CN101115985A - System and method for spectroscopic analysis of single particles - Google Patents

Abstract

The invention encompasses analyzers and analyzer systems that include a single particle analyzer, methods of using the analyzers and analyzers systems to analyze samples, either for single particles, e.g., protein molecules, or for multiple particles (multiplexing), methods of doing business based on the use of the analyzers or analyzer systems of the system, and electronic media for storing parameters useful in the analyzers and analyzer systems of the invention.

Description

The system and method for spectral analysis single-particle

Cross reference

The application requires in the U.S. Provisional Application No.60/613 that is entitled as " Continuous Wave SingleParticle Detector " of submission on September 28th, 2004,881, the U.S. Provisional Application No.60/624 that is entitled as " Sandwich Assay for Detection of Individual Molecules " that on October 27th, 2004 submitted to, the U.S. Provisional Application No.60/636 that is entitled as " Methods for DetectingIndividual Molecules; Particles; or Cells " that on Dec 13rd, 785 and 2004 submitted to, 158 right of priority, above-mentioned application is all complete to be incorporated herein by reference.

Background of invention

The progress that biomedical research, medical diagnosis, prognosis, monitoring and treatment selection, bio-terrorism detect the field relevant with the analyte concentration analysis with the various product analysis of other and small size has caused developing the sample analysis system that can detect the particle in the low concentration sample delicately.United States Patent(USP) Nos. 4,793,705 and 5,209,834 have put down in writing the Previous System of realizing utmost point Sensitive Detection.The invention provides in this field and further make progress.

Summary of the invention

In one aspect, the invention provides a kind of single-particle analyzer system.In certain embodiments, this system comprises that can gather a plurality of samples and sampling container and first automatically inquires after the sampling system that the fluid between the space is communicated with, with the analyzer that can detect single-particle, wherein said analyzer comprises the electromagnetic radiation source that is used to send electromagnetic radiation; Be set to receive first of electromagnetic radiation that this electromagnetic radiation source sends and inquire after the space; Be set to receive second of electromagnetic radiation that this electromagnetic radiation source sends and inquire after the space; Wherein said second inquires after space and described first inquires after the space fluid and is communicated with, and wherein inquires after space and second first and inquire after and have motive power between the space, causes particle to inquire after space and second first and inquires after between the space and move; With first inquire after first electromagnetic radiation detector that the space can be operatively connected, be used to measure first electromagnetic signature of particle; With second inquire after second electromagnetic radiation detector that the space can be operatively connected, be used for measuring at least one of second electromagnetic signature of particle and first electromagnetic signature of particle.In certain embodiments, described electromagnetic radiation source is the continuous wave electromagnetic radiation source, as light emitting diode or continuous wave laser.In the other embodiment, this analytic system comprises sampling system, and wherein the carry-over rate (carryover) of sampling system is less than about 0.02%.

In certain embodiments, first and second inquire after the space has about 0.02pL separately to about 300pL, or about 0.05pL about 50pL extremely, or about 0.1pL volume of about 25pL extremely.In certain embodiments, at least the first and second volumes of inquiring after one of space are adjustable.

In certain embodiments, analyzer system of the present invention further comprises at least and first inquires after space and second and inquire after the 3rd electromagnetic radiation detector that one of space can be operatively connected, is used for measuring at least one of first electromagnetic signature of particle and second electromagnetic signature of particle.In certain embodiments, the motive power of analyzer is pressure, is for example provided by pump, vacuum source, hydro-extractor or their combination.

In some analyzer system of the present invention, fluid is communicated with pipeline or the passage that comprises in the microfluidic devices, and provides pressure by one or more pumps.

In certain embodiments, analyzer system of the present invention further comprise with second inquire after that the space fluid is communicated with, can reclaim basically all sample retrieval systems of samples, and/or sample preparation system, and/or analyze first and second electromagnetic signatures and report analysis result's data analysis system.In comprising the embodiment of sample preparation system, sample preparation system can carry out specimen preparation by centrifugal, filtration, chromatography, lysis, change pH, interpolation damping fluid, interpolation reagent, heating or cooling, irradiation, interpolation label, label combination, the unconjugated label of separation or their combination.In comprising the embodiment of data analysis system, analysis can comprise the existence of determining particle, the concentration that does not exist and choose wantonly, and based on described existence, do not exist and/or concentration is determined possible diagnosis, prognosis, therapeutic state or recommended therapy.

In one embodiment, the invention provides a kind of analyzer system, this system comprises that sampling container and first inquires after the sampling system that the fluid between the space is communicated with; Comprise that first inquires after the single-particle analyzer that the space is inquired after in space and second, wherein second inquire after space and first and inquire after the space fluid and be communicated with, and inquire after space and second first and inquire after and have motive power between the space, cause particle to inquire after space and second and inquire after between the space and move first; With described first and/or described second inquire after the detecting device that the space can be operatively connected, be used to detect the detectable feature (if present) of particle; Sample retrieval system can be from sampling receptacle to inquiring after spatial movement and return sampling receptacle by this system's sample, and do not contact other parts of this analyzer, and do not contact the cleaning buffer in this analyzer basically; And data-analyzing machine, it receives the input of self-detector, and analyze the existence of particle or do not exist, and based on described existence or do not exist and report the result.This system may further include sample preparation system.

In another embodiment, the invention provides a kind of single-particle analyzer system, comprising: can gather a plurality of samples and sampling container and first automatically and inquire after the sampling system that the fluid between the space is communicated with; Can detect monomolecular analyzer, this analyzer comprises the electromagnetic radiation source that is used to send electromagnetic radiation; Be set to receive electromagnetic radiation that described electromagnetic radiation source sends first inquire after the space; With first inquire after first electromagnetic radiation detector that the space can be operatively connected, be used to measure first electromagnetic signature of particle.

In another embodiment, the invention provides a kind of analyzer system, it comprises analyzer, first sample in adding analyzer and the volume of second sample are less than 5 μ l, and when the concentration of analyte flew mole less than 5, this analyzer can detect between first sample that adds in the analyzer and second sample analyte concentration difference less than 20%.In certain embodiments, this system further comprises the sampling system of the fluid connection that can gather automatically between a plurality of samples and sampling container and the analyzer.

On the other hand, the invention provides the single-particle analyzer.In certain embodiments, the invention provides a kind of single-particle analyzer, it comprises that at least one is used to send the continuous wave electromagnetic radiation source of electromagnetic radiation; Be set to receive first of electromagnetic radiation that described electromagnetic radiation source sends and inquire after the space, this first volume of inquiring after the space is about 0.02pL about 300pL extremely; Be set to receive second of electromagnetic radiation that described electromagnetic radiation source sends and inquire after the space, this second container of inquiring after the space is that about 0.02pL is to about 300pL, wherein second inquire after space and first and inquire after the space fluid and be communicated with, wherein inquire after space and second and inquire after and have electromotive force between the space, cause and use electric power can make particle inquire after space and second to small part to inquire after between the space and move first first; With first inquire after first electromagnetic radiation detector that the space can be operatively connected, be used to measure first electromagnetic signature of particle; With with second inquire after second electromagnetic radiation detector that the space can be operatively connected, be used for measuring at least one of second electromagnetic signature of particle and first electromagnetic signature of particle.In certain embodiments, described continuous wave electromagnetic radiation source is selected from light emitting diode and continuous wave laser.In certain embodiments, at least the first inquire after the volume of inquiring after one of space in space and second and be about 0.1pL about 25pL extremely.In certain embodiments, at least the first and second volumes of inquiring after one of space are adjustable.In certain embodiments, at least the first inquire after space and second and inquire after one of space and limit by one of the cross-sectional area of the electromagnetic radiation beam that receives from electromagnetic radiation source and sensing range of one of at least the first electromagnetic radiation detector and second electromagnetic radiation detector at least.In some embodiment of back, sensing range is determined by the gap width in adjacent with one of first electromagnetic radiation detector and the second electromagnetic radiation detector at least spatial filter.In certain embodiments, at least the first and second inquire after one of space is limited by housing at least in part, and this housing comprises and is selected from glass, quartz, melts the solid material that coagulates quartzy, plastics or its combination in any.In certain embodiments, at least the first inquire after space and second and inquire after one of space and limit by fluid boundary at least in part.

In certain embodiments, analyzer further comprises at least and first inquires after space and second and inquire after the 3rd electromagnetic radiation detector that one of space can be operatively connected, is used for measuring at least one of first electromagnetic signature of particle and second electromagnetic signature of particle.

In some embodiment of analyzer of the present invention, one of at least the first electromagnetic radiation detector and second electromagnetic radiation detector are selected from CCD camera, video input module camera, streak camera, bolograph, photodiode, photodiode array, avalanche photodide detecting device, photomultiplier detector and combination in any thereof.

In certain embodiments, this analyzer further comprises one of pump, vacuum source and hydro-extractor at least, is used to promote particle to inquire after space and second first and inquires after moving between the space.

On the other hand, the invention provides analytical approach.In one embodiment, the invention provides a kind of analytical approach, comprise and use the single-particle analyzer system to measure the existence of particle in the sample that obtains or do not exist from individuality, this single-particle analyzer system comprises: (a) can gather a plurality of samples and sampling container and first automatically and inquire after the sampling system that the fluid between the space is communicated with; (b) can detect the analyzer of single-particle, this analyzer comprises: the electromagnetic radiation source that (i) is used to send electromagnetic radiation; (ii) be set to receive first of electromagnetic radiation that described electromagnetic radiation source sends and inquire after the space; (iii) be set to receive second of electromagnetic radiation that described electromagnetic radiation source sends and inquire after the space; Wherein second inquire after space and first and inquire after the space fluid and be communicated with, and inquire after space and second first and inquire after and have motive power between the space, cause particle to inquire after space and second and inquire after between the space and move first; (iv) inquire after first electromagnetic radiation detector that the space can be operatively connected, be used to measure first electromagnetic signature of particle with first; (v) inquire after second electromagnetic radiation detector that the space can be operatively connected, be used for measuring at least one of second electromagnetic signature of particle and first electromagnetic signature of particle with second.

In some embodiment of the inventive method, described analyzer further comprises the data analysis system of analyzing described first and second electromagnetic signatures and reporting described analysis result; In some embodiment, this analysis further comprises based on described analysis result determines diagnosis, prognosis, therapeutic state and/or methods of treatment therein.

In some embodiment of the inventive method, this analyzer system further comprise with second inquire after that the space fluid is communicated with, can reclaim the sample retrieval system of whole described samples basically, and/or sample preparation system.

In some embodiment of the inventive method, electromagnetic radiation source is the continuous wave electromagnetic radiation source.

In some embodiment of the inventive method, first and second inquire after the space has about 0.02pL separately to about 300pL, or about 0.05pL about 50pL extremely, or about 0.1pL volume of about 25pL extremely.

In some embodiment of the inventive method, at least the first and second volumes of inquiring after one of space are adjustable.

In some embodiment of the inventive method, motive power comprises pressure.In some embodiment, provide pressure therein by the source that is selected from pump, vacuum source, hydro-extractor and their combination.

In some embodiment of the inventive method, individuality is an animal or plant, for example animal, for example mammal, for example people.

In certain embodiments, method of the present invention comprises to be analyzed the multiple particle in the sample, in some such embodiment, every kind of detected in multiple particle particle comprises label, and every kind of wherein detected particle is distinguished with other particle mutually according to being selected from label identity, label intensity, mobility or its combined feature.

In some embodiment of the inventive method, sample is selected from blood, serum, blood plasma, bronchoalveolar lavage fluid, urine, cerebrospinal fluid, hydrothorax, synovia, ascites, amniotic fluid, gastric juice, lymph liquid, interstitial fluid, tissue homogenate, cell extract, saliva, phlegm, ight soil, physiology secretion, tears, mucus, sweat, milk, seminal fluid, seminal fluid liquid, vaginal fluid, from ulcer and other surperficial rash, the liquid of blister and abscess and comprise normal, the extract of the tissue in pernicious and suspection is organized in, any other composition that maybe may contain the health of particle.In certain embodiments, sample is selected from blood, blood plasma or serum.In some embodiment, this method further comprises the particle in the described sample of mark therein, wherein analyzes described sample and comprises the existence of the particle that detects described mark or do not exist; Randomly also comprise and from described sample, remove unconjugated label, and/or from described individuality, obtain described sample, and/or analyze the particle that is selected from protein, nucleic acid, nanosphere, microballoon, dendrimer, chromosome, carbohydrates, virus, bacterium, cell and combination in any thereof, for example from protein, nucleic acid, virus, bacterium and combination in any thereof, select particle.In certain embodiments, this particle is selected from amino acid, nucleotide, lipid, sugar, granule toxin, peptide toxin, venom, medicine and combination in any thereof.

In some embodiment of the inventive method, sample is the blood serum sample that contacts with fluorescently-labeled intended particle specific antibody; Wherein said analysis comprises the existence of the particle of certification mark, does not exist and/or concentration.Therein in some embodiment, this method further comprises based on the existence of the particle of mark, does not exist and/or concentration is determined diagnosis, prognosis, therapeutic state and/or methods of treatment.In certain embodiments, biomarker is TREM-1.In certain embodiments, this method is finished in less than 1 hour.In certain embodiments, based on the existence of one group of biomarker, do not exist and/or concentration is determined diagnosis, prognosis, therapeutic state and/or methods of treatment.In certain embodiments, this method is finished in less than 2 hours.

In one embodiment, the present invention includes a kind of analytical approach, comprise existence, do not exist and/or concentration is determined diagnosis, prognosis, therapeutic state and/or methods of treatment based on particle in the sample that from individuality, obtains, wherein use to comprise that the analyzer system that can detect monomolecular analyzer determines described existence, do not exist and/or concentration, wherein said analyzer comprises that is at least inquired after a space.In some embodiment, described analyzer comprises that at least two are inquired after the space therein.In certain embodiments, described analyzer system comprises can detect monomolecular analyzer, and this analyzer comprises that at least one is used to send the continuous wave electromagnetic radiation source of radiation, and wherein at least one is inquired after the space and is set to receive described radiation.

In another embodiment, the invention provides and a kind ofly have cancer and the method for examination individuality in order to determine whether, comprise that the analysis of operational analysis device is from one or more cancer markers in the sample of individuality, in the concentration of every kind of label less than 1 picomole, and the volume of sample is during less than about 5 μ l, and the concentration that this analyzer can detect one or more labels between a kind of sample and the another kind of sample is less than 20% variation.In certain embodiments, this method further comprises with the value of described analysis result and known label relatively.In certain embodiments, individuality is the smoker, and cancer is a lung cancer.

In another embodiment, the invention provides a kind of method that detects particle, comprising: by electric power particle is moved to volume and inquire after in the space and volume is extremely second inquiring after in the space of about 300pL of about 0.02pL to first of about 300pL for about 0.02pL; To at least one continuous wave electromagnetic radiation source of sample application; Inquire after when interacting in the space first when particle and continuous wave electromagnetic radiation, inquire after first electromagnetic signature of measuring particle in the space first; With inquire after when interacting in the space second when particle and continuous wave electromagnetic radiation, inquire after one of first electromagnetic signature of measuring particle in the space at least and second electromagnetic signature second.Particle is that described method further comprises in some embodiment of first particle therein: make second particle move to first and inquire after space, second and inquire after space, the 3rd and inquire after space and the 4th and inquire after in the space at least two; Inquire after space, second when second particle and continuous wave electromagnetic radiation first and inquire after space, the 3rd and inquire after when interacting in space and the 4th at least one of inquiring after in the space, measure one of first electromagnetic signature of second particle and second electromagnetic signature of second particle at least.

On the other hand, the invention provides a kind of computer-readable storage medium, comprise that is used to an instruction that has user interface, comprises the multi-purpose computer of display unit, this group instruction comprises: (a) be used to import with the logic with value that two single-particle detecting device analytic samples of inquiring after the space are obtained; (b) show input value result's display routine with described display unit.In certain embodiments, this instruction further comprises the comparison program that is used for comparison input value and database; Wherein display routine further comprises the logic that is used to show the comparison program result.

On the other hand, the invention provides electric signal or the carrier wave between computing machine, propagated by the internet, comprise that is used to an instruction that has user interface, comprises the multi-purpose computer of display unit, this group instruction comprises computer-readable storage medium, be used to the instruction that has user interface, comprise the multi-purpose computer of display unit comprising one group, this group instruction comprises: (a) be used to import with the logic with value that two single-particle detecting device analytic samples of inquiring after the space are obtained; (b) show input value result's display routine with described display unit.In certain embodiments, this group instruction further comprises the comparison program that is used for comparison input value and database; Wherein display routine further comprises the logic that is used to show the comparison program result.

On the other hand, the invention provides a kind of method of carrying out business activity, comprise by entity and use two detecting devices of inquiring after the space that have can detect single-particle to obtain the sample determination results, report described result, and the expense of reporting the result to this entity pays.In certain embodiments, this entity is that clinical labororatory improves correction (Clinical Laboratory ImprovementAmendments (CLIA)) laboratory.In certain embodiments, this entity is not the CLIA laboratory.

On the other hand, the invention provides the device of the electronic transhipment of inquiring after space and particle to be detected (comprising single-particle) of having made up continuous wave irradiation source, two or more different pL sizes.

In one aspect, the invention provides the single-particle analyzer, it comprises that at least one is used to send the continuous wave electromagnetic radiation source of electromagnetic radiation; Be set to receive first of electromagnetic radiation that described electromagnetic radiation source sends and inquire after the space, this first container of inquiring after the space is about 0.02pL about 300pL extremely; Be set to receive second of electromagnetic radiation that described electromagnetic radiation source sends and inquire after the space, this second volume of inquiring after the space is that about 0.02pL is to about 300pL, wherein this second is inquired after space and first and inquires after the space fluid and be communicated with, wherein first inquire after space and second and inquire after and have electromotive force between the space, cause and use electric power can make particle inquire after space and second to small part to inquire after between the space and move first; With first inquire after first electromagnetic radiation detector that the space can be operatively connected, be used to measure first electromagnetic signature of particle; With with second inquire after second electromagnetic radiation detector that the space can be operatively connected, be used for measuring at least one of second electromagnetic signature of particle and first electromagnetic signature of particle.

According to a further aspect in the invention, analyzer further comprises at least and first inquires after space and second and inquire after the 3rd electromagnetic radiation detector that one of space can be operatively connected, is used for measuring at least one of first electromagnetic signature of particle and second electromagnetic signature of particle.In one aspect, this continuous wave electromagnetic radiation source is selected from light emitting diode and continuous wave laser.

According to a further aspect in the invention, one of at least the first electromagnetic signature and second electromagnetic signature be selected from emission wavelength, emissive porwer, burst (burst) size, the duration of bursting, fluorescence polarization and combination in any thereof.

In another aspect of this invention, at least the first inquires after space and second inquires after one of space and has about 0.05pL to about 50pL, and preferably about 0.10pL is the volume of about 25pL extremely.In one aspect, at least the first and second volumes of inquiring after one of space are adjustable.In a replacement scheme, at least the first inquires after space and second inquires after one of space and is limited by one of the area of section of the electromagnetic radiation beam that receives from electromagnetic radiation source and sensing range of one of at least the first electromagnetic radiation detector and second electromagnetic radiation detector at least.In one aspect, sensing range is determined by the gap width in adjacent with one of first electromagnetic radiation detector and the second electromagnetic radiation detector at least spatial filter.

In another replacement scheme, at least the first and second inquire after one of space is limited by the solid housing, and this housing comprises and is selected from glass, quartz, melts the solid material that coagulates quartzy, plastics or its combination in any.In another replacement scheme, at least the first inquires after space and second inquires after one of space and is limited by fluid boundary.

In another aspect of this invention, one of at least the first electromagnetic radiation detector and second electromagnetic radiation detector are selected from charge-coupled device (CCD) camera, video input module camera, streak camera, bolograph, photodiode, photodiode array, avalanche photodide detecting device, photomultiplier detector and combination in any thereof.On the other hand, this analyzer further comprises one of pump, vacuum source and hydro-extractor at least, is used to promote particle to inquire after space and second first and inquires after moving between the space.

On the other hand, the invention provides a kind of method that detects particle, comprise that utilizing electric power that particle is moved to volume inquires after in the space and volume is about 0.02pL second inquiring after in the space of about 300pL extremely for about 0.02pL to first of about 300pL; To at least one continuous wave electromagnetic radiation source of sample application; Inquire after when interacting in the space first when particle and continuous wave electromagnetic radiation, inquire after first electromagnetic signature of measuring particle in the space first; With inquire after when interacting in the space second when particle and continuous wave electromagnetic radiation, inquire after one of first electromagnetic signature of measuring particle in the space at least and second electromagnetic signature second.

According to a further aspect in the invention, second inquire after the space and comprise a plurality of spaces of inquiring after.In accordance with a further aspect of the present invention, particle is first particle, and described method comprises that further making second particle move to first inquires after space, second and inquire after space, the 3rd and inquire after space and the 4th and inquire after in the space at least two; Inquire after space, second when second particle and continuous wave electromagnetic radiation first and inquire after space, the 3rd and inquire after when interacting in space and the 4th at least one of inquiring after in the space, measure one of first electromagnetic signature of second particle and second electromagnetic signature of second particle at least.

In accordance with a further aspect of the present invention, this method comprises that further inquiring after space and second with first inquires after the space and be connected, and adds fluid before improved.According to a further aspect in the invention, this method further comprises from protein, nucleic acid, nanosphere, microballoon, dendrimer, chromosome, carbohydrates, virus, bacterium, cell and combination in any thereof and selects particle.In one aspect, the combination of particle is selected from protein, nucleic acid, virus and bacterium.Perhaps, this method further comprises from amino acid, nucleotide, lipid, sugar, granule toxin, peptide toxin, venom, medicine and combination in any thereof and selects particle.

In another aspect of this invention, one of at least the first electromagnetic signature and second electromagnetic signature are produced by one of the intrinsic parameter of particle and extrinsic parameter of particle.In one aspect, this method further comprises with at least a label label particles, so that the extrinsic parameter to be provided.On the other hand, this label sends electromagnetic radiation, and is selected from dye marker, light scattering label and combination in any thereof.

At particle is another aspect of the present invention of first particle, described label is first label, described first particle and first label are included in the potpourri of multiple particle and multiple label, this method further comprises (a) first separate particles with at least a unconjugated label in the multiple label and multiple particle, and at least a unconjugated label in the multiple label can not be detected; (b) first particle in the multiple particle and reagent interact, and make first particle discharge first label that combines with it; (c) discharge the back from first particle and detect first label, directly detect first particle with this at first label.

In one aspect of the invention, first label that discharges from first particle is a particle, on the other hand, and with at least two kinds of diacritic these particles of label mark.In a replacement scheme, at least by direct this particle of mark of one of specificity and non-specific interaction, this interaction is selected from covalent bond, ions binding, hydrophobic effect combination, affine combination, hydrogen bonding, Van der Waals force attraction, co-ordination complex formation and combination in any thereof.In another replacement scheme, by coming this particle of indirect labelling with at least a binding partners incubation to form specific complex, wherein this binding partners comprises at least one label.

In one aspect, at least comprise one of specificity and non-specific interaction by the step with at least a binding partners incubation indirect labelling particle, this interaction is selected from covalent bond, ions binding, hydrophobic effect combination, affine combination, hydrogen bonding, Van der Waals force attraction, co-ordination complex formation and combination in any thereof.In a replacement scheme, particle is inquired after space and second first at least and is inquired after in one of space and the binding partners incubation.In another replacement scheme, before improved with particle and binding partners incubation.

On the other hand, binding partners is selected from polynucleotide/polynucleotide interaction, polynucleotide/polypeptide interacts and polypeptide/polypeptide interacts and combination in any.In one aspect, incubation described and at least a binding partners comprises the particle and the first binding partners incubation; The particle and the second binding partners incubation, wherein one of at least the first binding partners and second binding partners comprise at least one label.

