potential electrode surface electrogenerated chemiluminescence in conjunction with meaningful analyte in the measuring samples, this device comprises:
(a) analysis room, this analysis room limits the measuring samples volume and this volume has the charging drawing mechanism, and this analysis room further comprises electrode surface, and this electrode surface is exposed to described volume;
(b) make the equally distributed basically device of described compound on described electrode surface, this device is near described electrode surface;
(c) to the voltage source of described electrode application voltage;
(d) measure the optical detection device of the electrogenerated chemiluminescence on described electrode, produce;
Wherein make the equally distributed device of compound also comprise the device that produces the magnetic field that adapts with described electrode, the magnetic line of force in this magnetic field is paralleled with this electrode surface in described surface region basically, and the device that produces magnetic field, it comprises magnet a plurality of north-souths and that isolate by the nonmagnetic substance that is vertically fixed on below the described electrode.
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