In another aspect of this invention, particle being moved to first inquires after space and second and inquires after and comprise in the space this particle is used the separation mechanism that is selected from capillary gel electrophoresis, the electronic chromatography of capsule, isotachophoresis, magnetic field and combination in any thereof.In a replacement scheme, this method further comprises mobile in the opposite direction second particle with common side with first particle.In one aspect, particle is moved to first to be inquired after space and second and inquires after and comprise in the space and utilize electric power and the combination of another power is improved at least that described another power at least is selected from pressure gradient, gravity, surface tension, centrifugal force and combination in any thereof.

In another aspect of this invention, the mobility of particle depends on the interaction of electric power and particle physics parameter, and this physical parameter comprises one of intrinsic and extrinsic parameter at least.In one aspect, this method further comprises with at least a label label particles, so that the extrinsic parameter to be provided.In one aspect, this label can influence mobility, and is selected from electric charge label, electric charge/mass labels, magnetic mark thing and combination in any thereof.On the other hand, with at least two kinds of diacritic label label particles.

In a replacement scheme, at least by the direct label particles of one of specificity and non-specific interaction, this interaction is selected from covalent bond, ions binding, hydrophobic effect combination, affine combination, hydrogen bonding, Van der Waals force attraction, co-ordination complex formation and combination in any thereof.In another replacement scheme, by coming the indirect labelling particle with at least a binding partners incubation to form specific complex, wherein binding partners comprises at least one label.

In one aspect, at least comprise one of specificity and non-specific interaction by the step with at least a binding partners incubation indirect labelling particle, this interaction is selected from covalent bond, ions binding, hydrophobic effect combination, affine combination, hydrogen bonding, Van der Waals force attraction, co-ordination complex formation and combination in any thereof.In a replacement scheme, particle is inquired after space and second first at least and is inquired after in one of space and the binding partners incubation.In another replacement scheme, before improved with particle and binding partners incubation.

On the other hand, binding partners is selected from polynucleotide/polynucleotide interaction, polynucleotide/polypeptide interacts and polypeptide/polypeptide interacts and combination in any.In one aspect, incubation described and at least a binding partners comprises the particle and the first binding partners incubation; With the particle and the second binding partners incubation, wherein one of at least the first binding partners and second binding partners comprise at least one label.

In another aspect of this invention, the potpourri of different binding partners and particle incubation.On the other hand, particle is first particle in the potpourri of multiple particle and multiple label, with the first label mark, first particle in the multiple label, and wherein have any different with unconjugated label in unlabelled particle and the multiple label in first particle of the first label mark and the multiple particle.

On the other hand, with the second label mark, first particle in the multiple label, and wherein the ratio of the electromagnetic signature of the electromagnetic signature by measuring first label and second label can be distinguished in first particle and the multiple particle unconjugated label in the unlabelled particle and multiple label.In another aspect of this invention, detect first and second particles at least, this method further comprises counts first and second particles at least.In a replacement scheme, the step of counting first and second particles at least comprises the concentration that concentration and the external particles standard with concentration known by comparative sample come particle in the working sample.In another replacement scheme, the step of counting first and second particles at least comprises the concentration that concentration and the internal particle standard with concentration known by comparative sample come particle in the working sample.In another replacement scheme, the step of counting first and second particles at least comprises does not use external perimysium reference and internal standard and the concentration of particle in the working sample.

In another aspect of this invention, first and second particles are first and second particles in the potpourri of multiple particle and multiple label, be connected with first label in the multiple label on first particle, be connected with second label in the multiple label on second particle, first and second labels have different preset ranges separately, and wherein the difference of unconjugated label is the characteristic signal that unconjugated label produces in first particle and the multiple label, and the difference of first particle and second particle is the different range of first and second labels.

In another aspect of this invention, first and second particles are first and second particles in the potpourri of multiple particle and multiple label, first particle is with the first label mark, second label and first label can be distinguished, second particle the 3rd label mark that is substantially similar to first label, wherein first particle is different from second particle, be different from only with the particle in the multiple particle of the label mark that is substantially similar to second label in the multiple label, be different from unlabelled particle in the multiple label, be different from unconjugated label in the multiple label.In a replacement scheme, the first and the 3rd label sends electromagnetic radiation, the second and the 4th label influences mobility, and wherein first particle is different from second particle, be different from only with the particle in the multiple particle of the label mark that influences mobility, be different from unlabelled particle in the multiple particle, be different from unconjugated label in the multiple label.In another replacement scheme, second particle the 4th label mark that is substantially similar to second label, wherein the ratio of the electromagnetic radiation feature of the electromagnetic signature of first label and second label is different from the ratio of the electromagnetic radiation feature of the electromagnetic radiation feature of the 3rd label and the 4th label, and wherein distinguishes first particle and second particle by the difference between the label ratio of measuring first particle and second particle.

On the other hand, the electromagnetic signature of first label, second label, the 3rd label and the 4th label is a wavelength.In another replacement scheme, second particle the 4th label mark that is substantially similar to second label, wherein the summation of the electromagnetism intensity that sends of first label and second label is different from the summation of the electromagnetism intensity that the 3rd label and the 4th label send, and wherein the difference between the intensity summation of the intensity summation by measuring first and second labels and third and fourth label is distinguished first particle and second particle.

On the other hand, first particle is with the first label mark, and second particle is with the second label mark, and wherein the difference between the electromagnetic signature of the electromagnetic signature by measuring first particle and second particle is distinguished first particle and second particle.In one aspect, the electromagnetic signature of first particle and second particle is a wavelength.

In a replacement scheme, particle is first particle with intrinsic detectable feature, label is first label that influences mobility, second particle usefulness with intrinsic detectable feature influences the second label mark of mobility, and wherein distinguishes first and second particles by the difference of measuring mobility between first and second particles.In another replacement scheme, particle is first particle with intrinsic detectable feature, label is first label that influences mobility, second particle with intrinsic detectable feature is not labeled, and wherein distinguishes first and second particles by the difference of measuring mobility between first and second particles.

In another aspect of this invention, this method comprises that further comparison inquires after the electromagnetic signature of the particle of measuring in the space and in second electromagnetic signature of inquiring after the particle of measuring in the space first.In one aspect, this comparison step comprises electromagnetic signature and the background Electromagnetic Launching of distinguishing the particle of at least a mensuration by statistical analysis.On the other hand, this comparison step comprises the electromagnetic signature crosscorrelation of the particle that will measure, to measure particle's velocity.

With reference to quoting

All publications and the patented claim mentioned in this instructions all are incorporated herein by reference, and specifically and individually are incorporated herein by reference as each independent publication or patented claim.

Description of drawings

Fig. 1. the synoptic diagram of an embodiment of sample analysis of the present invention system.

Fig. 2. the synoptic diagram of an embodiment of method of the present invention.

Fig. 3. the synoptic diagram of the single-particle analyzer of one embodiment of the invention.

Fig. 4. be used for the synoptic diagram of capillary flow moving cell of the single-particle analyzer of one embodiment of the invention.

Fig. 5. have the synoptic diagram of single-particle analyzer of one embodiment of the invention of confocal arrangement.

The electrophoresis and the dilution curve of Fig. 6 .7.2kb dna fragmentation.A) cross correlation analysis of particle in the damping fluid blank.B) cross correlation analysis of particle in the 0.1fM sample.C) by the particle of crosscorrelation detection and the linear relationship of sample concentration.

Fig. 7. the typical curve of the TREM-1 that in measuring, detects for the sandwich molecular immune of single-particle analyzer system development.This setting-out line scope is 100-1500fM.

The typical curve of Fig. 8 .IL-6.A) IL-6 standard is according to R﹠amp; The dilution of D Systems kit obtains 0.1 to 10pg/ml linear response.B) the following IL-6 typical curve of 1pg/ml.C) from R﹠amp; The IL-6 typical curve of D Systems product document is used to use the mensuration of two amplification of signal steps.

Fig. 9. the typical curve that TSH detects in measuring based on the molecular immune of pearl.Acquisition target molecule on pearl, and target molecule and detection antibodies.Utilize pearl fixed target molecule, and remove unconjugated material.Detect complete pearl/target compound with the single-particle analyzer system.

Figure 10. in the sample that contains 10% human serum, add TSH.Sample uses in the sandwich catch assay for TSH, and analyzes in the single-particle analyzer system.The recovery that this mensuration is calculated is 108%.

Figure 11. the electrophoresis of dendrimer and dilution curve.A) cross correlation analysis of particle in the damping fluid blank.B) cross correlation analysis of particle in the 0.03fM sample.C) by the particle of crosscorrelation detection and the linear relationship of sample concentration.

Figure 12. the electrophoresis of dendrimer dilution curve.A) cross correlation analysis of photon in the damping fluid blank.B) cross correlation analysis of photon in the 0.03fM sample.

Figure 13. the electrophoresis and the dilution curve of bovine serum albumin(BSA) (" BSA ") in the presence of lauryl sodium sulfate (" SDS ").A) raw data of the particle that detects by crosscorrelation in the damping fluid blank.B) particle that detects by crosscorrelation in the 10fM sample.C) by the particle of crosscorrelation detection and the linear relationship of sample concentration.

Figure 14. the mobility of virion increases with the increase of electric current.

Figure 15. the detection of microorganism.A) Bacillus coli cells makes its specificity combination with the antibody incubation of mark.After removing unconjugated antibody, discharge the antibody of combination from cell, and measure.B) virion combines with dull and stereotyped, and with the antibody incubation of mark, washing discharges the label of combination, and measures.

Figure 16. mass labels.The electrophoretic mobility of organelle when combining with nucleic acid (PBXL-3) changes.A) the migration peak of independent PBXL-3 is at the 368ms place.B) the PBXL-3 migration peak that combines with nucleic acid is at the 294ms place.C) there is nucleic acid but the PBXL-3 that do not combine with nucleic acid moves at the 409ms place.

Figure 17. all use Alexa Fluor ^647 (" A647 "; Invitrogen, Carlsbad, California) discriminating of the virus of mark and nucleic acid.A) with the virus of coat protein antibody labeling be labeled in the potpourri of nucleic acid and be resolved to two peaks.B) the independent nucleic acid that is labeled.C) virus of independent antibody labeling.

Figure 18 .SDS electrophoresis.All use the discriminating of the protein and the nucleic acid of A647 mark.A) independent and protein antibodies.B) the independent nucleic acid that is labeled.C) at the protein that combines with labelled antibody be labeled in the potpourri of nucleic acid and be resolved to two peaks.

Figure 19. the discriminating of the label that discharges from protein target and target set nucleic acid.A) detection of the label that discharges from the protein target.Thyrotropic hormone (TSH) is fixed on 96 orifice plates, with the anti--TSH mark of A647 mark.Remove unconjugated reagent by washing.From dissociate the down antibody of A647 mark of TSH, in SMD, measure.Between the clean particle of the A647 that measures and original TSH concentration, observe linear relationship.B) differentiate that based on electrophoretic mobility the prediction of the label that discharges illustrates.C) differentiate that based on fluorescence intensity the prediction of the label that discharges illustrates.

Figure 20. the particle based on fluorescence intensity is differentiated.A) and B) brightness was mapped to the elapsed time, was used for detecting every kind of particle with two detecting devices.The monomolecular measured value of each some representative that illustrates on the figure.The scale of restriction x-axle (elapsed time) is to emphasize each molecule in the peak.Utilize the cutoff of 500 photons to divide subwindow and " secretly " to divide subwindow to separate with " bright ".The higher mean intensity of PBXL-3 molecular proportion pUC19 molecular emission.C) compare with the predicted value of determining by the spectral analysis undiluted sample by the PBXL-3 of molecule counting mensuration and the concentration of pUC19.

Figure 21. use detection and the discriminating of sandwich assay to particle.A) target protein (P) combines with pearl (B), also combines with label, and it can be detected.In the heterogeneous mensuration diagram of figure A, remove unconjugated label from sample, pearl-label-target compound carries out electrophoresis.B) measure in the diagram at the homogeneous of figure B, unsegregated sample carries out electrophoresis, pearl-label-target and the difference of unconjugated label.

Figure 22. use double-colored determination method to detect and differentiate the scheme of particle.Intended particle (T) combines with two kinds of labels (L).Every kind of label sends the electromagnetic radiation that difference can detect wavelength.A) sample carries out electrophoresis, and intended particle only is different from the particle with a kind of coloured label mark, and is different from unconjugated target and unconjugated label.B), add activator or antagonist that one of label particles combines with target with each the target-marking particle in two kinds of different labels.Sample carries out electrophoresis, and the particle with two kinds of labels is different from the particle with a kind of label that combines with competitor, and is different from unconjugated label.C) launch with wavelength 2 (λ 2) with catalysis (C) subunit of the ring AMP-dependant kinase A of FRET acceptor (A) mark with the non-activity tetramer of adjusting (R) subunit of FRET donor (D) mark.In the presence of cAMP, this tetramer dissociates, and single subunit will launch with wavelength 1 (λ 1).D) acceptor (R) and part (L) are used FRET acceptor (A) and donor (D) mark respectively, launch with λ 2 when being bonded to each other.The part of this mark is replaced with unlabelled part will make unconjugated tagged ligand with λ 1 emission.E) acceptor (R) and part (L) are used FRET acceptor (A) and donor (D) mark respectively, launch with λ 2 when being bonded to each other.The part of mark is replaced with competitor destroyed the receptor/ligand combination, will make unconjugated tagged ligand with λ 1 emission.F) with acceptor (A) and the complete substrate particle of quencher (Q) mark, not emission.Cutting this substrate with enzyme should will launch with the fragment of acceptor mark separately.

Figure 23. be used for the diagram of the mark of single-particle detection.A) with at least one particle target-marking particle of single dyestuff.B) with each at least one particle target-marking particle in two kinds of different dyes.

Figure 24. be used to detect and differentiate the diagram of the mark of at least two kinds of particles.A) particle of single dye marker of usefulness varying number.B) with the particle of each mark in two kinds of different dyestuffs or the two kinds of dyestuffs.C) with D) with the particle of two kinds of dye markers, wherein at least a dyestuff exists with multicopy.E) with particle with at least a mark in two kinds of dyestuffs of different fluorescence intensities.F) with a kind of dyestuff or influence the particle of the label mark of electrophoretic velocity.G) and H) particle of each personal a kind of dye marker.

Figure 25. be used for diagram based on the mark of electrophoretic velocity detection and discriminating.A) with the intrinsic detectable intended particle of different label marks that influences electrophoretic velocity.B) use the label label particles that can detect dye marker or influence electrophoretic velocity.

Figure 26. differentiate the diagram of two kinds of particles according to distinctive fluorescent emission intensity.A) with a kind of one or more copy target-marking particles of dyestuff, and according to each particle on the proportional fluorescent emission intensity of dye particles number that combines distinguish.B) distinguish with one or more copy target-marking particles of two kinds of dyestuffs, and according to the total intensity of fluorescent emission or the strength ratio of two kinds of different dyes.

Figure 27. the diagram of fluorescence polarization determination method.Intended particle with dye marker has different fluorescence polarizations, and this fluorescence polarization can be measured according to its rotational speed.Label particles has changed rotational speed with combining of acceptor, has changed the fluorescence polarization of detected particle subsequently.

Figure 28 .A) and B): the label of Shi Yonging, and present detection limit under various conditions.

Figure 29. be presented under every kind of concentration with the alkaline phosphatase of substrate reactions counting and inquire after the figure of the fluorescence-causing substance molecular number of analyzing on the spatial analysis device two.

New feature of the present invention is specifically described in claims.Will be better understood the features and advantages of the present invention with reference to following detailed description and accompanying drawing, following detailed description use the exemplary embodiment of the principle of the invention.

Detailed Description Of The Invention

The invention provides analyzer and analyzer system and use this analyzer and analyzer system carries out overdelicate detection, quantitatively and the method for differentiating to the particle of extremely low concentration.

Fig. 1 shows an embodiment of analyzer system of the present invention.The system that illustrates comprises that can gather a plurality of samples and sampling container and first automatically inquires after the sampling system that the fluid between the space is communicated with; Optional sample preparation system; Can detect the analyzer of single-particle, wherein said analyzer comprises that sample is by first inquiring after space and second and inquire after the space wherein, described space is set to accept electromagnetism (EM) radiation that electromagnetic radiation source sends, and can be operatively connected with dividing other electromagnetic radiation detector; With data analysis and reporting system.

This analyzer is small-sized, durable, accurate, is used to detect the interaction between single-particle, each particle and relates to single-particle or the incident of particle complex.This analyzer, analyzer system and relevant method can be used for producing and measuring the characteristic velocity of single-particle, electrophoretic velocity for example, for example utilize data cross to be correlated with to determine the speed of single-particle, thereby make the analysis noise minimization, and for example electrophoretic velocity and/or Electromagnetic Launching are differentiated particle according to its speed.This analyzer, analyzer system and relevant method have the ability of distinguishing at least a particle in the sample that contains multiple particle.In addition, this analyzer, analyzer system and relevant method can detect the plurality of target particle in the monocyte sample or the multiple appraisable feature of one or more intended particles with improvement.

For example, the embodiment of the following stated adopts electromagnetic radiation as the detection of particles means.In the field that single-particle detects, can adopt the detection system (for example laser induced fluorescence) that well known to a person skilled in the art based on optics usually.Yet, should be appreciated that also to comprise other particle detecting method in the scope of the present invention that for example, use chemiluminescence or radioactive label etc., not needing provides electromagnetic radiation to sample, and only need detect in these methods.

Device

In one aspect, the invention provides can test sample in the analyzer of each particle, wherein utilize motive power that described particle is moved by analyzer.In certain embodiments, analyzer comprises the single-particle detecting instrument, this single-particle detecting instrument uses continuous wavelength (CW) laser instrument as the EM radiation source, and comprises the space of inquiring after of two fluids connections, wherein utilizes pressure to make sample by inquiring after spatial movement.In certain embodiments, this analyzer comprises the single-particle detecting instrument, this single-particle detecting instrument uses continuous wavelength (CW) laser instrument as the EM radiation source, and comprises the space of inquiring after of two fluids connections, wherein utilizes electric power to make sample by inquiring after spatial movement.

On the other hand, the invention provides a kind of analyzer system.In certain embodiments, this system comprises the analyzer that can detect single-particle (for example unimolecule), wherein this detecting instrument comprise one be used for that sample is added the space of inquiring after that sampling system fluid in the analyzer is communicated with, be used to send the electromagnetic radiation source of electromagnetic radiation, wherein inquire after the space and be set to the EM radiation of sending in the received radiation source, with with first inquire after first radiation detector that the space can be operatively connected, be used to measure first electromagnetic signature of particle (for example molecule).In certain embodiments, this system comprises the analyzer that can detect single-particle (for example unimolecule), and wherein this detecting instrument comprises that two fluids are communicated with inquires after the space and be used for sample is added sampling system in the analyzer.In preferred embodiments, this sampling system is an automatic sampling system, does not need operating personnel to intervene promptly and can gather a plurality of samples.In certain embodiments, this system further comprises the sample recovering mechanism, utilize this mechanism can be after analysis recovery section or whole sample basically.In certain embodiments, this system further has specimen preparation mechanism, and it can be analyzed for the single-particle analyzer by partial or complete preparation sample.In certain embodiments, this system further is provided for control analysis and/or analyzes the computing machine of raw data, in other embodiments, is provided for reporting the annunciator of this analysis result.

Sample and particle

The invention provides and be used for high sensitivity, reliable, analyzer and the analyzer system that can repeatedly analyze the particle that a plurality of samples and sample may comprise.The invention provides the existence that detects particle, do not exist and/method of concentration.

Sample

Single-particle analyzer of the present invention or analyzer system can be analyzed any needs or need not handle promptly can inquiring after spatial movement and containing the sample that maybe may contain the particle that detects of detecting device that can enough this systems by this system.Include but not limited to from commercial Application sample, environmental sample, agriculture sample, bio-terrorism sample, be used for the sample of medical screening, diagnosis, prognosis or treatment and from the sample of or other research biomedical such as clinical or preclinical test etc.Sample can be from external or the body, or its combination.This system is particularly useful for the clinical sample that analysis is used for biomedical research, diagnosis or treatment.

For example the described mensuration of following examples can be with method of the present invention to carrying out such as biological samples such as biofluids.These liquid include but not limited to bronchoalveolar lavage fluid (BAL), blood, serum, blood plasma, urine, cerebrospinal fluid, hydrothorax, synovia, ascites, amniotic fluid, gastric juice, lymph liquid, interstitial fluid, tissue homogenate, cell extract, saliva, phlegm, ight soil, physiology secretion, tears, mucus, sweat, milk, seminal fluid, seminal fluid liquid, vaginal fluid, from the liquid of ulcer and other surperficial rash, blister and abscess with comprise normal, pernicious and suspect the extract of the tissue in being organized in, maybe may contain any other composition of the health of intended particle.Other similar sample as cell or tissue culture or nutrient solution, also is interested.

In certain embodiments, sample is a blood sample.In certain embodiments, sample is serum or plasma sample.In certain embodiments, sample is bronchoalveolar lavage fluid (BAL) sample.In certain embodiments, sample, for example blood, serum or plasma sample, need not further processing can use in the method for the invention.In the other embodiment, processing sample as described herein, for example, one or more intended particles of mark.Processing can be carried out before sample being added in the analyzer system of the present invention, perhaps can carry out after sample being added in this system.

For the particle of analyzing

The method of using analyzer of the present invention and analyzer system to detect at least a single-particle also is provided.A special feature of this single-particle analyzer be can sensing range particle widely.The particle that enough this analyzers of energy detect includes but not limited to the combination of molecule, supramolecular complex, organelle, pearl, molecule, the combination and the biosome of supramolecular complex.The example of the molecular particle that enough analyzers of the present invention of energy and correlation technique detect comprises: XC polymer, and as protein, nucleic acid, carbohydrates and organic and inorganic granule chemical individual.The latter's example includes but not limited to anti-autoimmunity deficit syndrome material, antibody, cancer-resisting substance, microbiotic, antiviral substance, enzyme, enzyme inhibitor, neurotoxin, opioids, hypnotic, antihistaminic, tranquillizer, anticonvulsive drug, muscle relaxant and anti-Parkinson medicine, antispasmodic and contraction of muscle agent, myotic and anticholinergic agents, immunodepressant (for example cyclosporin), the anti-glaucoma dissolved matter, anti parasitic and/or antiprotozoan dissolved matter, antihypertensive, antalgesic, alexipyretic and anti-inflammatory agent (as NSAID (non-steroidal anti-inflammatory drug)), local anesthetic, medicament for the eyes, prostanoid, antidepressants, antipsychotics, antiemetic, preparation, the special target agent, neurotransmitter, protein and cell response dressing agent.

Similarly, detectable chemical individual comprises small-particle, as amino acid, nucleotide, lipid, medicine, toxin, venom, substrate, pharmacophore and combination in any thereof.Protein also is significant in multiple therapeutic agent and diagnosticum, for example detects cell colony, blood group, pathogen, the immune response to pathogen, immune complex, carbohydrate, agglutinin, naturally occurring acceptor etc.Other examples of detectable particle comprise nanosphere, microballoon, dendrimer, chromosome, organelle, micelle and carrier particle.The example of organelle comprises the subcellular fraction particle, as nucleus, mitochondria, ribosomes and endosome.The example of biosome comprises virus, bacterium, fungal cell, zooblast, vegetable cell, eukaryotic, prokaryotic, archeobacteria cell and combination in any thereof.

The compound of the intended particle that also comprises the compound of the particle of forming by particle complex, the biosome that is combined with label, two or more nucleic acid and combine with one or more antibody or antibody fragment.The compound that detects the single-particle of two or more types comprises the particle that is selected from protein, acceptor, DNA, RNA, PNA, LNA, carbohydrates, organelle, virus, cell, bacterium, fungi, its fragment and combination thereof.Those skilled in the art should know how analyzer of the present invention and correlation technique are used to detect these and other particle according to a large amount of embodiment provided herein.

In one embodiment, can comprise synthetic or naturally occurring hormone, naturally occurring medicine, synthetic drug, pollutant, allergen, effector molecules (affecter) particle, growth factor, chemotactic factor (CF), cell factor, lymphokine, amino acid, oligopeptides, chemical intermediate, nucleotide and oligonucleotides with the chemical individual that analyzer and correlation technique detect.

Especially meaningfully detect microorganism and cell, comprise virus, protokaryon and eukaryotic, unicellular and multicellular organism cell, for example fungi, animal, mammal etc., and fragment.Method of the present invention also can be used to detect pathogen.Significant pathogen can be (but being not limited to) virus, for example herpesviral, Poxviruses, Togaviruses, arboviruse, picornavirus, orthomyxovirus, paramyxovirus, rhabdovirus, coronavirus, arenavirus and retrovirus.Also can comprise bacterium, include but not limited to Escherichia coli (Escherichia coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa), enterobacter cloacae (Enterobactercloacae), staphylococcus aureus (Staphylococcus aureus), enterococcus faecalis (Enterococcus faecalis), klepsiella pneumoniae (Klebsiella pneumoniae), salmonella typhimurium (Salmonella typhimurium), Staphylococcus epidermidis (Staphylococcus epidermidis), serratia marcesens (Serratia marcescens), Mycobacterium bovis (Mycobacterium bovis), methicillin resistant Staphylococcus aureus and proteus vulgaris (Proteus vulgaris).The example of pathogen is not limited to above-mentioned pathogen, and those skilled in the art should know that the microorganism of which kind of specific kind and parasite are particular importances in specific setting of the present invention or application.This paper provides further example.In addition, the tabulation of the non-limit of these biological and relevant diseases can be found in (for example) authorizes the U.S. Patent No. 5,795,158 of Warinner, and this patent is incorporated herein by reference.Significant clinically other particle can be inflammation (referring to, for example, people such as Lucey (1996) Clin Microbiol.Rev.9:532-62), cancer (referring to, for example, Sidransky (2002) Nat.Rev.Cancer 2:210-219; People such as Etzioni (2003) Nat.Rev.Cancer 3:243-52) or the biomarker of Alzheimer's (referring to, for example, Golde (2003) J.Clin.Invest.111:11-18).

In one embodiment, can detect and differentiate the particle of the several types in the same sample.Significant especially particulate example comprises antibody, infectious agent/nucleic acid/toxin, cancer cell/imbalance protein, mRNA/ corresponding proteins matter transcript, gene (DNA)/courier (RNA), gene (DNA)/protein, virus/toxin, bacterium/toxin, enzyme/substrate and the enzyme/product of infectious agent/anti-this infectious agent in the present invention uses.In certain embodiments, system of the present invention can analyze its existence, not exist and/or concentration and disease or disease system (constellation) relevant particle group.

Method described herein makes it possible to individually distinguish at least a particle in the sample that contains multiple particle.Do not need the particle that increases.Multiple particle comprises small-particle, nucleic acid (for example strand, two strands, DNA, RNA and heterozygote thereof), protein (for example peptide, polypeptide and protein), organic and inorganic molecule (for example metabolin, cell factor, hormone, neurotransmitter etc.) and biosome (for example virus and cell).In this regard, the sample that contains multiple particle can comprise multiple small-particle, multiple nucleic acid particle, multiple proteins particle, multiple organic and/or inorganic molecule and various kinds of cell and/or virus or above-mentioned various combinations.Therefore, can distinguish any particle in the sample, this sample comprises (i) nucleic acid, small-particle, organic/inorganic molecule or protein, (ii) nucleic acid and small-particle, (iii) nucleic acid and protein, (iv) protein and small-particle, (v) protein and organic/inorganic molecule, (vi) nucleic acid and organic/inorganic molecule, or (vi) nucleic acid, small-particle and protein and combination above-mentioned and cell/virus.

Except above-mentioned particle, can use these methods to differentiate to comprise such as with the particle of the compounds such as nucleic acid, antibody-antigenic compound, ligand-receptor compound, enzyme-substrate complex and protein-nucleic acid compound of label hybridization.

The single-particle analyzer

As shown in Figure 3, the analyzer of one embodiment of the invention gives Reference numeral 10 on the whole.Analyzer 10 comprises two continuous wave electromagnetic radiation sources 12, mirror 14, lens 16,20, two apertures 22 of 18, two micro objectives of capillary flow moving cell, two detector lens 24,26, two single photon detectors 28 of two detecting device light filters and the processor 30 that can be operatively connected with this detecting device.

In service, radiation source 12 alignment, so their light beam 32,34 is reflected away by the front 36 of mirror 14.Lens 16 focus on independent the inquiring after in the space (for example shown in Figure 4 inquire after space 38,40) of in the capillary flow moving cell 18 two with light beam 32,34.Micro objective 20 will be pooled on the aperture 22 from sample particle with from the light of the image of light beam 32,34.Aperture 22 covers the scattering from the wall of capillary flow moving cell 18.Detector lens 24 is compiled the light by aperture 22, and light is focused on the useful area of detecting device 28 after by detecting device light filter 26.Detecting device light filter 26 helps making noise signal (for example scattered light, surround lighting) to minimize, and makes the light maximization from particle.Processor 30 is according to the light signal of method processing as herein described from particle.In one embodiment, micro objective 20 is high numerical value aperture micro objectives.

Fig. 4 shows an embodiment of the capillary flow moving cell 18 of analyzer of the present invention.As shown in Figure 4, two light beams 32,34 from continuous wave electromagnetic radiation source 12 (Fig. 3) are optically focused on the target of interval preset distance (for example about 100 μ m). Light beam 32,34 is perpendicular to the length of the capillary flow moving cell 18 that is full of sample.Light beam 32,34 is to select to be used to excite the presetted wavelength operation of particular detection label.A plurality of diameters of inquiring after space 38,40 each free corresponding light bundle 32,34 of analyzer 10 (Fig. 3) determine, and/or are determined by the section of the corresponding light beam of selecting 32,34.Inquiring after space 38,40 is set to separately in each time interval of observing under the suitable sample concentration to inquire after at each and has only a particle in the space.Although light beam 32,34 can have other diameter that does not deviate from the scope of the invention, in one embodiment, the diameter of each light beam is about 5 μ m.

Sample is applied motive power.In one embodiment, motive power is pressure.In certain embodiments, motive power is for electric field improved in electrophoresis and that sample is applied.In certain embodiments, use motive combination, for example pressure and electric field.Under deposition condition, the particle with similar electric charge and quality moves by unit 18 with speed much at one.When particle passed through light beam, each fluorescent particles is excited at 32,34 o'clock through one-photon excitation.In less than 1 second, the detectable burst of light is sent in the particle decay that is stimulated.By in the time span of light beam, each particle repeats repeatedly to excite-launch circulation at it, make analyzer 10 (Fig. 3) at each particle by inquiring after space 38 or can detecting tens of extremely thousands of photons at 40 o'clock.The photon of two detecting devices 26 (Fig. 3) record fluorescent particles emission, time delay indication particle (or molecular complex) is inquired after the time of inquiring after space cost of space to second detecting device from a detecting device.Detecting device 26 record photon intensities.The signal that detecting device 26 is detected be divided into have the time road that can freely select wide evenly, time period arbitrarily.Determine contained number of signals in each time period.The several statistical analysis of one or combination are estimated the existence of particles.Like this, particle and the difference with background noise are at random come.

Fig. 5 shows the confocal arrangement of analyzer 50 of the present invention.Single micro objective 52 will combine from the light beam 32,34 in two continuous wave electromagnetic radiation sources 12, forms two and inquire after space (for example, shown in Figure 4 inquire after space 38,40) in capillary flow moving cell 18.But the dichronic mirror 54 that utilizes reflector laser can see through fluorescence separates fluorescence and laser.Any non-fluorescence that another one light filter 56 before detecting device 26 is eliminated in the detecting device.

Motive power

Utilize motive power to make particle by inquiring after spatial movement.In certain embodiments, being used for improved motive power is pressure.In certain embodiments, this pressure is provided by pump, pneumatic supply, vacuum source, hydro-extractor or their combination.In certain embodiments, this pressure is provided by pump.In certain embodiments, motive power is electric power.Also can use magnetic force (for example, being used to control moving of magnetic particle) or optical force.Also can make combination firmly.

Pressure

When utilizing pressure, for example pumping comes when improved, two inquire after space observation to particle between time delay be consistent with predictable, that is, can pre-determine time migration, this helps to distinguish particle and noise.Utilize other motive power, for example electrophoresis more is difficult to prediction drift, because the multiple material in the sample may move with multiple speed, this depends on their electrophoretic mobility.

In certain embodiments, utilize pump to provide pressure to come mobile example.Suitable pump is well known in the art, and for example, by such as Scivex, manufacturers such as Inc. make is used for pump such as purposes such as HPLC.For the less volume of pumping (for example, when sample concentration without limits the time), can use as United States Patent(USP) Nos. 5,094,594,5,730,187; 6,033,628; With 6,533,553 described miniflow pumps, these patent disclosures can pumping receive and rise or skin rises the device of the fluid volume of scope.Preferably with pump that sample contacts in all material all by making such as polyetheretherketone (PEEK), the material that melts quartz with fixed attention or sapphire equal altitudes inertia.

Standard pump has multiple size, can select suitable size to adapt to the sample size of expection and the needs that flow.In certain embodiments, use other pump of branch to carry out sample analysis and system's flushing.For example, analyze pump and can have about 0.000001mL, or about 0.001mL about 1mL extremely, or about 0.01mL about 0.2mL extremely to about 10mL, or about 0.005,0.01,0.05,0.1 or the capacity of 0.5mL.The capacity of flushing pump can be greater than analyzing pump, and for example about 0.01mL is to about 20mL, or about 0.1mL about 10mL extremely, or about 0.1mL about 2mL extremely, or about 0.05,0.1,0.5,1,5 or 10mL.The size of these pumps is just illustrative, those skilled in the art should know can according to application, sample volume, will pumping fluid viscosity, line size, flow velocity, temperature and other factors well known in the art select the size of pump.In certain embodiments, the pump of system is by the step motor drive that is easy to accurately control with the microprocessor utmost point.

In preferred embodiments, flushing pump and analysis series connection of pumps are used, and utilize specific non-return valve to control flow direction.Pipe design is for when analyzing pump absorption maximum sample, and this sample can not arrive this pump itself.Implementation method is to select to analyze pump and analyze intercapillary pipeline ID and length, makes piping volume greater than analyzing the throw of pump volume.

In certain embodiments, utilize the improved and sample of air pressure.Pneumatic supply and be controlled in the art field known.

Electric power and other

Be used for the improved electric field of electric power in order to produce, by electrode for example platinum electrode the high-voltage power supply (not shown) is connected with sample, for example, sample each end capillaceous can be placed an electrode.About 10 to about 1, and the voltage of 000V/cm may be suitable.

Electric power also can make up with other motive power.In some cases, can utilize other power that all particle's velocity in the sample are changed with identical degree.In certain embodiments, other power provides the difference label that different particle types or difference combine with particle and the method for unconjugated label.An example that can apply other power to sample is the pressure (and vacuum) that uses pump as mentioned above.In one embodiment, use syringe pump; But, also can use any controllable fluid delivery system to exert pressure to sample, as gravity sample introduction, positive displacement pump (positive displacement pump) or roller-type pump, and do not deviate from scope of the present invention.Be used for centrifugal force that fluid flows and relate to and inquire after the parts (not shown) that space 38,40 can be operatively connected, as the rotating disk (not shown).These parts can with the rotating disk unitary construction or as on being connected, placing with rotating disk, the module structure among contacting or being embedded in it.

In one embodiment, use magnetic resolution by in magnetic field, optionally keeping magnetic material.This technology also can be used for the non magnetic target of magnetic particle mark.In a kind of application of this technology, by target material is connected to come label particles with magnetic particle.This connection normally combines with specific binding partner by particle, and the coating coupling on this specific binding partner and the particle is provided for the functional group of coupling.Those skilled in the art will be appreciated that, this magnetic particle coupling is well known in the art, as following embodiment and can be from New EnglandBiolabs of Beverly, Massachusetts and Qiagen of Valencia, the kit that California obtains is described.The target material or the target that are connected with the magnetic mark thing are suspended in the fluid, then it are added in the chamber (not shown), are used for importing inquiring after in the space 38,40.When the magnetic gradient that chamber applies was passed in existence, the target of magnetic mark was retained in the chamber, if chamber contains matrix, it will combine with matrix.There is not the material of magnetic mark thing to pass through chamber.Then can be by changing magnetic field intensity or eliminating the material that magnetic field comes wash-out to keep.Can provide magnetic field with permanent magnet or electromagnet.To the selectivity of desired destination material by the specificity with the magnetic particle coupling combine-gametophyte provides.The chamber that applies magnetic field is provided with the matrix of materials with suitable magnetic susceptibility usually, with in chamber near the volume of stromal surface the part bring out strong magnetic field gradient.

In another embodiment, for example, sample is carried out electrophoresis by sample being placed the electrophoresis sample channel.The mobility of particle changes along with the character of particle in the sample fluid.The translational speed that electric power produces depends on the relative electric charge and the quality of single-particle.The type that is connected to the label (as electric charge/mass labels) on the particle can change moving of particle.In another embodiment, when having two or more particles, at least a particle can be inquired after space 38,40 by at least two and move with the direction opposite with other particle.Therefore, measured the electrophoretic mobility of the particle of every kind of detectable label.Based on the mensuration of the electrophoretic velocity of the particle of every kind of detectable label, can distinguish the single-particle in the sample that contains multiparticle.May be used among the present invention with almost any electrophoretic separation technique of immunoassays or the combination of nucleic acid hybridization labelling technique.

Electric power can with such as the combination of other motive power such as pressure, vacuum, surface tension, gravity and centrifugal force, differentiate particle.In one embodiment, when having two or more particles, can be used at difference selecting these power, cause at least a particle to inquire after space 38,40 by at least two and move to be different from other particle's velocity to different particles in the sample.Particle's velocity can be capable with the fluid levelling, and perhaps at least a particle can move with fluid with flowing antiparallel.In another embodiment, at least a particle has the antiparallel speed above rate of flow of fluid.In another embodiment, at least a particle and fluid stream vertically moves.In another embodiment, at least a particle moves with the array mode that is antiparallel to and flow perpendicular to fluid.

The EM radiation source

In the extrinsic label of particle or inherent feature is to interact such as light such as fluorescent marker or light scattering labels in embodiment of the present invention of (light-interacting) label or feature, utilizes EM radiation source irradiates label and/or particle.In for example using other embodiments of chemiluminescent labels, may needn't use the EM source to detect particle.Preferably use EM radiation source fluorescence excitation label.

Although only show two sources 12 among Fig. 3 and Fig. 5, should be appreciated that and only to use a continuous wave electromagnetic radiation source, and do not deviate from scope of the present invention.In addition, if only use a continuous wave electromagnetic radiation source 12, then this radiation source can be divided into the bundle of arbitrary number, and with the lead different spaces of inquiring after of arbitrary number of electromagnetic radiation.

In the embodiment depicted in fig. 3, each is inquired after space 38,40 and has independent continuous wave electromagnetic radiation source 12.Although only show two sources 12 among Fig. 3 and Fig. 5, can use the source of any number, and not deviate from scope of the present invention.In some cases, all continuous wave electromagnet sources electromagnetic radiation of launching identical wavelength.In the other embodiment, the electromagnetic radiation of different source emission different wave lengths.Can design heteroid source and inquire after the space.For example, in one embodiment, can utilize two or more same spaces of inquiring after of irradiation, continuous wave electromagnetic radiation sources with different emission, and this structure can extend to and inquires after the space more.In another embodiment, each inquires after the space with the electromagnetic radiation irradiation of different wave length.It will be appreciated by those skilled in the art that analyzer of the present invention can use illumination wavelength and the many different combination of inquiring after the space.

Do not deviate from scope of the present invention although can use other source, in one embodiment, source 12 is to produce about 200 to about 1, the continuous wave laser of 000nm wavelength.Such source 12 has small-sized, durable, relatively cheap advantage.In addition, they have the ability that produces bigger fluorescence signal than other light source usually.The object lesson in suitable continuous wave electromagnetic radiation source includes but not limited to: argon, krypton, helium-neon, helium-cadmium type laser instrument, and tunable diode lasers (red in infrared region), wherein each all has the possibility that makes doubling frequency.Laser instrument provides Continuous irradiation, and does not use auxiliary electron or mechanical hook-up, as shutter, interrupts its irradiation.LED is the irradiation source of another low cost, high reliability.Progress in superbright LED with high absorption cross section and quantum yield and dyestuff supports that LED is applied to single-particle to be detected recently.These laser instruments can use separately or with use such as other combination of light sources such as mercury-arc lamp, element arc lamp, Halogen lamp LED, arc discharge, plasma discharge, light emitting diode or its combinations.

The suitableeest laser intensity depends on the photobleaching feature of single dyestuff and crosses and inquire after the required time span in space (comprise particle rapidity, inquire after the distance between the space and inquire after the size in space).In order to obtain peak signal, need be with the maximum intensity irradiation sample of the dyestuff that can not cause the high number percent of photobleaching.When finally inquiring after the space for crossing to particle, preferred intensity bleached less than 5% dyestuff.

In one embodiment, inquire after space 38,40 by the cross-sectional area of corresponding light beam 32,34 and one section light beam decision in the detecting device visual field.In one embodiment of the invention, inquiring after space 38,40 is 0.02pL to 300pL.In one embodiment of the invention, inquiring after space 38,40 is 0.02pL to 50pL.In another embodiment, inquiring after space 38,40 is about 0.1 to about 25pL.Preferably inquire after the space and be about 1pL.It will be appreciated by those skilled in the art that and to select to inquire after space 38,40 at the peak performance of analyzer.Although show the minimum space of inquiring after background noise is minimized, inquire after the space greatly and have and to analyze the advantage of low concentration sample within reasonable time.In one embodiment of the invention, it is enough big to inquire after the space, can be the particle of about 1000fM to about 1 narrow mole (zM) by detectable concentration.In one embodiment of the invention, it is enough big to inquire after the space, can be the particle of about 1000fM to about 1 vast mole (aM) by detectable concentration.In one embodiment of the invention, it is enough big to inquire after the space, can be the particle of about 10fM to about 1 vast mole (aM) by detectable concentration.In many cases, inquire after the space greatly and allow the particle of detectable concentration, and need not other pre-concentration device or technology less than about 1fM.It should be recognized by those skilled in the art that and onlyly inquire after that the space size depends on brightness, the level of background signal of the particle that will detect and the concentration of the sample that will analyze.

Can limit the size of inquiring after space 38,40 by the optical system of regulating analyzer.In one embodiment, can regulate the diameter of light beam 32,34, change the volume of inquiring after space 38,40.In another embodiment, can change the visual field of detecting device 26.Therefore, can regulate source 12 and detecting device 26, to inquire after space 38,40 internal radiations and to detect single-particle.In another embodiment, the width in the crack 22 (Fig. 3) in the visual field of decision detecting device 26 is variable.This structure allows to inquire after the space near changing in real time, with the sample that compensation more or less concentrates, guarantees to exist simultaneously the low possibility of two or more particles in inquiring after the space.

Also can provide the physical constraint of inquiring after the space by solid walls.In one embodiment, when sample fluid was included in the kapillary, wall was one or more unit 18 walls.In one embodiment, unit 18 is made by glass, but under the situation that does not deviate from scope of the present invention, also can use and see through about 200 to about 1, other materials of the light of 000nm or higher scope, as quartzy, melt and coagulate quartz and organic material, as Teflon, nylon, plastics, for example Polyvinylchloride, polystyrene and tygon, or its combination in any.Although can use other cross sectional shape (for example triangle, cylindrical) under the situation that does not deviate from scope of the present invention, in one embodiment, capillary flow moving cell 18 has square cross section.In another embodiment, inquiring after the space can be limited by etched passage (not shown) in the chip (not shown) at least in part.

Inquiring after space 38,40 connects by fluid.In one embodiment, this fluid is a water-based.In other embodiments, this fluid is the combination of non-aqueous fluid or water and non-aqueous fluid.In addition, this fluid also can contain reagent, ionic composition or the screening agent (sieving agents) of regulating pH, as solubility macroparticle or polymkeric substance or gel.Be expected to inquire after and valve or other device can be set between the space temporarily interrupt the fluid connection.The space is considered to be communicated with by fluid temporary transient inquiring after of interrupting.

In another embodiment of the present invention, inquire after laminar flow (be also referred to as sheath stream) big or small retrained of space 38,40 by specimen material in the dilution volume.Inquiring after space 38,40 can also can be limited by the combination of sheath stream with the irradiation source size or the detecting device visual field by independent sheath current limit.Sheath stream can be set to multiple mode, comprises following listed:

1. specimen material is the internal material of concentric laminar flow, and the dilution volume externally.

2. the dilution volume is in a side of sample volume.

3. the dilution volume is in the both sides of specimen material.

4. the dilution volume does not still surround specimen material fully on a plurality of sides of specimen material.

5. the dilution volume is fully around specimen material.

6. the dilution volume is with one heart fully around specimen material.

7. specimen material is a discontinuous internal material in the series, and the dilution volume drips specimen material around each fully.

Those skilled in the art will be appreciated that analyzer in some cases comprises 3,4,5,6 or the how different space of inquiring after.

Detecting device

In one embodiment, light (for example ultraviolet light, visible or infrared light) is detected electromagnetic radiation.Detecting device 26 can be caught amplitude and the duration that photon that fluorescent particles for example sends is burst, and is translated into electric signal.Can utilize such as pick-up units such as CCD camera, video input module camera, streak cameras and produce image with continuous signal.In another embodiment, can use the device that produces continuous signal such as bolograph, photodiode, photodiode array, avalanche photodide detecting device and photomultiplier detector etc.Also can use the combination in any of above-mentioned detecting device.In one embodiment, use avalanche photodide to detect photon.

The specificity optical system between space 38,40 and the respective detection device 26 thereof is inquired after in utilization, can detect several different characteristics of the electromagnetic radiation of emission, comprising: emission wavelength, emissive porwer, the size of bursting, burst duration and fluorescence polarization.

It will be appreciated by those skilled in the art that one or more detecting devices 26 can be arranged on each and inquire after 38,40 places, space, and single detecting device 26 can be set to detect any feature of electromagnetic radiation of sending listed above.

In case particle is labeled and makes it can detect (if perhaps particle has the internal characteristics that it can be detected), then can under the situation that does not deviate from scope of the present invention, use any suitable testing agency well known in the art, for example the photomultiplier and the combination thereof of CCD camera, video input module camera, streak camera, bolograph, photodiode, photodiode array, avalanche photodide detecting device and generation continuous signal.In one embodiment, use avalanche photodide to detect photon.Can detect the different characteristic of electromagnetic radiation, comprise: emission wavelength, emissive porwer, the size of bursting, the duration of bursting, fluorescence polarization and combination in any thereof.

The counting and distinguish

Method as herein described allows to illustrate particle, because they once have one by inquiring after the space.Can be according in the number of particles of the time span inside counting of setting with determine the concentration of sample by the sample volume of inquiring after the space.Inquiring after the space when comprising the entire cross section of intact sample stream, calculation sample concentration only need be at the population of time span inside counting and the volume by the sample flow xsect.When inquiring after the space, can calculate the concentration of particle by the typical curve that the control sample that utilizes normal concentration produces less than sample flow.In another embodiment, the concentration that can determine particle by the particle relatively measured and internal particle standard.If the known sample dilutability can calculate the concentration of particle in the initial sample.

The data analysis of detected particle comprises crosscorrelation.In one embodiment, directly with the photon signal crosscorrelation.In this case, utilize at least two detecting devices to detect the fluorescence signal (photon) that sends from least two samples of inquiring after the space.The signal that detecting device is detected respectively is divided into random time section (bin), and each time period has the time span of selecting in advance (bin width).Although can use other bin width under the situation that does not deviate from scope of the present invention, in one embodiment, selecting the bin width is that about 10 μ s are to about 5ms.Preferred bin width is 1ms.Determine the number of signals that each time period comprises.For each time period, carry out cross correlation analysis in the time period scope of second detecting unit of selecting from first detecting unit.The cross correlation analysis result is carried out at least a statistical analysis, and/or the result is carried out analysis of threshold.Several statistical analysis of described statistical analysis or at least a combination are used for estimating the existence of particle.Like this, based on the existence of coherent signal at least two detector channels with particle with at random with background noise difference come.

In one embodiment, before the cross correlation analysis data, at first the analyzing and testing signal is measured noise level, and selects the above signal of threshold value.In one embodiment, determine noise level by average signal on a large amount of bin.In other embodiments, determine background level according to average noise level or root mean square noise.In other cases, select typical level of noise or statistical value.In most of the cases, the expectation noise is followed the Poisson distribution.

Determine to differentiate the threshold value of true signal (peak, flex point, point) and noise.Must be noted that when selecting threshold value the false positive number of signals that random noise is produced is minimum, and the true signal number of getting rid of is minimum.Select the method for threshold value to comprise the fixed value of determining that noise level is above, and based on the Distribution calculation threshold value of noise signal.In one embodiment, threshold setting is the standard deviation that is higher than the fixed qty of background level.Suppose that noise is that Poisson distributes, use the false positive number of signals of this method in can the estimating experiment time-histories.The signal that two detecting devices are identified carries out cross correlation analysis then.

The time migration of cross-correlated signal provides the switching time between the respective detection device, therefore determines particle's velocity based on the spacing of detecting device, for example electrophoretic velocity.In some cases, according to the fact detection particle of time migration corresponding to the known time skew.In the other situation, by the unknown offset detection particle of measuring according to population distribution.

In another embodiment, can be to carrying out cross correlation analysis from the data of two above detecting devices (for example 3,4,5,6 or 6 above detecting devices), these detecting devices are being inquired after on the relative position in space or are being detected on the wavelength different.In this case, can carry out cross correlation analysis to data from the different detecting devices of combination in any.For example, each of inquiring after the space at two is provided with three detecting devices, and each detecting device detects emitting at different wavelengths (R respectively, G, in situation B), R1 is relevant with R2, G1 is relevant with G2, and B1 is relevant with B2, causes having the time migration of the particle of the wavelength emission that single detecting device detects.Also can carry out other cross correlation analysis combination, for example R1 is relevant with G1, R1 and B1 is correlated with, G1 is correlated with B1 overlapping group.The result of cross correlation analysis will indicate the frequency of the particle of double-tagging.The various combination of cross correlation analysis can be used for mutually according to speed and mark (color) difference particle.In addition, use the many character that will measure single-particle in the potpourri to cross correlation analysis more accurately.

In another embodiment, use a kind of analytical approach, this method is carried out cross correlation analysis to the data of coming self-detector with the combination in any of different positions and/or wavelength.Therefore, it should be recognized by those skilled in the art that by the label combination of using the Electromagnetic Launching (as dye marker) to change particle or particle mobility (as electric charge/quality or magnetic mark thing) and can distinguish multiple particle in the potpourri.

Method as herein described can single difference contains at least a particle in the sample of multiple particle.Multiple particle comprises small-particle, nucleic acid (for example strand, two strands, DNA, RNA and heterozygote thereof), protein (for example peptide, polypeptide and protein), organic and inorganic molecule (for example metabolin, cell factor, hormone, neurotransmitter, chemistry or biological reaction product etc.) and biosome (for example virus and cell).In this regard, the sample that contains multiple particle can comprise multiple small-particle, multiple nucleic acid particle, multiple proteins particle, multiple organic and/or inorganic molecule and various kinds of cell and/or virus or above-mentioned various combinations.Therefore, can distinguish any particle in the sample, this sample comprises (i) nucleic acid, small-particle, organic/inorganic molecule or protein, (ii) nucleic acid and small-particle, (iii) nucleic acid and protein, (iv) protein and small-particle, (v) protein and organic/inorganic molecule, (vi) nucleic acid and organic/inorganic molecule, or (vii) nucleic acid, small-particle and protein and combination above-mentioned and cell/virus.This method intended particle in the sample that do not need to increase.

Except above-mentioned particle, use these methods to differentiate to contain such as with the particle of the compounds such as nucleic acid, antibody-antigenic compound, ligand-receptor compound, enzyme-substrate complex and protein-nucleic acid compound of label hybridization.

In certain embodiments, analyzer of the present invention or analyzer system can be lower than 1 nanomole by detectable concentration, or 1 picomole, or 1 fly mole, or the analyte of 1 vast mole or 1 narrow mole, the biological example label.In certain embodiments, the concentration of biomarker is lower than 1 nanomole in sample, or 1 picomole, or 1 fly mole, or 1 vast mole or 1 narrow mole, and the volume of each sample is during less than about 100,50,40,30,20,10,5,2,1,0.1,0.01,0.001 or 0.0001 μ l, this analyzer or analyzer system can detect a sample with respect to the analyte of another sample or multiple analytes (for example one or more biomarkers) concentration less than about change of 0.1,1,2,5,10,20,30,40,50,60 or 80%.In certain embodiments, when the concentration of analyte is lower than about 1 picomole, and the volume of each sample is when being lower than about 50 μ l, and this analyzer or analyzer system can detect analyte concentration between first sample and second sample less than about 20% change.In certain embodiments, fly mole when the concentration of analyte is lower than about 100, and the volume of each sample is when being lower than about 50 μ l, this analyzer or analyzer system can detect analyte concentration between first sample and second sample less than about 20% change.In certain embodiments, fly mole when the concentration of analyte is lower than about 50, and the volume of each sample is when being lower than about 50 μ l, this analyzer or analyzer system can detect analyte concentration between first sample and second sample less than about 20% change.In certain embodiments, fly mole when the concentration of analyte is lower than about 5, and the volume of each sample is when being lower than about 50 μ l, this analyzer or analyzer system can detect analyte concentration between first sample and second sample less than about 20% change.In certain embodiments, fly mole when the concentration of analyte is lower than about 5, and the volume of each sample is when being lower than about 5 μ l, this analyzer or analyzer system can detect analyte concentration between first sample and second sample less than about 20% change.In certain embodiments, fly mole when the concentration of analyte is lower than about 1, and the volume of each sample is when being lower than about 5 μ l, this analyzer or analyzer system can detect analyte concentration between first sample and second sample less than about 20% change.

Analyzer system

Except single-particle analyzer described herein, the present invention also provides analyzer system, this analyzer system also can comprise sampling system, sample retrieval system, sample preparation system except that the single-particle analyzer, be used to control such as analysis CALCULATION OF PARAMETERS machine such as flow velocity and/or comprise computing machine and/or analyze the data analysis and the reporting system of raw data, and the annunciator that is used to report this analysis result.

In certain embodiments, this analyzer system comprises that can gather a plurality of samples and sampling container and first automatically inquires after the sampling system that the fluid between the space is communicated with; Can detect monomolecular analyzer, this analyzer comprises that (i) is used to send the electromagnetic radiation source of electromagnetic radiation; (ii) be set to receive first of electromagnetic radiation that described electromagnetic radiation source sends and inquire after the space; (iv) inquire after first electromagnetic radiation detector that the space can be operatively connected, be used to measure first electromagnetic signature of particle with first.In certain embodiments, as mentioned above, this analyzer comprises that further can detect second of single-particle inquires after window.

Sampling system

In certain embodiments, analyzer system of the present invention comprises and is used for the sample equal portions are added in the single-particle analyzer for the sampling system of analyzing.Can use any mechanism that can add sample.Can utilize the vacuum that pump produces or be applied to the pressure of liquid is pushed on the sample in managing, perhaps, attract sample by being used for that sample is added any other mechanism in the sampling pipe.Usually, but optional, sampling system is introduced the sample of known sample volume in the single-particle analyzer; In the existence or non-existent some embodiment that detect one or more particles, the size of accurately knowing sample is not critical.In preferred embodiments, sampling system is gathered monocyte sample or a plurality of sample automatically.Join in this intrasystem embodiment at the sample with known volume, sampling system provides sample more than about 0.0001,0.001,0.01,0.1,1,2,5,10,20,30,40,50,60,70,80,90,100,150,200,500,1000,1500 or 2000 μ l for analysis.In certain embodiments, sampling system provides the sample that is less than about 2000,1000,500,200,100,90,80,70,60,50,40,30,20,10,5,2,1,0.1,0.01 or 0.001 μ l for analysis.In certain embodiments, sampling system provides about 0.01 to 1500 μ l for analysis, or about 0.1 to 1000 μ l, or about 1 to 500 μ l, or about 1 to 100 μ l, or about 1 to 50 μ l, or the sample of about 1 to 20 μ l.In certain embodiments, sampling system provides about 5 μ l to 200 μ l for analysis, or about 5 μ l are to about 100 μ l, or the sample of about 5 μ l to 50 μ l.In certain embodiments, sampling system provides the sample of about 10 μ l to 200 μ l or about 10 μ l to 100 μ l or about 10 μ l to 50 μ l for analysis.In certain embodiments, sampling system provides about 0.5 μ l sample to about 50 μ l for analysis.In certain embodiments, sampling system provides the sample of about 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150,200,500,1000 or 2000 μ l for analysis.In certain embodiments, sampling system provides the sample of about 50 μ l for analysis.In certain embodiments, sampling system provides the sample of about 25 μ l for analysis.In certain embodiments, sampling system provides the sample of about 10 μ l for analysis.Sampling system can provide the sample volume greater than actual analysis.For example, sampling system can provide about 25 μ l, or about 20 μ l, or about 15 μ l, or about 10 μ l samples, wherein only analyzes about l to about 5 μ l.

In certain embodiments, sampling system be provided at may be different between sample and the sample sample volume.In these embodiments, sample volume can be arbitrary sample volume as herein described, and as expected, may be along with every kind of sample or sample sets and change.

Accuracy between the accuracy of the sample volume of sampling system and sample and the sample volume is required as analyzing at hand.In certain embodiments, the accuracy of sampling volume depends on employed pump, typically uses less than about 50,40,30,20,10,5,4,3,2,1,0.5,0.1,0.05 or 0.01% the CV of sample volume and represents.In certain embodiments, the sample of sampling system and the degree of accuracy between the sample are used less than about CV of 50,40,30,20,10,5,4,3,2,1,0.5,0.1,0.05 or 0.01% and are represented.In certain embodiments, the interior accuracy of the mensuration of sampling system is by representing less than about CV of 10,5,1,0.5 or 0.1%.In certain embodiments, the interior accuracy of the mensuration of sampling system shows the CV less than about 5%.In certain embodiments, between the mensuration of sampling system accuracy by representing less than about CV of 10,5 or 1%.In certain embodiments, accuracy shows CV less than about 5% between the mensuration of sampling system.

In certain embodiments, sampling system provides low carry-over rate, and its advantage do not need to be extra washing step between sample.Therefore, in certain embodiments, the carry-over rate is less than about 1,0.5,0.1,0.05,0.04,0.03,0.02,0.01,0.005 or 0.001%.In certain embodiments, the carry-over rate is less than about 0.02%.In certain embodiments, the carry-over rate is less than about 0.01%.

In certain embodiments, sampling system sampling loop.In these embodiments, a plurality of samples are attracted in the pipeline continuously, and each all passes through damping fluid " plug " and other sample separation.Generally read sample one by one, between do not need the flushing.Flushing once when finishing in the loop.For example, can in the independent hole of microtiter plate, reclaim every kind of plug.

Sampling system can be fit to the application standard determinator, for example 96 hole microtiter plates, or preferred 384-orifice plate.In certain embodiments, this system comprise 96 orifice plate steady arms and be used for sample hose is dipped in the hole and the hole outside mechanism, for example, provide the mechanism that moves along X, Y, Z axle.In certain embodiments, sampling system provides a plurality of sampling pipes, for example, and a plurality of pipes in 8 holes of row " suction " from the microtiter plate.In certain embodiments, with all samples of a detecting device analysis from a plurality of pipes; In other embodiments, a plurality of Single Molecule Detection device can be connected with sample hose.Before the sampling system sampling, can prepare sample by being included in the step of in the plate hole sample being operated, perhaps can in analyzer system, prepare sample, perhaps both some combinations.

Sample reclaims

A very useful feature of the embodiment of analyzer of the present invention and analyzer system is that not needing to consume sample can analyze.When specimen material has this particular importance in limited time.Recovery sample also makes people carry out other analysis or analysis again to it.Limited and/or the expectation application of analytic sample again for sample size, for example legal medical expert, drug screening and clinical diagnostic applications, the advantage of this feature it will be apparent to those skilled in the art that.

Therefore, in certain embodiments, analyzer system of the present invention further is provided for the sample retrieval system of recovery sample after analysis.In these embodiments, this system comprises mechanism and the method with sample sucks in the analyzer, (for example by same path) sampling receptacle (for example sample hose) is returned in analysis then.Because there is not sample destroyed, and sample do not enter any valve or other pipeline, and its keeps not contaminated.In addition because all material in the sample path all is the height inertia, for example PEEK, melt and coagulate quartz or sapphire, little pollution from the sample approach.Use can accurately be controlled the volume of attraction by the pump (particularly analyzing pump) of step motor control and it is pushed back.This allows with dcq buffer liquid recovery sample fully or almost entirely, even if having, also is extremely low dilution.Therefore, in certain embodiments, after analysis, reclaim above about sample of 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%.In certain embodiments, the sample of recovery is not diluted.In certain embodiments, the diluted sample of recovery is lower than about 1.5 times, 1.4 times, 1.3 times, 1.2 times, 1.1 times, 1.05 times, 1.01 times, 1.005 times or 1.001 times.

Reclaim for sampling and/or sample, can use and any fluid sample be transferred to mechanism the analyzer from sampling receptacle.In certain embodiments, analyze entrance point capillaceous and be connected with the short pipe of a length, for example PEEK manages, and this pipe can be immersed in such as in the sampling receptacles such as test tube or sample well, perhaps can remain on the waste canister.When coming in the cleaning device original sample by flushing, this pipe is positioned on the waste canister, draws flushing waste.When drawing sample, this pipe is placed into sample well or in vitro.In general, draw sample fast, release lentamente then, simultaneously the particle in the observation sample.Perhaps, in certain embodiments, in drawing a round-robin part, sample is drawn lentamente at least; Can be when slowly drawing analytic sample.Afterwards can the fast return sample and get express developed.In certain embodiments, can be in inwardly (absorptions) and extroversion (extraction) circulate analytic sample, this has improved the counting statistics of for example little dilute sample, and has confirmed result etc.Preserve sample if desired, then it can be returned in the original same sample well, perhaps in another hole.If do not need to preserve sample, this pipeline is positioned on the waste canister.

Sample preparation system

Specimen preparation comprises the required step of primary sample that preparation is used to analyze.These steps can comprise, for example, below one or more: separating step, as centrifugal, filtration, distillation, chromatography, concentrate, lysis, change pH, add damping fluid, add thinning agent, add reagent, heating or cooling, interpolation label, label in conjunction with, crosslinked with irradiation, separate unconjugated label, deactivation and/or remove interfering compound and for preparing required any other step of sample with the analysis of single-particle analyzer.In certain embodiments, processing blood is with separated plasma or serum.Also can carry out other mark, remove unconjugated label serum or plasma sample, and/or dilution step.

As known in the art, can carry out the preparation of sample, wherein, for example, in one or more particles, add label with homogeneous or heterogeneous form.In homogeneous system, from sample, do not remove unconjugated label.In certain embodiments, come one or more intended particles of mark by adding one or more labelled antibodies that can combine with one or more intended particles.In Heterogeneous systems, increase one or more steps of removing unconjugated label.In certain embodiments, also adopt the separating step that for example uses capture antibody fixed target particle.Therefore, in certain embodiments, the homogeneous preparation may further comprise the steps: 1) add and suspect the sample that contains intended particle; 2) add to detect (for example mark) antibody.In certain embodiments, heterogeneous preparation may further comprise the steps: 1) add capture antibody; 2) washing; 3) sealing; 4) add the sample that suspection contains intended particle; 5) washing; 6) add to detect (for example mark) antibody; 7) washing; 8) discharge the molecule (may the needs neutralization) of combination according to the method difference.

In certain embodiments, analytic system comprises sample preparation system, and this sample preparation system provides some or all required process of single-particle analyzer sample for analysis.This system can carry out the above listed any or all of step that is used for specimen preparation.In certain embodiments, use the sample preparation system section processes sample of analyzer system; Therefore, in certain embodiments, sample can be outside analyzer system section processes, for example,, and within analyzer, use the sample preparation system section processes by centrifugal, for example the mark sample, mix etc. with damping fluid.In certain embodiments, blood sample is handled beyond analyzer system, so that serum or plasma sample to be provided, will further be handled by sample preparation system in this sample adding analyzer system, with one or more intended particles of mark, and randomly remove unconjugated label.

In certain embodiments, analytic system sampling preparation system, this sample preparation system provides and will for example prepare blood sample, saliva sample, urine samples, cerebrospinal fluid sample, lymph sample, BAL sample, biopsy samples, forensic samples, bio-terrorism sample etc. fully by the preparation fully of the sample of this systematic analysis.In certain embodiments, this analyzer system sampling preparation system, this sample preparation system provides some or all specimen preparations.In certain embodiments, initial sample is a blood sample, further handles with analyzer system.In certain embodiments, sample is serum or plasma sample, further handles with analyzer system.Serum or plasma sample can further be handled, for example the label that can combine with one or more intended particles by contact; Can remove or not remove unconjugated label then, and use sample.

In certain embodiments, outside the analytic system or within the specimen preparation assembly in analytic system, on one or more microtiter plates,, carry out specimen preparation as on 96 orifice plates.The reservoir of reagent, damping fluid etc. can be by pipeline or other suitable structure well known in the art and flat board the hole intermittently fluid be communicated with.Can in 96 orifice plates or pipe, prepare sample respectively.Sample separation, label combination and (in case of necessity) label separating step can carry out on a plate.In certain embodiments, discharge the particle of preparation then on the slave plate, with sample transfer in pipe, in order to collecting in the sample analysis system.In certain embodiments, the institute of preparation sample all carries out on a plate in steps, and analytic system directly obtains sample from this plate.Although utilize 96 orifice plates to describe this embodiment, should be appreciated that and to use any container that holds one or more samples and be suitable for preparing sample.For example, can use the standard microtiter plate in 384 holes or 1536 holes.More generally, in certain embodiments, sample preparation system can hold and prepare and surpasses about 5,10,20,30,40,50,60,70,80,90,100,200,300,500,1000,5000 or 10,000 kind of sample.In certain embodiments, can gather a plurality of samples for the multi-parser systematic analysis.Therefore, in certain embodiments, from sample preparation system, gather 2 kinds of samples, or surpass about 2,3,4,5,7,10,15,20,50 or 100 kind of sample, and on various product analyzer system parallel analysis.

Also can use micro-fluidic system to carry out specimen preparation, and as the part of sample preparation system component analysis device system, therefore the particle that contains enough high concentrations especially for suspection detects the sample of the less sample of needs.The principle and the technology of miniflow operation are known in the art.Referring to, for example U.S. Patent No. 4,979, and 824; 5,770,029; 5,755,942; 5,746,901; 5,681,751; 5,658,413; 5,653,939; 5,653,859; 5,645,702; 5,605,662; 5,571,410; 5,543,838; 5,480,614; 5,716,825; 5,603,351; 5,858,195; 5,863,801; 5,955,028; 5,989,402; 6,041,515; 6,071,478; 6355,420; 6,495,104; 6,386,219; 6,606,609; 6,802,342; 6,749,734; 6,623,613; 6,554,744; 6,361,671; 6,143,152; 6,132,580; 5,274,240; 6,689,323; 6,783,992; 6,537,437; 6,599,436; 6,811,668 and disclosed PCT patented claim WO9955461 (A1).Sample can continuous or parallel preparation, uses for list or multi-parser system.

Preferably, sample comprises damping fluid.Damping fluid can be outside analyzer system and sample mix, perhaps can be provided by specimen preparation mechanism.Although can use any suitable damping fluid, but preferred damping fluid has low fluorescence background, is inertia to the particle of detectable label, can keep working pH, and in motive power is in the electrodynamic embodiment, has the ionic strength that is suitable for electrophoresis.Buffer concentration can be any suitable concentration, and for example about 1 to about 200mM.Can use any Laemmli buffer system Laemmli, as long as it provides dissolubility, function and the detectability of target molecule.Preferably, for the application of using pump, damping fluid is selected from phosphate, glycinate, acetate, citrate, acidify salt (acidulate), carbonate, imidazoles, triethanolamine, glycine amide, borate, MES, Bis-Tris, ADA, aces, PIPES, MOPSO, Bis-Tris propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, tromethamine, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS and CABS.A kind of particularly preferred damping fluid is pH 7.4 phosphate buffered saline (PBS)s that contain 0.1%Tween 20, but imidazole buffer salt solution, BBS and tris buffer saline also can be accepted.Preferably, damping fluid is selected from Gly-Gly, bicine, tricine, 2-morpholino ethyl sulfonic acid (MES), 4-morpholino propane sulfonic acid (MOPS) and 2-amino-2-methyl-1-propanol hydrochloric acid (AMP).A kind of particularly preferred damping fluid is a 2mM Tris/ borate, and pH 8.1, but Tris/ glycocoll and Tris/HCl also can accept.

For the application of using electrophoresis, preferred buffer is selected from Gly-Gly, bicine, tricine, 2-morpholino ethyl sulfonic acid (MES), 4-morpholino propane sulfonic acid (MOPS) and 2-amino-2-methyl-1-propanol hydrochloric acid (AMP).A kind of particularly preferred damping fluid is 2 mM Tris/ borates, and pH 8.1, but Tris/ glycocoll and Tris/HCl also can accept.Can contain the zwitter-ion that concentration is up to 2M in the electrophoresis sample.This does not improve the electric current of electrophoresis system, and makes the interaction with capillary surface reduce to minimum.

Use for some, damping fluid desirably further is included in the sieving matrix that uses in this method embodiment.Although can use any suitable sieving matrix, the sieving matrix of expectation has low fluorescence background, and can specificity provide the particle of detectable label to rely on the delay of size.Can there be (for example about 0.1% to about 10%) with the concentration of any appropriate in sieving matrix.Can use any suitable molecular weight (for example about 100,000 to about 10,000,000).The example of sieving matrix comprises poly-(oxygen ethene) (PEO), poly-(vinylpyrrolidone) (PVP), straight chain polyacrylamide and derivant (LPA), hydroxymethyl-propyl cellulose (HPMC) and hydroxyethyl cellulose (HEC), they all are soluble in water, and have unusual low viscosity under dilute concentration (0.3%wt/vol).In addition, these polymer solutions are higher than their entanglement (entanglement) threshold value, and are easy to preparation, filtration and filled capillary pipe.In the Tris/ borate buffer solution, add 0.2%LPA (5,000,000-6,000,000mw) can in one minute electrophoretic separation, differentiate IgG and 1.1kb nucleic acid fragment (referring to, for example, the following examples 7a).

In some cases, the inherent characteristic of intended particle has produced the electromagnetic signature that can measure.In other situation, before detecting with analyzer, can be with detectable label target-marking particle.Detectable label can be, for example, and luminous marker, or light scattering label.In one embodiment, detectable label is a luminous marker.Although can use other luminous marker in the case without departing from the scope of the present invention, useful luminous marker comprises fluorescent marker, chemiluminescent labels and bioluminescence marker thing.In addition, also can monitor fluorescent quenching.In addition, can use other light scattering label in the case without departing from the scope of the present invention.Useful light scattering label comprises metal, as gold, silver, platinum, selenium and titanyl compound.

In order to be detected, particle must produce, and perhaps can produce electromagnetic radiation.Electromagnetic radiation is the inherent characteristic of particle or the extrinsic characteristic of particle.The example of inherent characteristic comprises fluorescence and light scattering, but particle may have more than one the inherent characteristic that it can be detected that makes.The extrinsic characteristic is provided by the label that is connected on the particle.With particle localization before inquiring after in the space 38,40, use label afterwards or simultaneously.

Preferred testing tool is a fluorescent marker.The example of fluorescent marker is found in HANDBOOK OF FLUORESCENT PROBES AND RESEARCH PRODUCTS (R.Haugland, 9th Ed., Molecular Probes Pub. (2004)).Detectable also can be produced by the combination in any of the intrinsic of particle and extrinsic characteristic.

The method of label particles is well known to a person skilled in the art.Label is connected to adopts any known method on the particle, comprise direct connection or use binding partners to connect.In some cases, labeling method is nonspecific.For example, known all nucleic acid of certain methods mark and regardless of its specific nucleotide sequence.In other situation, mark is specific, and wherein the oligonucleotides of mark combines with the target nucleic acid sequence-specific.

Label of the present invention comprises dye marker, electric charge label, mass labels, quantum dot (Quantum Dots) or pearl, magnetic mark thing, light scattering label, polymeric dye and is connected to dyestuff on the polymkeric substance.Dyestuff comprises to material increases the big compounds that color maybe can produce luminous or fluorescence.Dyestuff can absorb or send the light of specific wavelength.Dyestuff can embed or be non-covalent or be covalently bound on the particle.Dyestuff itself can constitute probe, as the probe of the little groove structure, cruciform, annular or other conformation assembly that detect particle.Dyestuff can comprise BODIPY and ALEXA dyestuff, Cy[n] dyestuff, the SYBR dyestuff, ethidium bromide and relevant dyestuff, acridine orange, dimerization cyanine dye such as TOTO, YOYO, BOBO, TOPRO, POPRO and POPO and derivant thereof, Bb, OliGreen, PicoGreen and relevant dyestuff, cyanine dye, fluorescein, LDS 751, DAPI, AMCA, Cascade Blue, CL-NERF, dansyl, the dialkyl amido cumarin, 4 ', 5 '-two chloro-2 ', 7 '-the dimethoxy fluorescein, 2 ', 7 '-dichlorofluorescein, DM-NERF, Yihong, erythrosine, fluorescein, Hydroxycoumarin, isosulfan blue, lissamine rhodamine B, peacock green, methoxy coumarin, the naphthyl fluorescein, NBD, the Oregon is green, PyMPO, Pyrene, rhodamine, Rhodol is green, 2 ', 4 ', 5 ', 7 '-tetrabromo sulphonyl fluorescein, tetramethylrhodamin, texas Red, the X-rhodamine, the Dyomic series dye, the Atto-tec series dye, Coumarins, phycobniliprotein (phycoerythrin, phycocyanin, allophycocyanin), green, yellow, red and other fluorescin and quantum dot (Quantum Dots).Those skilled in the art should know other operable within the scope of the present invention dyestuff.This is not the tabulation of limit, and acceptable dyestuff comprises all dyestuffs that can be used for detecting label particles of the present invention known now or that know in the future.By containing fluorescent marker, as fluorescent particles, fluorescence coupling antibody etc., the rayed sample that can absorb with fluorescent particles, and detect the light that sends with the light determinator.

Operable in the present invention light scattering label comprises metal, as gold, silver, platinum, selenium and titanyl compound.Those skilled in the art will be appreciated that other microballoon or pearl also can be used as the light scattering label.In another embodiment of the present invention, label influences the electrophoretic velocity and/or the separation of the intended particle of the identical or different size that can not pass through electrophoretic separation.These labels are called as electric charge/mass labels.The connection of electric charge/mass labels has changed the electric charge of intended particle and the ratio of translation frictional resistance to be enough to influencing its electrophoretic mobility with mode of separating and degree.

In another embodiment, label has changed electric charge or quality, or the combination of electric charge and quality.According to the spatial diversity of their behaviors in electric field or according to the speed difference of their behaviors in electric field, the electric charge/mass labels that combines with particle can with unconjugated particle or the difference of unconjugated label.

Also can utilize the paramagnetic microballon or the nanosphere label particles of glycocalyx.Authorize the U.S. Patent No. 4,452,773 (being hereby incorporated by) of Molday and put down in writing the preparation of Armco magnetic iron-glucose pearl, and the general introduction of the whole bag of tricks of the suitable particle that is connected with biomaterial of preparation is provided.Polymer coated being described in that is used for the magnetic particle that uses in the high gradient magnetic partition method authorized in the Deutsche Bundespatent No.3720844 of Miltenyi and the U.S. Patent No. 5,385,707 can find these two pieces of complete being incorporated herein by reference of patent.U.S. Patent No. 4,770,183 have put down in writing the method for preparing paramagnetic beads.

Being connected pearl really with particle, blanking method is not to implement key of the present invention, a large amount of replacement schemes known in the art.Connect common interaction by particle and specific binding partner, the coating coupling on this binding partners and the pearl, and be provided for interactional functional group.Antibody is the example of binding partners.Antibody can with member's biological example of high-affinity coupling system plain coupling, particle and for example avidin coupling of another member.Also can use the speciesspecific epitope's of identification first antibody second antibody in the present invention, for example anti-mouse Ig and the mouse Ig of the Chinese People's Anti-Japanese Military and Political College.Coupling method allows to use the single magnetic coupling individuality with multiple particle, for example antibody, avidin etc. indirectly.

In an application of this technology that Cohen people (1988) PNAS 85:9660-3 such as () Cohen describes, intended particle can with the coupling of magnetic mark thing, and be suspended in the fluid of (not shown) in the chamber.When the magnetic field that chamber provides was crossed in existence, the target of magnetic mark was stayed in the chamber.There is not the material of magnetic mark thing to pass through chamber.Then can be by changing magnetic field intensity or eliminating the material that magnetic field comes wash-out to keep.Its chamber that applies magnetic field is provided with the matrix of materials with suitable magnetic susceptibility usually, in chamber, to bring out strong magnetic field gradient near part in the volume of stromal surface.This allows to keep the particle of suitable weakly magnetization, and this method is called as high gradient magnetic and separates.

In another embodiment of the present invention, the extrinsic characteristic that particle can be detected is provided by at least two kinds of labels.For example, with two or more label target-marking particles, every kind of label is because the emission that detects under one or more wavelength is different from the emission of other label and difference.In this example, distinguish particle and free label according to the ratio of the emission that detects at two or more wavelength place.In another example, with two or more labels and at least two kinds of label label particles in the emission of same wave strong point.In this example, the fluorescence intensity difference particle that produces according to detected two kinds of being connected, three kinds or the emission of multiple label with each particle.

In another embodiment, dyestuff has identical or overlapping excitation spectrum, but has diacritic emission spectrum.The preferred dyestuff of selecting makes them have the emission spectrum that is different in essence, and preferably has the emission maximum value of being separated by greater than about 10nm, preferably has the emission maximum value of being separated by greater than about 25nm, more preferably is separated by greater than the emission maximum value of about 50nm.When needs utilized instrumental method to distinguish two kinds of dyestuffs, a plurality of light filters and diffraction grating can detect emission spectrum separately independently.Also can improve the instrument discriminating by the dyestuff of selecting to have narrow bandwidth rather than wide bandwidth, still, these dyestuffs must have the emission of high-amplitude or exist with enough concentration, so that the loss of integrated signal intensity can not damage input.

In an example, the fluorescence that second label can quencher first label causes the fluorescence signal of the particle of double-tagging to disappear.The right example of suitable fluorescence/quencher comprises 5 ' 6-FAMTM/3 ' Dabcyl, 5 ' Oregon green  488-X NHS ester/3 ' Dabcyl, 5 ' texas Red -X NHS ester/3 ' BlackHole QuencherTM-1 (Integrated DNA Technologies, Coralville, IA).

In the another one example, FRET (fluorescence resonance energy transfer) (" FRET ") can be used two kinds of labels, and FRET is the interaction that depends on distance between the excited state of two kinds of dye particles.In this case, excite from donor and transfer to the acceptor particle, and donor ballistic phonon not.Donor and acceptor particle must be closely near (for example in about 100 ).Suitable donor/acceptor is to comprising fluorescein/tetramethylrhodamin, IAEDANS/ fluorescein, EDANS/dabcyl, fluorescein/QSY7 (R.Haugland, " Molecular Probes; " Ninth edition, 2004) and well known to a person skilled in the art that other is many right.

In order to help detecting and/or differentiate, can as dye marker and mass labels, come label particles with more than one label.For example, can be with two kinds of antibody labeling protein, a kind of is unlabelled, as quality or mass label, another kind has dye marker.Can with dye marker only distinguishing with the protein of antibodies of protein and another kind of similar size be come according to (for example) slower speed (because the quality of the antibody of combination or mass improve cause) in addition when using electrophoresis then as motive power.

For the particle of accurate certification mark, the particle of mark must be distinguished mutually with unconjugated label.Those skilled in the art are familiar with the method for many realization this point.For example, in heterogeneous mensuration, before analysis with the separate particles of unconjugated label and mark.In one embodiment, this mensuration is that homogeneous is measured, and the combinatory analysis sample by electrophoresis and single-particle fluoroscopic examination comprises unconjugated label.In this case, be chosen as the particle of mark and the deposition condition that unconjugated label provides friction speed.

All nucleic acid of the common mark of non-specific mark of nucleic acid, and regardless of specific nucleotide sequence.Those skilled in the art are familiar with various nucleic acid marking technology commonly used.These methods comprise: intercalative dye, as TOTO, ethidium bromide and iodate third ingot, be used to form co-ordination complex the ULYSIS kit, be used to mix the ARES kit of the chemical reactivity nucleotide analog that can easily be connected and mix the nucleotide analog that contains biotin for the label with the streptavidin combination is connected with label.The nucleotide analog that enzyme process mixes mark is to well known to a person skilled in the art another kind of method.

The technology of non-specific labelled protein also well known to a person skilled in the art.Can use several chemical reaction acidic amino acids on protein surface, primary amine for example is as lysine.In addition, also can partly go up the interpolation label to the carbohydrates of protein.Also developed the isotype specific reagent that is used for labelled antibody, as Zenon mark (Haugland, 2004).

In one embodiment, the specified particle in the mark potpourri.Specific marker can be by realizing that with intended particle and the binding partners combination that is labeled wherein binding partners and intended particle interact by complementary mating surface specificity.Adhesion between the gametophyte can be covalent interaction or noncovalent interaction, as hydrophobic, hydrophilic, ion with hydrogen bonding, Van der Waals force attract or co-ordination complex forms.The example of binding partners comprises that the activator of cell-membrane receptor and antagonist, toxin and venom, antibody and virus epitopes, hormone (for example opioid peptides, steroids etc.) and hormone receptor, enzyme and zymolyte, co-factor and target sequence, medicine and drug targets, oligonucleotides and nucleic acid, protein and monoclonal antibody, antigen and specific antibody, polynucleotide and complementary polynucleotide, polynucleotide and polynucleotide are in conjunction with albumen; Biotin and avidin or streptavidin, enzyme and enzyme cofactor; With agglutinin and specificity carbohydrates.Can be used as the illustrative acceptor that binding partners works and comprise naturally occurring acceptor, for example thyroid binding globulin, agglutinin, the range protein (cluster of differentiation or CD particle) found at cell surface etc.CD divides known and protein the unknown on the subrepresentation eukaryotic surface, and for example, CD4 is the molecule that mainly defines helper T lymphocyte.

In one embodiment, sample be coated with can with the pearl or the microballoon reaction of the binding partners of intended particle reaction.Any unconjugated component separating of pearl and sample detects the pearl of the particle that contains combination with analyzer of the present invention.The pearl of fluorescent dye is particularly suitable for these methods.For example, use the fluorescent bead of oligomerization sequence bag quilt to combine, and behind suitable separating step, allow to detect target sequence with target complementary series specificity.

In one embodiment, a kind of method that detects particle adopts sandwich assay, uses monoclonal antibody as binding partners.First antibody is connected with the surface, as capture antibody.Add sample then, have can by the particle of the epi-position of antibody recognition will with lip-deep antibodies.The unconjugated particle of flush away only stays the particle of specificity combination basically.In conjunction with particle/antibody then can with the detection antibody response that contains detectable.After incubation makes detection antibody and particle reaction, the detection antibody of flush away non-specific binding.Particle and detection antibody can discharge from the surface, detect with analyzer of the present invention.Perhaps, have only the antibody capable of detection to discharge, and detect, thus the indirect detection particle.Perhaps, have only with the label that detects antibodies and can discharge, and detect, thus the indirect detection particle.

A kind of variation is to use the part that can be discerned by cell receptor.In this embodiment, part and surface combination, thus catch the cell of expression specificity acceptor, and utilize the ligand-labeled cell of mark.Acceptor can be a surface immumoglobulin.Can determine the existence of the cell of specificity combination by this way.Therefore, have the target ligand with the acceptor complementation of surface combination, can detect the cell that has these ligand specificities' immunoglobulin (Ig).In another embodiment, can use antibody with the part of surface combination with part and surperficial non-covalent the connection.

Data aggregation, analysis and report

For example, use the personal computer (not shown in Fig. 1) that standard or customized software are housed, produce the velocity histogram at the peak of each fluorescence types that exists in the show sample, will be relevant by the data cross that the detection of particles signal is formed.Applying to sample in the embodiment of electric field, each particle depends on the distinctive electric charge of particle, size and shape the switching time between detecting device.Also the computer operation analyzer be can utilize, flow velocity, operation sampling, sample recovery, specimen preparation etc. for example controlled.

This system also can comprise the data report device that is used for report data and/or analysis result.Can use any means well known by persons skilled in the art for this purpose.Before report; can further analyze raw data (for example population, cross-correlation data, wavelength of fluorescence, fluorescence intensity etc.) with suitable software, with the combination of possible identity, the concentration of particle in the indication sample, detected particle, relevant with particular disorder normal, unusually or the specificity level compare level, the existing, not existing and/or the possible particle source of the possible diagnosis of concentration, detection and any other analysis that before report, can carry out data of detected particle based on one or more particles.Can use any mechanism that suitable report is provided as the data report device.The example of the non-exclusionism of data report device is included in demonstration on the TV monitor, printout, is used for the data transmission of long-range demonstration or printout, for example by internet, acoustic notifications etc.

Method

On the other hand, the invention provides analytical approach, comprise and use a kind of detection system that the sample that obtains from individuality is analyzed, this detection system has at least two can detect the monomolecular space of inquiring after, and wherein analysis comprises the existence of particle in the working sample or do not exist.In certain embodiments, described individuality is plant or animal; In certain embodiments, described individuality is a mammal; In certain embodiments, described individuality is the people.Detection system can be as herein described any.In certain embodiments, detection system can use the CW laser instrument as electromagnetic radiation source.In certain embodiments, detection system has two and inquires after the space, and for example the volume of each is about 0.02pL about 300pL extremely, or about 0.02pL to 50pL, or about 0.1 to about 25pL.In certain embodiments, use the plural space of inquiring after.Use the space of inquiring after more than 3,4,5,6 or 6 in certain embodiments.Can use two other embodiments of inquiring after the detecting device in space that have as described herein in embodiments of the present invention.

In certain embodiments, the multiple different particle (multichannel) of analytic sample.In certain embodiments, analysis is from a plurality of samples of a plurality of individualities.In the other embodiment, analyze sample from a plurality of individualities.

The present invention also provides a kind of analytical approach, comprise based on the existence of picking up from particle in the individual sample, do not exist and/or concentration is determined diagnosis, prognosis, therapeutic state (for example progress of monitor therapy and/or effect) and/or methods of treatment, wherein use to have two single-particle detecting devices of inquiring after the space and determine the existence of particle, do not exist and/or concentration." diagnosis " used herein comprise and use testing result examination individuality, determining the neurological susceptibility to disease or pathological condition, or the existence of disease or pathological condition and/or the order of severity, and comprises the shortage or the existence of the neurological susceptibility of determining disease or pathological condition.In certain embodiments, analyze and to comprise the existence of determining polytype particle in the sample, do not exist and/or concentration.These methods may further include to obtaining determining of the individual of sample and/or its representative (for example health care supplier) report diagnosis, prognosis, therapeutic state, monitoring and/or treatment.The single-particle detecting device can be arbitrary embodiment as herein described, comprises analyzer and analyzer system.This detection system can use the CW laser instrument as electromagnetic radiation source.In certain embodiments, inquire after the space for two and have about 0.02pL separately to about 300pL, or about 0.02pL to 50pL or about volume of 0.1 to about 25pL.In certain embodiments, use the plural space of inquiring after.In certain embodiments, use the space of inquiring after more than 3,4,5,6 or 6.

Fig. 2 provides the explanation of an embodiment of method of the present invention.Use has two can detect the monomolecular sample (in certain embodiments, using the CW laser instrument as the EM radiation source) of inquiring after the detection system analysis in space from individual (for example people), obtains analysis result.In certain embodiments, the result can be the existence of one or more intended particles, not exist and/or concentration; In certain embodiments, further analysis result is to provide determining etc. of the determining of diagnosis, prognosis, curative effect, treatment type.In certain embodiments, report is informed this individuality or its representative.

The present invention also provides and utilizes Computer Analysis data of database analytical approach.This database comprises the analysis result of one or more samples, and this analyzer has at least two single-particle detecting devices of inquiring after the space to carry out, and it comprises the existence of determining particle in the sample, does not exist and/or concentration.In certain embodiments, analyze and to comprise the existence of determining polytype particle in the sample, do not exist and/or concentration.Sample can obtain from any source described herein.In certain embodiments, in such as biological studies such as clinical testing or preclinical test research or fundamental researchs, obtain sample.The single-particle detecting device can be any embodiment described herein.Detection system can utilize the CW laser instrument as electromagnetic radiation source.In certain embodiments, inquire after the space for two and have about 0.02pL separately to about 300pL, or about 0.02pL to 50pL, or about volume of 0.1 to about 25pL.In certain embodiments, use the plural space of inquiring after.Use the space of inquiring after more than 3,4,5,6 or 6 in certain embodiments.

In some aspects, the invention provides a kind of computer-readable recording medium, as CD, it comprises that one group is used to have user interface, comprise the instruction such as the multi-purpose computer of display units such as television indicator or print unit, and the instruction of this group comprises the logic that is used to import with having the value that two single-particle detecting device analytic samples of inquiring after the space obtain; The optional comparison program that is used for comparison input value and database; With the display routine that shows input value and/or comparison program result with described display unit.In another embodiment aspect this, the invention provides electric signal or the carrier wave between computing machine, propagated by the internet, comprise that one group is used to have user interface, comprise the instruction such as the multi-purpose computer of display units such as television indicator or print unit, wherein the instruction of this group comprises and is used to import with detecting unimolecule and having the logic of the value that two detection system analytic samples of inquiring after the space obtain; The comparison program that is used for comparison input value and database; With the display routine that shows the comparison program result with described display unit.

Because the sensitivity and the reliability of detection system, at short notice high accuracy and accuracy ground analyze one or more intended particles in a large amount of samples existence, do not exist and/or concentration.For example, method of the present invention can be used for fast, determine result of study reliably, delicately, for example include but not limited to clinical before and the biomedical research result of clinical testing.Method of the present invention is also useful in (for example) clinical diagnosis, prognosis, monitoring and methods of treatment are determined.In these embodiments, this method can further comprise the step of diagnosis, prognosis, monitoring or the treatment determined to individual or its representative's report analysis result of collected specimens or by this analysis result.

" individuality " can be any source of sample (normally biological sample).In embodiments, individuality is a biosome, preferred animal, and more preferably mammal, optimum is chosen.Animal comprises that domestic animal, match animal, pet, research uses the animal and human.In certain embodiments, individuality is the people, and in certain embodiments, the people suspects the patient suffer from such as pathology diseases such as infectious disease or non-infective diseases, or at the experimenter of one or more illness examinations.In certain embodiments, to the heredity tendentiousness of disease and/or at the expression examination individuality of hereditary variation or unusual proteins associated matter or other label.In certain embodiments, individuality is to suspect the people with virus or infected by microbes.In certain embodiments, individuality is plant or other biosome.In certain embodiments, individuality is abiotic individuality.

In certain embodiments, method of the present invention comprises analyzing and picks up from individual sample.In certain embodiments, this method comprises the step from individual collected specimens.In some cases, in research is used, whole individuality, for example whole biosome or one group of biosome (for example bacterial clump) can comprise sample.In the other situation, more typically, sample is a part of picking up from individual individuality.Sample can be this paper previously described any.Therefore, for example, sample can be a biofluid, for example blood, serum, blood plasma, bronchoalveolar lavage fluid, urine, cerebrospinal fluid, hydrothorax, synovia, ascites, amniotic fluid, gastric juice, lymph liquid, interstitial fluid, tissue homogenate, cell extract, saliva, phlegm, ight soil, physiology secretion, tears, mucus, sweat, milk, seminal fluid, seminal fluid liquid, vaginal fluid, from the liquid of ulcer and other surperficial rash, blister and abscess with comprise normal, pernicious and suspect the extract of the tissue in being organized in, maybe may contain any other composition of the health of particle.In certain embodiments, sample is a blood sample.In certain embodiments, sample is a plasma sample.In certain embodiments, sample is a blood serum sample.

Detect its exist, do not exist and/or the sample of concentration in particle also as described herein.The particle of any kind described herein can detect with method of the present invention, and can be used for purpose described so far, and following purpose in greater detail.In certain embodiments, particle is molecule, supramolecular complex, organelle, biosome, cell and combination in any thereof.In certain embodiments, one or more particles are biosomes, for example virus, bacterium, fungal cell, zooblast, vegetable cell, eukaryotic, prokaryotic, archeobacteria cell and combination in any thereof.In certain embodiments, biosome is virus, for example herpesviral, Poxviruses, Togaviruses, arboviruse, picornavirus, orthomyxovirus, paramyxovirus, rhabdovirus, coronavirus, arenavirus and retrovirus.In certain embodiments, this particle is a bacterium, for example, Escherichia coli, Pseudomonas aeruginosa, enterobacter cloacae, staphylococcus aureus, enterococcus faecalis, klepsiella pneumoniae, salmonella typhimurium, Staphylococcus epidermidis, serratia marcesens, Mycobacterium bovis, methicillin resistant Staphylococcus aureus and proteus vulgaris.In certain embodiments, one or more particles are the molecules that are selected from down group: amino acid, peptide, protein, nucleotide, oligonucleotides, nucleic acid, DNA, RNA, monose, disaccharides, oligosaccharides, carbohydrates, lipid, hormone, cell factor, chemotactic factor (CF), lymphokine, venom, toxin, naturally occurring medicine, synthetic drug, pollutant, allergen, effector molecules particle, growth factor, metabolic intermediate, substrate, pharmacophore, inorganic molecule, organic molecule and combination in any thereof.

In certain embodiments, sample is being added this sample of detection system pre-treatment.In certain embodiments, sample does not add processing and promptly adds in the detection system; In these embodiments, sample can not need further to handle promptly to detect, and perhaps can handle sample in detection system before with systematic analysis.Can as described in this paper other parts, before or after adding, handle.

In certain embodiments, sample preparation comprises with fluorescently-labeled particle specific antibody label particles.In certain embodiments, with the multiple particle in the multiple fluorescently-labeled antibody labeling monocyte sample, every kind of antibody is specific for a kind of particular type of intended particle.In certain embodiments, the particle of mark is a biomarker.Biomarker include but not limited to inflammation, infected by microbes, pathological state label, presentation markup thing, grow label etc.In certain embodiments, detecting it exists, does not exist and/or the particle of concentration is the label of infected by microbes.An example of the label of infected by microbes is the triggering acceptor (TREM-1) of expressing on the myeloid cell cell, this is a kind of label of finding in body fluid, bacterial indicator or fungal infection, sample resist-the TREM antibody treatment with fluorescently-labeled before adding or after adding.

Analyzer of the present invention and analyzer system are particularly suitable for the multichannel analysis, that is, and and the particle in the test sample more than a type.Can make the sample demultiplexing in order to following method, this method comprises: 1) sample is divided into multiple sample, analyzes the particle of one or more types in every duplicate samples; 2) different particles are used different labels, for example, different particles is used different label colors, number, intensity etc.; Or 3) different particles use different mobilities, for example in electrophoresis.In certain embodiments, analyze polytype particle in the simple sample.The particle types number can surpass 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,40,50,75 or 100 kind in the simple sample.The particle kind number can be less than 200,100,50,40,30,20,19,18,17,16,15,14,13,12,11,10,9,8,7,6,5,4 or 3 kind.In certain embodiments, the particle types number is about 2 to about 20, or about 2 to about 5, or about 2 to about 10, or about 10 to about 20 kinds.

The method of difference particle types is as described herein.Particularly, these methods can use composite marking signal intensity (for example different particles being used the label of varying number), label identity (for example different particles being used different labels) and the label mobility (for example, when motive power is electric power, different particles are used different mobilities), or its combination.

Method of the present invention comprise have in the test sample common relevant or provide the polytype particle (i.e. " group ") of information needed existence, do not exist and/or concentration." group " comprises one group of particle that can detect its existence by determination method of the present invention as used herein.Particle can have makes them perhaps may need mark just can detect with the inherent feature of system of the present invention detection.Therefore, method of the present invention comprises makes sample contact suitable multiple label, be used to detect one or more members of one group of particle existence, do not exist and/or concentration.This particle group can be used for, and for example, bio-terrorism sample analysis, medical inspection, diagnosis, prognosis, monitoring and/or treatment are selected; Biomedical research, legal medical expert, agricultural analysis and commercial Application.For example, described group may be relevant with the diagnosis of particular type, for example, the biological group of infectiousness, be used for mark group, or be used to estimate the function of various systems, for example endocrine group such as diseases such as angiocardiopathy, cancer or specific types of cancer, diabetes, arthritis, Alzheimer's; Described group can be relevant with bio-terrorism, for example may be the group of bio-terrorism biology or toxin; Described group can be used for medical screening, for example, and specific genetic polymorphism relevant with specified disease or pathological state or that be correlated with normal or extraordinary situation or the relevant histone matter of sudden change; Described group can be relevant with prognosis, and for example relevant with specific types of cancer label group can be used for the recurrence and/or the progress of cancer after the treatment of determining section or complete resection cancer.Described group also can be used for the examination blood sample, can comprise a large amount of infectious agents and/or will carry out the antibody of examination to blood.Similarly, method of the present invention can the analysis list sample, detects a large amount of abuse materials, surrounding material or any in veterinarily important material.An advantage of the invention is that it allows layout to suffer from the battery of tests that syndromic individuality carries out to suspection, thereby detects this syndromic paathogenic factor simultaneously.Other field that can the use group comprises research.

Figure 28 shows can be at the example that is used for the label group that group that all kinds use uses, and is described below.

The group that is used for the bio-terrorism sample analysis can comprise each mechanism as known to the skilled person threaten list in the tabulation surpass one or more of 30 kinds of pathogen and toxin.The public health personnel seldom see wherein most of pathogen in suspicious specimen, thus they be difficult to Rapid identification they.In addition, many pathogenic infection symptom can not occur immediately in infected individuality, and symptom occurs postponing to grow to several days, have limited control disease and the individual selection of treatment.Shortage can fast detecting and the actual monitoring network of identifying multiple pathogen in the current threat tabulation or toxin mean the significant deficiency of the terrified ability of antibiont.

With the biological threat agent sensor of " Detect to Protect/Warn " program run preferably 1) can in 1-2 hour time window, detection of biological threaten agent, thereby provide time enough that incident is responded, 2) extremely low maintenance cost, thereby continuous monitoring when needed, with 3) have enough selectivity, in fact eliminated false positive.U.S. Bio-Watch plan relates to the disease prevention and control center of Ministry of Energy, Environmental Protection Agency (EPA) (EPA) and U.S. sanitary and Department of Welfare.Finally, this plan can detect biological attack and the report attack in 24 hours that surpasses 120 Garies.

The autonomous pathogen detection of Bio-Watch plan utilization system (Autonomous PathogenDetection System (" APDS ")), this system is to air sampling, detects, and the machine of the file cabinet size of reporting the result.APDS is integrated flow cytometer and PCR in real time augmentation detection instrument has sample collection, specimen preparation and fluidics, and the compact that can detect multiple pathogen and/or the toxin simultaneously instrument of operation automatically is provided.This system designs for the fixed position, in this position continuous monitoring air sample and the existence of report particular organisms agent automatically.APDS has the ability that can reach 100 kinds of different reagent and contrast in the monocyte sample of measuring at subway system, transportation base, the big group of office and conference centre.The latest developments of biosensors, APDS-II uses pearl to catch immunoassay and the compact flow cytometer is identified multiple biostimulation thing simultaneously.The present invention is not subjected to the restriction of the requirement identical with the APDS system, and identical detection can quicker, cheaply, accurately be provided.

In addition, the present invention medical science, medical inspection, diagnosis, prognosis, monitoring and/or treatment is selected and biomedical research in have many others application.In certain embodiments, the present invention can be used to detect controlled drug and material, therapeutic dose monitoring, health status, be used to transplant donor coupling, gestation (for example by detecting human chorionic gonadotrophin or alpha-fetoprotein) and disease detection, for example endotoxin, cancer antigen, the pathogen etc. of purpose.

In certain embodiments, those skilled in the art can use the present invention and detect chemistry and biologic artifact and medicine, include but not limited to anti-autoimmunity deficit syndrome material, cancer-resisting substance, microbiotic, antiviral substance, enzyme, enzyme inhibitor, neurotoxin, opioids, hypnotic, antihistaminic, tranquillizer, anticonvulsive drug, muscle relaxant and anti-Parkinson medicine, antispasmodic and contraction of muscle agent, myotic and anticholinergic agents, immunodepressant (for example cyclosporin), the anti-glaucoma dissolved matter, anti parasitic and/or antiprotozoan dissolved matter, antihypertensive, antalgesic, alexipyretic and anti-inflammatory agent (as NSAID (non-steroidal anti-inflammatory drug)), local anesthetic, medicament for the eyes, prostanoid, antidepressants, antipsychotics, antiemetic, preparation, the special target agent, neurotransmitter, protein and cell response dressing agent.Protein also is significant in multiple therapeutic agent and diagnosticum, for example detects cell colony, blood group, pathogen, the immune response to pathogen, immune complex, carbohydrate, agglutinin, naturally occurring acceptor etc.

In certain embodiments, usage flag thing group is carried out clinical diagnosis, for example diagnoses infectious disease or inflammation.For example, can the mark sample, to detect antigen or the antibody relevant in the monocyte sample with arbitrary infectious agent, these infectious agents include but not limited to bacterium, virus, fungi, mycoplasma, rickettsia, Chlamydia, prion and protozoan, measure the autoantibody relevant with autoimmunity disease, measure the pathogen of sexually transmitted disease, perhaps mensuration and relevant analytes such as pulmonary disease, disorder of gastrointestinal tract, cardiovascular disorder, neurological disorder, muscle skeleton illness, skin disease.The group that is used for clinical diagnosis can comprise that other is used for the label of the existence of the illness relevant with specified disease or pathological state, for example markers of inflammation thing.

For example, diagnosis of sepsis disease can be used the various combinations of following diagnostic marker: inflammation biomarker TREM-1; Inflammation biomarker IL-6 and IL-8; Inflammation biomarker IL-10 and IL-12, optional IL-18; The fungal infection biomarker; For Escherichia coli, for example for one or more pathogen labels of multiple specific bacterial strain; For staphylococcus aureus, for example for one or more pathogen labels of multiple specific bacterial strain; For Candida albicans, for example for one or more pathogen labels of multiple specific bacterial strain; For enteric bacteria, for example for one or more pathogen labels of multiple specific bacterial strain; And other clinical marker thing and optional negative control.

In certain embodiments, clinical diagnosis can be only based on a kind of label, and for example TREM-1 is used for determining pyemic existence or not existing, and perhaps for the lung sample, is used for determining the existence of pneumonia or not having (for example lung ventilator patient).Also can diagnose with blood plasma, serum or BAL sample.Diagnosis can be based on the comparison of the value that obtains from analytic sample with the value of normal or unusual (for example ill) colony.

In certain embodiments, can use a group echo thing that is used to diagnose CAP, it is following arbitrary or whole combination: inflammation biomarker TREM-1; Inflammation biomarker IL-6 and IL-8; Inflammation biomarker IL-10 and IL-12 and optional IL-18; Virus infections biomarker SAA; For streptococcus pneumonia (Streptococcus pneumoniae), for example for one or more pathogen labels of multiple specific bacterial strain; For Respiratory Syncytial Virus(RSV), for example for one or more pathogen labels of multiple specific bacterial strain; For haemophilus (Haemophilus), for example for one or more pathogen labels of multiple specific bacterial strain; For mycoplasma (Mycoplasma), for example for one or more pathogen labels of multiple specific bacterial strain; And other clinical marker thing and optional negative control.For example, the group that is used for bacterial pathogens is conspicuous for those skilled in the art; Referring to, for example, people 2003.J Microbiol Methods 53:245-52 such as Dunbar.

The detection of infectious disease and diagnosis need to detect multiple antibody usually; Therefore, also can use the group evaluation specific antibody that is used to detect following combination, for example: adenovirus, Bordetella pertussis (Bordetella pertussis), campylobacter jejuni (Campylobacter jejuni), Chlamydia pneumoniae (Chlamydia pneumoniae), chlamydia trachomatis (Chlamydia trachomatis), cholera toxin, cholera toxin b, hair shape clostridium (Clostridium piliforme) (Tyzzer ' s), cytomegalovirus, diphtheria toxin, infectious ectromelia virus (Ectromelia virus), EDIM (young mouse epidemic diarrhea), encephalitozoon cuniculi (Encephalitozoon cuniculi), Epstein-BarrEA, Epstein-Barr NA, Epstein-Barr VCA, the HBV core, the HBV coating, HBV surface (Ad), HBV surface (Ay), the HCV core, HCV NS3, HCV NS4, HCV NS5, helicobacter pylori (Helicobacter pylori), hepatitis A, hepatitis D, HEV orf2 3KD, HEV orf2 6KD, HEV orf3 3KD, HIV-1 gp120, HIV-1 gp41, HIV-1 p24, HPV, HSV-1 gD, HSV-1/2, HSV-2 gG, HTLV-1/2, Flu-A, Flu-A H3N2, influenza B, Leishmania donovani (Leishmania donovani), Lyme disease, LCV, mycoplasma pneumoniae (M.pneumoniae), Mycobacterium tuberculosis (M.tuberculosis), parvovirus, mumps, mycoplasma pulmonis (Mycoplasma pulmonis), parainfluenza 1, parainfluenza 2, parainfluenza 3, parvovirus, mouse pneumonia virus, poliovirus, polyomavirus, reovirus-3, RSV, rubella, measles, sendai virus, schizotrypanum cruzi (T.cruzi), Spirochaeta pallida (T.pallidum) 15kd, Spirochaeta pallida p47, tetanus toxin, safe tired Er Shi murine encephalomyelitis virus, toxoplasm and varicella zoster.

The isotype group can be used for such as the detection of antibody mediated immunity defective diseases such as Huppert's disease, HIV infection, solid organ knurl or chronic liver disease, sign etc.The researchist also can use these groups to measure the aggregate level of some isotype in specified diseases, as the bacterial infection of various IgG defectives relevant with reaction/non-reactiveness, enhancing or unusual allergic reaction, autoimmunity disease, GI disease, malignant tumour, chest syndrome or recurrence.Described group can comprise for example combination of IgA, IgE, IgG1, IgG2 α, IgG2 β, IgG3, IgM and light chain (κ or γ).

In order to detect phosphate transferase activity for the useful multiple different protein kinases of doctor and researchist, one group of substrate protein can mix with ATP, the antibody of contact (for example) different colours coding contacts (for example) biotinylated report antibody and streptavidin-phycoerythrin conjugate then then.Then can be with its each reaction of unique markers tests.Described group can comprise following combination, for example: Akt, Akt/PKB (always), Akt/PKBpS473, ATF2 (Thr71), Erk-2, Erk1 (Thr202/Tyr204), Erk1/Erk2 (Thr202/Tyr204), Erk2 (Thr202/Tyr204), GSK-3 β, IkappaB-α pS32, IkappaB-α is total, JNK (pTpY183/185), JNK is total, JNKp (Thr183/Y182), MAPKAP K2, p38 (always), p38 MAPKpT180/pY182, p53 (always), p53 pS15, PKB-α, PKC, SAPK1, SAPK1a/JNK2, SAPK4, STAT1 pY701, STAT1 is total, STAT3 (Tyr705) and ZAP-70.This system can detect the protein of modification, for example, and by modifying and the protein of phosphorylation with specific antibodies.This system can detect one or more modifications of the molecule of one or more types.

In order to detect relevant with tissue reconstruction unusual and morbid state, cancer for example, the group of the matrix metalloproteinase of enzyme (MMP) family is useful, can comprise MMP-1, MMP-12, MMP-13, MMP-2, MMP-3, MMP-7, MMP-8 and MMP-9.

Other group comprises cancer biomarker group, for example combination of alpha-fetoprotein, PSA, cancer antigen 125 and carcinomebryonic antigen; Cardiac marker, for example, the combination of creatine kinase-MB, Endothelin 1, PAP, SGOT and TIMP-1; The label that is used for Alzheimer's.

The allergen group, for example the multiple analyte allergic reaction detects and uses, and for example can use different allergens, and these allergens are as the target of allergen specific antibody; Second marker molecules uses anti human IgE to finish this reaction.The allergenic example that is used for this group comprises chain lattice spore (mould), Bermuda grass (Bermuda Grass), cat soft flocks, the white of an egg, breast, mite (Mite Pternoyssinus), mountain cdear (Mountain Cedar), short hogweed, timothy grass and wheat (food).Can utilize similar method test example such as autoimmune antibody, wherein use antigen, as ASCA, beta-2 microglobulin, centromere B, chromatin, ENA Profile 4 (SSA, SSB, Sm, RNP), ENA Profile5 (SSA, SSB, Sm, RNP, Scl-70), ENA Profile 6 (SSA, SSB, Sm, RNP, Scl-70, Jo-1), histone, histone h1, histone H2A, histone H2B, histone H 3, histone H 4, HSP-27 pS82, HSP-27 is total, HSP-32, HSP-65, HSP-71, HSP-90a, HSP-90b, Jo-1, PCNA, PR3, PR3 (cANCA), ribosomes P, RNP, RNP-A, RNP-C, SCF, Scl-70, serum amyloid protein P, SLEProfile 8 (SSA, SSB, Sm, RNP, Scl-70, Jo-1, ribosomes-P, chromatin), Sm, Smith, SSA, SSB, streptolysin O and TPO.

Other group can be used for measuring blood vessel (for example people's blood vessel takes place) takes place, comprises, for example, the combination of IL-8, bFGF, VEGF, angiogenin and TNF.Can utilize other group to measure cell activation (for example people's cell activation), can comprise, for example, the combination of IL-8, β FGF, VEGF, angiogenin and TNF; The group that is used for B cell activation (for example human B cell activation) can comprise, the for example combination of CD79b (Ig β), BLNK, Btk, Syk and PLCy, the group that is used for T cell activation (for example human T-cell's activation) can comprise, for example, the combination of TCRz, SLP-76, ZAP-70, Pyk2, Itk and PLC γ.

The label group that is used for inflammation (for example human inflammation) can comprise, for example, and the combination of IL-8, IL-1 β, IL6, IL10, TNF and IL-12p70, and other well known to a person skilled in the art cell factor or biomarker.The group that is used for chemotactic factor (CF) (for example human chemokine) can comprise, for example, the protein 10 of IL-8, RANTES, KC (mouse), interferon gamma monokine, monocyte chemoattractant protein-1, macrophage inflammatory protein 1-α, macrophage inflammatory protein 1-β and the interferon-of inducing-induce.The group that is used for apoptosis (for example human apoptosis) can comprise, for example, and the combination of the PARP of cutting, Bcl-2 and active caspase (caspase)-3 albumen.The group that is used for people's anaphylatoxin can comprise, for example, and the combination of anaphylatoxin C4a, 3a and 5a.The group that is used for allergic reaction medium (for example people's allergic reaction medium) can comprise, for example, the combination of IL-3, IL-4, IL-5, IL-7, IL-9 (mouse), IL-10, IL-13 (mouse), eotaxin (CCL11), granulocyte colony stimulating factor and granulocyte macrophage colony stimulating factor.

For research and diagnosis, cell factor can be used as the label of a large amount of illnesss, disease, pathological state etc., and can be included in several different groups.It is current that its coordination of cell factor/chemotactic factor (CF) or inharmonic being adjusted in more than 100 kinds are arranged is significant clinically.For the specified disease process is associated with the change of cytokine levels, desirable method need be analyzed the various kinds of cell factor of every kind of sample.The exemplary cells factor of using in current that use in the mark group and the group that can use in the inventive method and composition includes but not limited to BDNF, CREB pS133, CREB is total, DR-5, EGF, ENA-78, the eotaxin, fatty acid binding protein, basic FGF, G-CSF, GCP-2, GM-CSF, GRO-KC, HGF, ICAM-1, IFN-α, IFN-γ, IL-10, IL-11, IL-12, IL-12 p40, IL-12 p40/p70, IL-12 p70, IL-13, IL-15, IL-16, IL-17, IL-18, IL-1 α, IL-1 β, IL-1ra, IL-1ra/IL-1F3, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, JE/MCP-1, KC, KC/GROa, LIF, lymphocyte chemotactic factor (LCF), M-CSF, MCP-1, MCP-1 (MCAF), MCP-3, MCP-5, MDC, MIG, MIP-1 α, MIP-1 β, MIP-1 γ, MIP-2, MIP-3 β, OSM, PDGF-BB, RANTES, Rb (pT821), Rb (always), Rb pSpT249/252, Tau (pS214), Tau (pS396), Tau (always), tissue factor, TNF-α, TNF-β, TNF-RI, TNF-RII, VCAM-1, VEGF.

Can for example be diabetes or thyroid gland label also in the clinical labororatory or the useful endocrine label of life science personnel, the foundation group.The example of endocrine label comprises adiponectin, dextrin, C-peptide, calcitonin, CRF, FGF-9, GLP-1, hyperglycemic factor, growth hormone, insulin, leptin, lipoprotein (a), phylaxin, T3, T4, TBG, thyroglobulin and TSH.The metabolic marker thing also is useful in research or clinical practice, comprises aPoA-I, apolipoprotein A-1, apolipoprotein A-1 I, apolipoprotein B, apoC-II, apoC-III, apo E, β-2 glycoprotein, 1 Collagen Type VI, 2 Collagen Type VIs, 4 Collagen Type VIs, 6 Collagen Type VIs, glutathione S-transferase, islet cells and tTG (chylous diarrhea).

Clinical labororatory and researchist need inquire after a plurality of nucleotide sequences usually simultaneously, for example, are used for tissue typing by detecting a plurality of target allele; Suitable group can comprise HLAI class and II class, 1 group of HLAI class monoclonal antibody original antibody, 2 groups of HLAI class monoclonal antibody original antibodies, PRAI class, PRAI class and II class, PRA II class, SSOI class HLA-A, SSO I class HLA-B, SSO I class HLA-C, SSO II class DP, SSO II class DQB1, SSO II class DRB1 and SSO II class DRB3,4,5 combination.

A kind of gestation group can comprise, for example, and for the detection of human chorionic gonadotrophin, hepatitis B surface antibody, rubella virus, alpha-fetoprotein, 3 ' estradiol and other target substance in the conceived individuality.

Be to be understood that, the sensitivity of analyzer of the present invention allows design and finishes the label and the label group that can't design and finish so far, make simply the answer that is/denys in order to exist (for example tumour or genetic abnormality) not only to the label of abnormal level, and can analyze more accurately, for example determine the outbreak of illness in early days, and compare the normal range of label and the level of in individuality, finding more accurately.Present assay method usually just could the certification mark thing when pairing pathologic condition reaches the stage that treatment is effectively impossible or validity is extremely low.For example, current detection level for multiple cancer only detects on the FA level of cancer.Method of the present invention not only allows early detection, and can set up the baseline values of the label of normal individual, and these labels exist in normal individual, but there is pathological conditions in unusual high or low level indication.

Therefore, the present invention includes based on the detection of one or more biomarkers and/or the method for quantitative early detection disease or symptom.In general, from individual as people such as the concentration of biomarker in the samples such as blood, blood plasma or blood serum sample and be considered to normal or unusual value and compare.Can utilize analyzer of the present invention and analyzer system to measure the level of the biomarker of normal and ill colony, this level far below at present detect and diagnosis in the level used, for example this level be at present can be quantitative 0.1,0.01,0.001 or 0.0001 times of level.Therefore, can set up database to the normal and abnormal level of particular disorder, and can the examination individuality, the detection of illness is greatly early than reaching up to now.In addition, also may have the database of normal and exceptional value, but for examination was individual, existing method may not conventionally be implemented, determine that with enough sensitivitys ground the label value of individuality is whether in normal range.For example, the method for IL-6 concentration is merely able to the IL-6 that detectable concentration is low to moderate about 5pg/ml in most of existing working samples; The normal range of IL-6 value is about 1 to about 10pg/ml; Therefore, existing method can only detect the IL-6 on normal range top.On the contrary, analyzer of the present invention and analyzer system allow detectable concentration to be lower than about 0.1pg/ml, are lower than the IL-6 of normal range value 1/10th in other words.Therefore, analyzer of the present invention and analyzer system can produce biomarker for example IL-6 more extensive, more have nuance database, also allow according within the normal range and outside biomarker carry out examination, thereby allow early detection.

This early detection method of the present invention can be used for detect existing one or more biomarkers, perhaps find the outbreak of this biomarker and illness or make progress relevant, and any disease or the illness that can obtain its numerical data base.

As an example, the diagnosis of cancer depends on the rough test that uses tumor growth usually, and as showing tumour itself, this is coarse, perhaps must reach high level before this method can detect, for example in the actual clinical condition.When detecting, tumour grows into enough sizes that intervention can not take place usually before shifting.For example, need diameter of tumor＞1cm by the X ray detection of lung cancer, CT scan needs diameter of tumor＞2-3mm.In addition, also can use the biomarker of tumor growth, still, tumour has been in late period in the time of can detecting under the detectable level of existing clinical technology to biomarker usually.In addition, (for example intervening, for operation, chemotherapy or the radiation therapy that dwindles or excise one or more tumours) after, usually can not enough measure tumor marker delicately, determining whether cancer recurs, when the focus of remnants proceeds to the degree that further intervention can not be successful.Use analyzer of the present invention, system and method, can detect beginning, (for example because the lower metastatic potential) of tumor growth and intervene the recurrence of tumor growth when more possibility is successful.

Therefore, the invention provides 1) screening can't be used to because of contents level is too low to diagnose or the method for the biomarker of monitoring of diseases up to now; 2) based on 1) in the method for detection examination seizure of disease of biomarker that find or at present known, the level of this label is far below now possible; With 3) method of the monitoring course of treatment or experimental therapy validity, have ability far above current possible detection effect (comprising the recurrence of disease).

This comprises the existence that detects individual illness, does not have, develops possibility or progress degree methods.This illness can be pathologic or non-pathologic (for example aging, gestation).The example of pathological disorders comprises common pathological disorders, inflammation for example, and it is relevant with a large amount of particular pathologies illnesss, for example diabetes, heart disease, arthritis, cancer etc.Pathological disorders also comprises multiple particular disorder, includes but not limited to cancer, inflammatory disease and/or autoimmunity disease, enterogastric diseases, skin disease, sacred disease, hereditary disease, infectious disease, aging, allergic reaction etc.Cancer includes but not limited to lung cancer, cancer of the stomach, cancer of pancreas, cancer of the esophagus, oophoroma, breast cancer, prostate cancer, carcinoma of urinary bladder, colon cancer and the carcinoma of the rectum.This method comprises uses analyzer of the present invention or analytic system analysis from one or more disease markers in the sample of individuality, wherein this analyzer or analytic system can be lower than 1 nanomole by detection level, or 1 picomole, or 1 fly mole, or 1 vast mole, or one or more labels of 1 narrow mole.In certain embodiments, when the concentration of biomarker is lower than 1 nanomole, or 1 picomole, or 1 fly mole, or 1 vast mole, or 1 narrow mole the time, and when the volume of sample during less than about 100,50,40,30,20,10,5,2,1,0.1,0.01,0.001 or 0.0001 μ l, this analyzer or analytic system can detect between a kind of sample and the another kind of sample one or more marker concentrations less than about change of 0.1,1,2,5,10,20,30,40,50,60 or 80%.In certain embodiments, when the concentration of analyte is lower than about 1 picomole, and when the volume of every kind of sample during less than about 50 μ l, this analyzer or analytic system can detect analyte concentration between first sample and second sample less than about 20% variation.In certain embodiments, fly mole when the concentration of analyte is lower than about 100, and when the volume of every kind of sample during less than about 50 μ l, this analyzer or analytic system can detect analyte concentration between first sample and second sample less than about 20% variation.In certain embodiments, fly mole when the concentration of analyte is lower than about 50, and when the volume of every kind of sample during less than about 50 μ l, this analyzer or analytic system can detect analyte concentration between first sample and second sample less than about 20% variation.In certain embodiments, fly mole when the concentration of analyte is lower than about 5, and when the volume of every kind of sample during less than about 50 μ l, this analyzer or analytic system can detect analyte concentration between first sample and second sample less than about 20% variation.In certain embodiments, fly mole when the concentration of analyte is lower than about 5, and when the volume of every kind of sample during less than about 5 μ l, this analyzer or analytic system can detect analyte concentration between first sample and second sample less than about 20% variation.In certain embodiments, fly mole when the concentration of analyte is lower than about 1, and when the volume of every kind of sample during less than about 5 μ l, this analyzer or analytic system can detect analyte concentration between first sample and second sample less than about 20% variation.This method also can comprise relatively the value of value that this analysis obtains and known biomarker, to determine existing, not existing or the progress degree of disease; And then this method can comprise informs individual this comparative result, or based on the described relatively more definite course of treatment, prognosis or diagnosis.In certain embodiments, this method comprises analyzes a plurality of samples of gathering from individuality usually in certain time-histories, be identified for the concentration change degree and/or the rate of change of one or more labels of the particular disorder that detected, and with this intensity of variation and/or rate of change and normal and/or exceptional value relatively.Should be appreciated that the combination that in measuring the illness existence, do not exist or making progress, also can use absolute value and rate of change etc. with the skill level that improves.

In an example, the invention provides the existence of lung cancer in a kind of examination individuality, the method that does not exist or make progress.In certain embodiments, described individuality can be to have the people who suffers from the lung cancer highly dangerous; Such individuality comprises and is exposed to the individuality that causes the lung cancer thing, and for example smoking or occupational expose, as are exposed to asbestos, and about 50,55,60,65,70,75 or the individuality more than 80 years old, or accepts the individuality of existing lung cancer or other treatment of cancer.Individuality can be asymptomatic.The present invention includes use can detection level be 1 nanomole, or 1 picomole, or 1 fly mole, or 1 vast mole, or the analyzer analysis of one or more labels of 1 narrow mole is from the lung cancer of one or more lung cancer in phlegm, BAL, blood, serum or the plasma sample of individuality and/or one or more particular types label of small cell carcinoma for example.In certain embodiments, detection level is lower than 1 picomole.In certain embodiments, when the concentration of biomarker less than 1 nanomole, or 1 picomole, or 1 fly mole, or 1 vast mole, or 1 narrow mole, and when sample volume during less than about 100,50,40,30,20,10,5,2,1,0.1,0.01,0.001 or 0.0001 μ l, this analyzer or analytic system can detect between a kind of sample and another sample one or more marker concentrations less than about variation of 0.1,1,2,5,10,20,30,40,50,60 or 80%.In certain embodiments, when the concentration of biomarker during less than 1 picomole, this analyzer or analytic system can detect less than in one group of sample of 5 μ l less than 20% concentration change.This method also can comprise relatively the value of value that this analysis obtains and known biomarker, existing, not existing or the progress degree with lung cancer in definite individuality; In addition, this method also can comprise informs individual this comparative result, or based on the described relatively more definite course of treatment, prognosis or diagnosis.In certain embodiments, this method comprise more in time from the same individual biomarker level value that obtains with from another individual biomarker level value that obtains, and determine diagnosis, prognosis, possibility degree or the progress degree of lung cancer as mentioned above.

In addition, method of the present invention allows the biomarker group of discovery and use sensitivity raising to determine the detection consequence of (for example) treatment results and/or treatment.For example, dwindle or eliminate in the treatment of cancer of cancerous tissue relating to, know whether and when cancer is useful and with which kind of speed recurrence.The sensitivity of the inventive method allows to obtain such information at the more early stage of recurrence and with higher degree of accuracy, thereby allows taking measures in early days in palindromia.Certainly, the examination for seizure of disease in the original normal individuality also is like this.

In addition, should be appreciated that the sensitivity of analyzer of the present invention and method and demultiplexing allow to use method of the present invention to carry out polytype clinical, research, and agricultural and commercial Application.Therefore, can design the biomarker group and be used for replication, for example be used for examination and other research application.Because sensitivity for analysis is higher than the sensitivity for analysis that uses up to now in extensive biomedical research, changes so can detect, and find and use new biomarker than the lower level label of now possible level.In certain embodiments, the new biomarker that had not used before can using in the biomarker group perhaps can use the lower biomarker of previously used sensitivity in of the present invention group.

Therefore, in certain embodiments, method of the present invention comprises with having two samples of inquiring after the single-particle detecting device analysis in space from individuality, wherein can detect in the monocyte sample at least 1,2,3,4,5,6,7,8,9,10,15,20,30,40,50,100 or the particle (if present) of type more than 100 kind, its sensitivity is lower than 1nM, 1pM, 100fM, 10fM, 5fM, 4fM, 3fM, 2fM, 1fM, 0.5fM, 0.1fM or 0.01fM, 0.001fM, 0.0001fM, 0.00001fM or 0.000001fM.Every type particle may have different detection levels.

Because method of the present invention has improved the sensitivity and the ability of multichannel sample, the volume of the sample that needs in this method can correspondingly reduce.At sample is that the volume of sample can be less than 1000,500,200,100,75,50,40,30,20,10,5,4,3,2,1,0.1,0.01,0.001 or 0.0001 μ l in certain embodiments of the present invention of biofluid such as body fluid (for example serum).Can be 1 to the number of the dissimilar particles of this sample analysis, perhaps greater than more than 1,2,3,4,5,6,7,8,9,10,15,20,30,40,50,100 or 100.In certain embodiments, sample volume is extremely about 500 μ l of about 1 μ l, or about 10 μ l are to about 200 μ l, or about 10 μ l are to about 100 μ l, or about 10 μ l are to about 50 μ l, or about 50 μ l, the number of the particle types of being analyzed is about 1 to about 50, or about 1 to about 20, or about 1 to about 10.

In certain embodiments, the analysis of sample was carried out in the specific period.In some cases, in one day, perhaps about 12,8,4,2,1,0.5,0.4,0.3,0.2,0.1,0.05,0.01 or less than 0.01 hour in analyze.In certain embodiments, the invention provides to use and have the method that two single-particle detecting devices of inquiring after the space are analyzed a plurality of samples, wherein average each sample is in less than 1 hour, or less than in 0.5 hour, or less than 0.2 hour, or less than 0.1 hour, or less than 5,4,3,2,1,0.5 or 0.1 minutes inner analysis.In one embodiment, the invention provides a kind of use and have the methods that two single-particle detecting devices of inquiring after the space are analyzed clinical sample, wherein average each sample is less than 1, or less than 0.5, or less than 0.1 hour inner analysis.

In one embodiment, the invention provides a kind of definite individuality and whether suffer from the method for disease, comprise use have two inquire after the single-particle detecting device analysis in space from the existence of biomarker in the sample of individuality, do not exist or concentration, wherein the analysis of sample is less than 2 hours, or 1 hour, 0.5 hour, or carry out in 0.25,0.1 or 0.01 hour.This method also comprises from individuality and obtains sample, and/or to individual report analysis result.In one embodiment, the invention provides the method that comprises to individual or its representative's report analysis result of collected specimens, comprise use have two inquire after the single-particle detecting device analysis in space from the existence of TREM-1 in the sample of individuality, do not exist or concentration, wherein the analysis of sample is carried out in less than 1 hour.

In certain embodiments, report the existence that detects particle, do not exist and/or concentration to the individual of collected specimens or to the health worker of the individuality of being responsible for collected specimens.In certain embodiments, based on the existence of particle, do not exist and/or concentration is diagnosed, the course of treatment of prognosis, monitoring and/or suggestion.In certain embodiments, to course of treatment that the health worker of the individuality of individuality, its representative or the responsible collected specimens of collected specimens reports diagnosis, prognosis, monitoring and/or suggestion.

Should be appreciated that method of the present invention also can utilize this information to come evaluation study or clinical or preclinical test to providing information such as individualities such as researchist or professional medical-care personnel.For example, obtain the hereditary information of disease and produced research and clinical analysis field with the correlativity of key gene sudden change.Hereditary information (promptly measuring the foranalysis of nucleic acids of hereditary variability) and protein group information (promptly measuring the analysis of the actual protein of hereditary variability expression) are all useful in research and clinical setting.Usually, research or the diagnostic method based on the information of suddenling change about key gene need use polymerase chain reaction (PCR) and version thereof.The sensitivity of this method allows to detect the catastrophic event of nucleic acid or protein or nucleic acid and protein, and does not need amplification of nucleic acid.In addition, utilize method of the present invention to detect its existence of expressing the multiple proteins relevant easily, do not exist and/or the variation of concentration, thereby can screen the effect of (for example) institute test agent fast, delicately pathological condition with specific hereditary configuration and/or pathological condition.Can detect simultaneously and/or the quantitatively a large amount of different labels in the monocyte sample, for example protein.

Other industry of the present invention and environmental applications comprise production run control, environmental monitoring and food security.For example, can analyze from such as environment such as soil, water or air sources or from such as waste streams, water source, supply line or the pollution of the sample of industrial source such as producing batch.The example of possible pollutant comprises pesticide, petroleum product, industrial dust and biosome.Many identical pollutants are to be concerned about in the food supply, biosome particularly, and as the fungi in the grain, the bacterium in meat, game, agricultural product, the dairy products.Commercial Application for example comprises from bio-reactor or food fermentation process as brewageing the quality control of fermentation medium.

On the other hand, the invention provides business method.In one embodiment, the invention provides a kind of method of carrying out business activity, comprise by entity and use two detecting devices of inquiring after the space that have can detect single-particle (for example unimolecule) to obtain the sample determination results, report described result, and the expense of reporting the result to this entity pays.In certain embodiments, this detecting device can be arbitrary embodiment as herein described.In certain embodiments, this entity is that clinical labororatory improves correction (Clinical Laboratory Improvement Amendments (CLIA)) laboratory.In certain embodiments, this entity is not the CLIA laboratory.Sample can be the sample of any type of the enough single-particle detecting device analyses of energy.In certain embodiments, sample is from individuality.Individuality can be the individuality of any type as herein described.In certain embodiments, individuality is the patient (for example animal, for example people) that examination, diagnosis, prognosis, monitoring and/or definite methods of treatment are carried out in expectation.In certain embodiments, individuality is an individuality (for example animal, for example people) of participating in clinical testing or preclinical test research.In certain embodiments, sample comes from the individuality of the part of conduct such as projects such as biomedical research, agricultural research, educational research, bio-terrorism research.In certain embodiments, can be by the individual of reception result report, for example the individuality of health care personnel and/or collected specimens is to the entity of analyzing, for example pay in the CLIA laboratory, perhaps can pay to the individuality that receives report from the entity of analyzing by the individual of collected specimens, perhaps pay, perhaps pay to the two or its some combination to this entity itself.In another embodiment, the invention provides a kind of method of carrying out business activity, comprise that the health care supplier uses two measurement results of inquiring after the detecting device acquisition in space from the sample of individuality that have that can detect single-particle, reports described result to individual or its representative; And the expense of paying described result's report by this individuality.

Embodiment

Following embodiment is illustrative, is not to limit remaining disclosure.The dna fragmentation that combines with nucleic acid, particle, protein, virus and organelle are pumped or carry out electrophoresis.

The following analysis of data; The adjacent bin that operational analysis software will contain photon is grouped into the photon that derives from particle and bursts.For the experiment of using electrophoresis, with the photon that the derives from particle crosscorrelation of bursting, to determine their electrophoretic velocity (time migration that in two detecting devices, detects).

Detection-the electrophoretic velocity and the dilution curve of embodiment 1.DNA fragment

Be used for proving the electrophoresis of nucleic acid with the 7.2kb fragment of the DNA of A647 mark.With SmaI single restriction site place digestion M13mp18 RFI DNA (New England Biolabs, Beverly, MA).(Eugene OR) illustrates the 7.2kb fragment that obtains with the AlexaFluor647 mark according to the manufacturer for Molecular Probes, Inc. to use ULYSIS  nucleic acid marking kit.According to its concentration at the DNA of the absorbance measurement Alexa at 260nm place Fluor mark.According to its concentration at the absorbance measurement Alexa at 650nm place Fluor.The mark degree is 190 dyestuffs of each dna particle.Prepare serial dilutions by stock solution, produce 0.03 to 100fM concentration range.Each sample is added in the analyzer of the present invention electrophoresis 4 minutes.Fig. 6 shows the histogrammic example of particle crosscorrelation of the sample that contains 0-0.1fM DNA.When 0.1fM, detect the peak of 9 particles at the 142ms place, and the background sample-there is 1 particle at the 78ms place.Proved in the population that detects and be up between the sample concentration of 100fM and had linear relationship.

Embodiment 2. is for the sandwich assay of biomarker

A kind of biomarker that the definite TREM-1 of nearest report is bacterium or fungal infection (referring to, for example, people such as Bouchon (2000) J.Immunol.164:4991-5; Colonna (2003) Nat.Rev.Immunol.3:445-53; People such as Gibot (2004) N.Engl.J.Med.350:451-8; People such as Gibot (2004) Ann.Intern.Med.141:9-15).Used sandwich assay formats to develop mensuration (Sandwich Assay for Detection ofIndividual Molecules, US temporary patent application No.60/624,785) to TREM-1.The mensuration reagent that is used for the TREM-1 detection can be buied (R﹠amp commercial; D Systems, Minneapolis, MN).This is determined in 96 orifice plates and carries out.Use monoclonal antibody as capture agent, use another kind of monoclonal or polyclonal antibody to detect.Detect antibody AlexaFluorA647  mark.

The mensuration program is as follows:

With the capture antibody bag by flat board, wash 5 times,

2. with the 1%BSA among the PBS, 5% sucrose seals,

3. be added in the target that dilutes in the serum, incubation washs 5 times,

4. add and detect antibody, incubation washs 5 times,

5. add 0.1M glycocoll pH 2.8, discharge the mensuration composition of combination on the slave plate.

6. sample is transferred to check-out console from disposable plates, make the pH of sample be neutrality, and on the single-particle analyzer system, analyze.

Fig. 7 shows the typical curve of the TREM-1 that uses this mensuration generation.This 100-1500 that is determined at mensuration flies in the scope of mole to linear.Added R﹠amp recently; The ELISA of D Systems measures.The typical curve that the ELISA of report measures is between the 60-4000pg/ml.This embodiment points out us can measure 100fM (4.7pg/ml) routinely in typical curve, allows the mensuration of about 10 times of sensitivities.

Also use the commercial reagent (R﹠amp that buys; D Systems) developed the sandwich assay that is used to detect IL-6.This scheme substantially as above for as described in the TREM-1, difference is that target dilution and capture antibody are to as R﹠amp; D Systems is described.Detecting antibody is the R﹠amp that uses AlexaFluor  647 marks; The antibody of D Systems.This mensuration allows to detect the IL-6 (Fig. 8 A and B) that is lower than 0.5pg/ml.The detection limit that calculates is 0.06pg/ml.Be the detection of 0.5 to 10pg/ml IL-6 for normal level, this level of sensitivity is splendid.Compare with other commercial multichannel mensuration that comprises IL-6 of buying, this system provides the remarkable improvement of detection level.With R﹠amp; The mensuration of D Systems is compared, detection limit roughly the same (Fig. 8 C), but this system has the advantage of multichannel, and do not rely on amplification step, and R﹠amp; The mensuration of D Systems needs these two.Single-particle analytic system data realize quantitatively by each molecule in the counting low concentration solution with different being of ELISA data, rather than carry out bulk molecule and measure.The former is more accurate than the latter.

Embodiment 3. is based on the mensuration of pearl

Measure the identical microtiter plate form of use, wherein utilize frosting fixed target molecule for above-mentioned two kinds.The single-particle analytic system is also mated with the mensuration of using microballoon or pearl to carry out in solution, realizes combining form and not individual the separating of combination.Fig. 9 shows the result based on the mensuration of pearl who detects thyrotropic hormone (TSH).This data declaration sandwich assay can directly be transferred to based on the form of pearl and by this system and use.(Miltenyi Biotec, Auburn CA) resist-TSH capture antibody bag quilt with biotinylated superparamagnetism streptavidin microballon.By the dilution of in phosphate buffer, catching 0-200fM TSH with excessive 4 ℃ of incubations of microballon.Collection has the microballon of the TSH that catches, goes up washing at high gradient magnetic separating column (Miltenyi Biotec).Remove pearl from post, (OR) the anti-TSH of mark detects antibody 37 ℃ of following incubations 2 hours for Molecular Probes, Eugene with AlexaFluor  647.Collection has and the pearl that catches the detection antibody that TSH combines, and with the phosphate buffer washing, removes from post.On the particle analysis post, analyze these pearls, fly to produce on mole TSH scope linear response (Fig. 9) at the 50-200 that measures.

The detection of target protein in embodiment 4. human serums

Developed and the above similar sandwich assay in order to detect intraserous target.Measure for this, in the sample that contains 10% human serum, add the TSH of known quantity.Add the TSH specific antibody of mark, remove unconjugated label, analytic sample on the single-particle analyzer system.The results are shown among Figure 10, prove that the TSH of all addings in this mensuration is recovered.

The detection of the inferior fM concentration of embodiment 5. nucleic acid polymers

3DNA ^TMDendrimer is the particle that the nucleic acid particle by many branches, interconnection constitutes.Use is so that (Hatfield, 4 layers of dendrimer of A647 mark PA) prove the electrophoresis of particles available from Genisphere Inc..The manufacturer points out that when 20ng/ μ l concentration, each dendrimer of this dendrimer contains about 37,000 deoxynucleotides of useful about 375 kinds of dye markers.Use the volumetric molar concentration of these manufacturers' explanation calculation sample.Prepare serial dilutions by stock solution, produce 0.01 to 33fM concentration range.Each sample is added in the analyzer of the present invention electrophoresis 4 minutes.

Figure 11 shows and contains 0 and the histogrammic example of particle crosscorrelation of the sample of 0.03fM dendrimer.When 0.03fM, 163 and the peak at 227ms place in detect 17.6 particles altogether, and the background sample has 1 particle at the 119ms place.Proved in the population that detects and be up between the sample concentration of 33fM and had linear relationship.

Figure 12 shows 0 and the example of the adjacent photon of the sample of the 0.03fM cross-correlation result of bursting.The latter is presented at the 206ms place and detects the photon peak, and background sample detection to 0 photon.

The detection of embodiment 6.BSA-SDS electrophoresis and dilution curve

Prove the electrophoresis of protein with the bovine serum albumin(BSA) (BSA) of A647 mark.According to manufacturer's explanation, with succinimide ester (Molecular Probes, Inc., Eugene, OR) the covalent labeling BSA of A647 carboxylic acid.By (Millipore Corporation, Bedford MA) go up ultrafiltration not the Alexa Fluor and the Separation of Proteins of coupling at Microcon YM-30 film.According to its concentration, Alexa Fluor is proofreaied and correct in the contribution at 280nm place at the BSA of the absorbance measurement A647 at 280nm place mark.According to its concentration at the absorbance measurement Alexa at 650nm place Fluor.The mark degree is 1.9 Alexa Fluors of each protein granule.Carry out serial dilution by stock solution, produce 0.03 to 30fM concentration range.Each sample is added in the analyzer of the present invention electrophoresis 4 minutes.

Figure 13 shows the histogrammic example of particle crosscorrelation of the sample that contains 0-10fM protein.When 10fm, detect the peak of 30 particles at the 427ms place, and the background sample have 4 particles between 250-600ms.Proved in the population that detects and be up between the sample concentration of 30fM and had linear relationship.

Detection-the electrophoretic mobility of embodiment 7. viruses

The anti-GP8 antibodies of M13K07 and A647 Zenon mark proves the detection of virus.Anti--GP8 antibody is with ZenonTM IgG labelling kit (Molecular Probes, Inc., Eugene, OR) mark 5 minutes at room temperature.3.7pM M13K07 (New England Biolabs, Beverly, MA) with the anti-GP8 antibody of 110pM mark in 1xPBS 4 ℃ be incubated overnight.By making reactant liquor cross S-400HR spin post twice, from free antibodies, be purified into the bacteriophage of mark.To be added on the analyzer of the present invention electrophoresis 4 minutes from the eluent dilution of S-400 post.

Figure 14 shows the histogrammic example of particle crosscorrelation.The mobility of virion increases with electric current and increases.In addition, although the concentration of virus is all identical under each condition, as expection, the population that (1 μ A) detected in 4 minutes in moving the slowest sample is minimum, increases when fair speed.

The detection of embodiment 8. bacteriums and virus

Developed the mensuration of utilizing immunoassay to measure whole biosome such as virus or bacterium.Figure 15 shows the measurement result that is used for detecting microorganism.Utilization detects e. coli k12 JM109 based on the determination method of pearl.Cell and antibody (rabbit polyclonal) the room temperature incubation 1 hour that is coupled on the AlexaFluor  647.Bacterial suspension is centrifugal by 0.2 micron filter membrane, to separate unconjugated antibody and the antibody that is attached on the cell.With cell washing 8 times, be resuspended in the buffer release liquid (0.1M glycocoll pH 2.8) and incubation 10 minutes.It is centrifugal by filter membrane to discharge solution, neutralization, and in the particle analysis system, analyze.In the mensuration that virus detect to be used, by incubation at room temperature, (M13 bacteriophage particles MA) combines with micro titer plate well is passive for New England Biolabs, Beverly to make stock solution from dilution.Liquid in the sucking-off hole sealed 30 minutes.Anti-M13 antibody adds in the hand-hole with Zenon (Molecular Probes) mark and with 1000ng/ml.Dull and stereotyped incubation 1 hour, washing, the material with 0.1M glycocoll pH 2.8 discharges combination neutralizes, and analyzes on the single-particle analyzer system.The mensuration that is used for producing data among Figure 15 does not all have in order to reduce background, to make and detect maximization or minute is minimized and optimize.Optimization should reduce detection limit, and obtains the result in 2 hours.

Selecting Escherichia coli is the antibody and the test that can obtain to detect it as the reason of model.Escherichia coli are multifarious biologies of a kind of abundant research, and its bacterial strain is mainly according to serotype and difference.This sign completely is reflected in has developed lot of antibodies for distinguishing multiple serotype, and numbering surpasses 700 kinds (www.textbookofbacteriology.net) now.For measuring development, this be advantage be again shortcoming.Advantage has been that many candidate's antibody are available, and finds that the probability of high-quality antibody is bigger.Shortcoming is very carefully to solve the specificity problem.It is desirable to, can select only to detect target serotype and not detect the antibody of other serotype right.In fact, catching and/or detect antibody may be by being made up of one group of antibody of extremely low cross reactivity with target strain specific reaction and with other strain.Special concern be non-pathogenic strain as the ubiquitous common pollutant of clinical sample.Select optimum antibody may need very long still conventional screening process.Analyzer of the present invention and analyzer system high-speed significantly accelerated finishing of this screening process.If use one group of antibody,, thereby provide the even detection of every kind of relevant strain with the concentration of every kind of antibody of balance.Except the cross reactivity of detection and non-pathogenic e. coli strains, also studied the cross reactivity of other pathogen such as staphylococcus, enteric bacteria, candida albicans and pseudomonad strain.Developed the mensuration of these pathogen of specific detection.When carrying out clinical testing and before, can specific detection and difference target pathogen in order to ensure each mensuration, carry out a large amount of cross reactivities and detect.

The concentration of bacterium is lower in the expection sample, even in the sample from the active stage sepsis patient.Although because each bacterium contains many binding sites for specific antibody, make this mensuration have built-in " amplification ", may need another amplification step.Verified another amplification of signal step is possible in these immunoassays, and adapts with the detection that utilizes analyzer of the present invention.Amplification realizes by the enzymatic activity with the alkaline phosphatase that detects antibody coupling.For example, (Switzerland) alkaline phosphatase of coupling combines with the biotin of fixing for Roche, Basel with streptavidin.Behind the unconjugated enzyme of flush away, add non-fluorescence alkaline phosphatase substrate, with sample incubation a few minutes.Count each molecule of the fluorescence-causing substance of antibody-enzyme conjugates generation then with analyzer.

In the model experiment of the type, with the concentration of alkaline phosphatase dilution for known 0-150 molecule/200 μ l, (9H-(1 with substrate then, 3-two chloro-9,9-dimethyl acridine-2-ketone-7-yl) di(2-ethylhexyl)phosphate ammonium salt [DDAO-phosphate, Molecular Probe]) reaction, the latter is cut into fluorescence-causing substance.Reaction system was 37 ℃ of following incubations 60 minutes.Cessation reaction is as described hereinly analyzed two inquiring after in the spatial analysis device.Figure 29 shows the fluorescence-causing substance molecular number of counting when each enzyme concentration.From 200 μ l samples, detect and be less than 10 enzyme molecules.Detect for relevant clinical in directly bacterium or virus and to measure when sensitive inadequately, perhaps hope will measure sensitivity expand to before not during the degree of sign, use this basic skills.

The enzymatic amplification of sort signal can be used for detecting as mentioned above single bacterium, perhaps can be used for detecting any analyte that can combine with enzyme-ligand conjugates, and wherein unconjugated enzyme-part is removed or deactivation.In order to detect the Escherichia coli in the blood, limit reagent and condition, wherein the Escherichia coli in the blood sample are identical with the bacterium behavior of incubation growth, make it possible to accurately determine concentration according to typical curve.This mensuration can disclose the existence than the target organism of the more more number of culture indication, detects that live and dead biology because should measure.The existence of therapeutic antibiosis element does not influence the Bacteria Detection of this system in the expection patient sample.Blood contains many molecules that can interference measurement antibody combine with bacterial target.Importantly determine to make required association reaction maximization and make other minimized condition.

Embodiment 9. usefulness quality status stamp substance markers change the electrophoretic velocity of protein complex

(Martek BiosciencesCorp., Columbia MD) mix with biotin labeled 1kb PCR fragment (b-NA) PBXL-3 of streptavidin mark (PBXL-3/SA), with the detection of proof binding interactions.PBXL-3/SA and b-NA (800pM) etc. volumetric molar concentration are containing the 10mM Tris of 0.1% casein hydrolysate as carrier, and the room temperature incubation is at least 1 hour among the 0.5mM EDTA pH 8.1.Also carry out independent PBXL-3/SA and the contrast incubation of PBXL-3/SA with the identical 1kb fragment (NA) that does not contain biotin.Behind the incubation, sample 2mM Tris, 10,000 times of 0.1mM EDTApH 8.1 dilutions to final concentration is 8fM.Sample is added in the analyzer of the present invention electrophoresis 4 minutes.

Figure 16 shows the histogrammic example of particle crosscorrelation.Under the situation that does not contain nucleic acid, the migration peak of organelle (PBXL-3/SA) is (figure A) at 368 places.Move when combining with nucleic acid comparatively fast, 294ms (figure B) is changed at visible peak.Only when combining with organelle, just shifts nucleic acid, because its existing in reaction (not conforming to the biotin labeling thing) causes the migration peak of organelle at the 409ms place (figure C).

Embodiment 10. differentiates the electrophoresis of two kinds of particles-do not sieve according to mobility

Anti--GP8 the antibodies of M13K07 and A647 Zenon mark is to prove the discriminating of virus and nucleic acid.With three times of excessive Zenon A647 at room temperature mark anti--GP8 antibody 5 minutes.3.7pM M13K07 (based on the plaque forming unit of New England Biolabs report) and the mark of 110pM anti--GP8 antibody room temperature incubation 1 hour in 1xPBS.Reactant liquor is stored under 4 ℃ spends the night.Cross S-400HR spin post twice, the bacteriophage of purifying mark from free antibodies by making reactant liquor.With 10,000 times of elution samples dilutions, be added on the analyzer of the present invention electrophoresis 4 minutes.Nucleic acid samples is the 1kb PCR fragment that derives from M13K07 and use the A647 mark.All samples is all analyzed under the total concentration of 8fM.

Figure 17 shows the histogrammic example of particle crosscorrelation.When having the combination of PCR nucleic acid fragment and virus/antibody, 245 and 296ms place be resolved to two peaks (scheming A).The migration peak of independent labeling nucleic acid is (figure B) at the 302ms place.C (is schemed) at the 222ms place in independent migration peak with virus antibodies.

Embodiment 11. differentiates that according to mobility two kinds of particle-utilizations have the SDS electrophoresis of linear polyacrylamide

The IgG sample of A647-mark and 1.1kb PCR product are containing 0.2% linear polyacrylamide (LPA, 5,000,000-6,000,000MW), the 18mM tris of 0.01% lauryl sodium sulfate and each 1 μ g/ml bovine serum albumin(BSA), Ficoll  and polyvinylpyrrolidone, the 18mM glycocoll prepares among the pH8.6.Sample pump is delivered in the analyzer kapillary, turned off pump, apply electric field (300V/cm).Determine the crosscorrelation of particle as the function of time migration.The data set of collecting one minute is analyzed.

Figure 18 shows the histogrammic example of particle crosscorrelation.Figure A shows the sample that only contains the IgG that concentration is 26fM and use the A647 mark, and it shows the peak of a crosscorrelation incident at the 75ms place, and 75ms is that IgG inquires after the required time of transfer between the space at two.Figure B shows and only contains the sample that concentration is the PCR product of 10fM and usefulness A647 mark, shows the peak of crosscorrelation incident at the 220ms place, and 220ms is that the PCR product is inquired after the required time of transfer between the space at two.Figure C shows and contains IgG and PCR product that is respectively 13fM and 5fM and the sample of using the A647 mark, the peak that shows two crosscorrelation incidents, one at the 75ms place, another proves that at the 215ms place this mensuration can be based on distinguishing this two kinds of molecules their different switching time in analyzer under described condition determination.

The detection of the label of the indirect detection of embodiment 12. particles-discharge from intended particle

Biotinylated resisting-thyrotropic hormone (TSH) antibody is fixed on 96 orifice plates of streptavidin bag quilt the unconjugated antibody that flush away is excessive.Xiang Kongzhong is added in and contains 1% bovine serum albumin(BSA) and 0.1%Tween ^Anti--TSH the antibody of TSH antigen in 20 the phosphate buffer and A647 mark.Flat board is incubation under vibration.Sucking-off liquid, hole flushing three times.By with 0.1M glycocoll-HCl, pH 2.8 incubations are from the dissociate antibody of A647 mark of the sandwich body of TSH.Collect the antibody of free A647 mark, dilute, and analyze by SMD.Visible label that discharges and the linear relationship between the original object particle concentration among Figure 19 A.

It will be appreciated by those skilled in the art that and to use similar method mark and release mark thing from the nucleic acid.People such as Matray have instructed mark and the method (Matray, 2004) of release mark thing on protein and the nucleic acid.Those skilled in the art also should be appreciated that the label that discharges from target protein and nucleic acid potpourri separate with differentiate basic identical with original object.Figure 19 B and C show the possible method of the label of two kinds of releases of two kinds of operational analysis device differences.

Embodiment 13. differentiates two kinds of particles according to intensity

With respect to nucleic acid---with the linear pUC19 of Alexa Fluor  647 marks, intrinsic fluorescence albumen composition PBXL-3 time per unit is launched more multi-photon.According to ULYSIS  nucleic acid marking kit (Molecular Probes, Inc., Eugene, the Alexa Fluor  647 mark pUC19 DNA of scheme Oregon).Phosphate buffer (PBS) (10mM sodium phosphate, 150mM NaCl, pH 7.2) middle 0.01% casein hydrolysate (the Sigma-Aldrich Corp. that adds, St.Louis, Missouri), be used for preparing the independent protein of serial dilution (2.5,5,7.5,10 and 20fM), independent nucleic acid or their potpourri.By sample being moved in 4 minutes by analyzer with 1 μ L/min pumping.

Analyze data by crosscorrelation will be carried out greater than the detection signal of 4 standard deviations of average background.Figure 20 A and B show the figure of the cross-correlated signal of protein complex and independent nucleic acid.The scope of elapsed time is limited in the incident (seeing Figure 20 A and B) that only shows in the peak, and emphasizes the different peculiar fluorescence intensity of protein complex and nucleic acid.The luminance level of selecting 500 photons is separated the high strength window of protein complex and the low-intensity window of nucleic acid as cut off.Make and differentiate two kinds of molecular speciess in this way, measure the protein complex of series concentration and the event number that detection of nucleic acids arrives.Typical curve is to use two brightness windows to protein and nucleic acid mapping, and definite slope of a curve.

In three different potpourris, differentiate protein complex and nucleic acid based on fluorescence intensity.Utilization is detected molecular number in the potpourri of PBXL-3 and pUC19, based on the slope of typical curve, calculates the concentration of every kind of composition.The protein of mensuration and the concentration and the predicted value of nucleic acid are compared, prove, can determine the concentration (Figure 20 C) of sample composition by detected molecular number and typical curve in the comparative sample.In addition, the concentration of measuring by molecule counting is very consistent with the concentration that the undiluted stock solution that is used to prepare sample by extensive spectroscopic assay records.

Embodiment 14. is used to measure the biologicall test of single-particle character

The mensuration that is usually used in biomone comprises sandwich ELISA mensuration, when can detecting and catch and detect two epi-positions of antibodies, this method exists, but they are only limited to two epi-positions usually, lack sensitivity (being generally pM level or higher), and can not detect the particle that has only an epi-position.FRET (fluorescence resonance energy transfer) (FRET) method is used in competition assay usually, exists when also detecting two epi-positions, also lacks sensitivity.Mass spectroscopy need cut into macroparticle usually to be had enough volatile fragment and analyzes, and in whole sample colony modification is averaged.

SMD analyzer of the present invention provides the major advantage that can use in biologicall test: high sensitivity, can be individually rather than generally measure particle or molecular complex, differentiate particle based on electrophoretic velocity, and can in unitary determination, monitor a plurality of wavelength.These advantages detect the multiple electromagnetic signature of intended particle by analyzer and the unique function of its electrophoretic velocity of mensuration determines.

14A. sandwich assay

In heterogeneous mensuration form (Figure 21 A), contain the detection solution and the pearl reaction of using intended particle specific antibody bag quilt of intended particle.On pearl, catch target, the unconjugated material of flush away.Pearl-target compound then with the fluorescent marker incubation of specificity combining target, produce the sandwich body of mark.With the sandwich body of analyzer certification mark of the present invention, and measure its electrophoretic velocity.In homogeneous mensuration form (Figure 21 B), form the detection solution that contains pearl-target compound in the same manner, but do not remove unconjugated material.Utilize the electrophoretic velocity of sandwich body of independent target and label to distinguish them.

L4B. double-colored discriminating

Second kind use to be adopted and to measure form with homogeneous and detect two labels on the single target particle simultaneously.Use this method, unconjugated label not be attached to target on label separate.Sample can be in analyzer of the present invention electrophoresis, this analyzer is inquired after the place, space first and is had the detecting device that is used for first emission wavelength, inquires after the place, space second and has the detecting device that is used for second emission wavelength.Inquire after at two and to detect particle in the space, but only counting has the particle of the spectral fingerprint of two kinds of labels.Utilize different electrophoretic velocities to differentiate the label of unconjugated and combination.Figure 22 A shows an example of double-colored determination method.

Can utilize the homogeneous sandwich assay to measure the posttranslational modification pattern of single protein granule.Label reaction with protein granule and every kind of modified specificity of a plurality of potential decorating sites.Every species specificity label has unique fluorescence spectrum.For example, reaction mixture is moved by each a plurality of detecting device of inquiring after the space, the spectral fingerprint of record protein-marker complex (the photon ratio in the different wave length passage) (seeing Figure 26 B) by electrophoresis.In addition, can measure the electrophoretic velocity of various marked members.This chance owing to intended particle and unconjugated label in the single passage meets and has reduced background.Identified and identical particle combination that from the spectral fingerprint of multi-detector the label that therefore corresponding posttranslational modification takes place is right on single-particle.

Because this method obtains the data of single-particle, therefore the mensuration than the average modification level of protein colony provides more information.Average measurement can not be distinguished the protein of single and multiple modification.For example, by obtaining the method for the level of on average modifying, can't come having the potpourri of a kind of protein of modifying a kind of protein of 1 and having modification 2 and the protein of unmodified and the combination difference of protein with two kinds of modifications.Analyzer of the present invention can clearly be distinguished these particles.

The example of the modification that can analyze in this way comprises the glycosylation pattern of relatively reorganization and native protein, measure the phosphorylation level of a plurality of site of single protein, in proteolysis maturation and protein degradation, detect precursor and product simultaneously, the range protein that produces of the various combination by its constituent relatively, and the combination of modifying, relevant as range protein and phosphorylation state.

14C. measure in conjunction with agonist/antagonist

The third is applied in detecting the particle of two kinds of marks in the mensuration of the material that influences the label combination.Every kind of particle label composite marking of spectroscopy uniqueness.Have the particle of the spectral fingerprint of any one or two kinds of labels by counting, determine combination and mark unconjugated label under the situation of the antagonist that has activator or competition combination.Figure 22 B shows an example of this mensuration.The application of this mensuration comprises at it influences the screening of medicaments compound to what catalysis and regulatory enzyme subunit, nucleic acid and its transcription factor, acceptor and part and enzyme combined with its substrate.

14D. use the competitive assay of FRET

1.cAMP measure ring AMP (cAMP) dependant kinase

Under the situation that does not have cAMP to exist, there is catalysis (C) and regulates the non-activity tetramer (C2R2) of (R) subunit.When cAMP in conjunction with the time, the tetramer dissociates, and discharges active catalytic subunit.In mensuration to cAMP, can be with a pair of FRET fluorophore mark catalysis and regulator subunit.For example, can use donor fluorophore mark regulator subunit, with acceptor fluorophore mark catalytic subunit.Under the situation that does not have cAMP, donor and acceptor are closely close, and energy is transferred to acceptor from donor, the acceptor ballistic phonon.When having cAMP, donor and acceptor are closely close, and acceptor is ballistic phonon not.Analyzer of the present invention provides detection sensitivity for this technology that is generally used for large volume mensuration on the single-particle level.Figure 22 C shows an example of double-colored mensuration.

2. competitive part is in conjunction with mensuration

Can use donor and acceptor fluorophore labeled receptor (R) and part (L).When part and receptors bind, donor and acceptor are closely close, the acceptor ballistic phonon.When having unlabelled part (for example, in sample to be analyzed), can replace the part of mark from acceptor, donor and acceptor are no longer closely close, and acceptor is ballistic phonon not.Can produce calibration curve, this curve is associated the unmarked part of known quantity with the acceptor number of ballistic phonon.Can be according to the ligand level in the sample acceptor population sample estimates of calibration curve and ballistic phonon.Figure 22 D shows the example that simple FRET measures.

3. measure in conjunction with agonist/antagonist

As at competitive part in conjunction with in measuring, can use donor and acceptor fluorophore labeled receptor (R) and part (L).When part and receptors bind, donor and acceptor are closely close, the acceptor ballistic phonon.In the receptor-ligand potpourri, add potential sample in conjunction with activator or antagonist.Activator in the sample has increased the amount with the tagged ligand of receptors bind, and has increased the quantity of the acceptor particle of ballistic phonon.Antagonist in the sample has reduced the part number of ballistic phonon.In drug discovery and exploitation, can utilize this method at potential curative effect examination library of compounds.SMD method of the present invention is particularly useful in interacting at screening low concentration, high-affinity, because it has high sensitivity.Figure 22 E shows the example that competitive FRET measures.

4. enzyme velocity determination

Can utilize at the opposite side of cleavage site and measure hydrolytic enzyme activities with the substrate of quencher and acceptor particle mark.The fluorescence of intact substrate particle (quencher and acceptor are closely close) changes (quencher and acceptor are no longer close) when hydrolysis.For example, the peptide substrates that contains target protein enzyme restriction enzyme site can at one end be used the fluorescence quencher mark, at other end acceptor mark.Since closely near quencher, the photon that the emission of intact substrate peptide is few.The more photon of (product) peptide emission of cutting.Occurrence rate according to the peptide particle that cuts is determined percent hydrolysis.Figure 22 F shows the example that enzyme FRET measures.The advantage of SMD method of the present invention is and can measures enzymatic activity dynamics to single-particle, rather than the average activity of hundreds of or thousands of particles in total body measurement.

It will be apparent to those skilled in the art that analyzer of the present invention also can be used for the wherein similar mensuration of binding partners usefulness different colours fluorophore mark.

Embodiment 15. is used to detect the labelling strategies of single goal particle

It should be recognized by those skilled in the art that and to use many policy tag intended particles to make it can be detected in particles mixture or differentiate.Label can connect with any known method, comprises the interactional method of non-specific or specificity of usage flag thing and target.Label can provide detectable signal or influence the mobility of particle in electric field.In addition, can directly or by binding partners realize mark.It below is the example of operable labelling strategies in the present invention.

15A. single-particle detects (seeing Figure 23):

Can be with a plurality of copies (Figure 23 B) label particles of a plurality of copies (Figure 23 A) of a kind of dyestuff, a kind of dyestuff, two kinds of dyestuffs or two kinds of dyestuffs, based on different emissive porwers and/or emission wavelength can detect this particle and with unconjugated label difference.

15B. the electromagnetic signature according to more than one particles detects and differentiates (referring to Figure 24)

Based on emissive porwer, can be with a kind of particle of a plurality of copy marks of dyestuff and with the particle difference of the less copy number mark of identical dyestuff come (Figure 24 A).According at two wavelength, rather than the emission at a wavelength place, can will distinguish come (Figure 24 B) with the particle of two kinds of dye markers and particle only with a kind of dye marker.By measuring the different proportion of two kinds of dyestuffs, can will distinguish come (Figure 24 C and D) with the particle of one or more copy marks of two kinds of dyestuffs and particle with the less copy number mark of two kinds of dyestuffs.Based on their emission wavelength, the difference (Figure 24 E) of the fluorescence total intensity of sending according to each particle can be distinguished the particle with one or more copy marks of two kinds of dyestuffs with different fluorescence intensities.Based on their emission spectrum and/or electrophoretic velocity, with dyestuff and influence electrophoretic velocity the label mark particle can with only distinguish come (Figure 24 F) with the particle that moves the label mark.Based on the difference of the electromagnetic signature of two kinds of dyestuffs, can will distinguish come (Figure 24 G and H) with a kind of particle of one or more copy marks of dyestuff and particle with one or more copy marks of different dyes.

15C. the electrophoretic velocity according to more than one particles detects and differentiates (seeing Figure 25)

Based on they different electrophoretic velocities, can will distinguish come (Figure 25 A) with the particle of the label mark that influences electrophoretic velocity and with the particle of the different label marks that influence electrophoretic velocity.Based on their emission spectrum and/or electrophoretic velocity, the particle with dye marker can be distinguished come (Figure 25 B) with the intrinsic detectable particle with the label mark that influences electrophoretic mobility.

Embodiment 16. differentiates two kinds of particles according to the characteristic strength of fluorescent emission

The ability of high sensitivity and single detection particle is an advantage in analysis list particle modification level.For example, the protein that can will degrade with multiple ubiquitin mark.In sample, add the fluorescent marker that is used for ubiquitin, make it to combine, and utilize electric power to make it pass through detecting device with target.Electrophoretic velocity is different from the label of combination, and is proportional for ubiquitin reference numerals on the photon number of each detection of particles and the protein.Therefore, this mensuration provides the information that distributes about ubiquitin number of labels on each simple protein particle, rather than average.Figure 26 A and B show the example of possible experiment.

Embodiment 17. fluorescence polarizations (FP) are measured

Can use the fluorophore label particles.When using the particle of polarized light excitation labeling, the light that sends also can be polarized.The polarisation of light degree of sending is the function of the fluorescence lifetime of the mobility of label and fluorophore.When the particle of mark combined with big acceptor particle, its mobility reduced, and its polarization strengthens.In comprising competitive part combination, substrate combination and the multiple mensuration such as enzymatic activitys such as protein kinases, the electric charge that can utilize label-particle-acceptor centering is as detection system.

Conventional FP measures the polarization of all samples particle in the detection of aggregation thing, has limited dynamic range.(P is the polarization unit that calculates by the fluorescence intensity of measuring parallel with excitation plane (F1) and vertical (F2) to the actual range of polarization value normally about 10 to about 300mP in the mensuration, and be difficult to detect the change of the arbitrary end value of this scope P=(F1-F2)/(F1+F2) wherein).SMD analyzer of the present invention is once counted a particle for having high or low polarization, rather than average polarization is provided.Figure 27 shows the example that detects by fluorescence polarization.

Others

Above-described detailed description is in order to help those skilled in the art to implement the present invention.Yet, this description and the present invention of asking for protection on scope, be not subjected to specific embodiments disclosed herein and/or aspect restriction because these embodiments and aspect are the explanations of several embodiments of the present invention and aspect.Anyly be equal to embodiment and/or aspect also within the scope of the invention.In fact, except shown in this paper and describe, according to top description, various changes of the present invention will be conspicuous for those skilled in the art, and not deviate from the spirit or scope of discovery of the present invention.These changes also drop within the scope of claim.

When introducing key element of the present invention or its preferred aspect, " one " and " described " meaning is to have one or more key elements.Term " comprises ", " comprising " and " having " comprise, and the meaning is the key element that also may have other except listed key element.

The list of references of quoting

All publications of quoting among the application, patent, patented claim and other list of references all are incorporated herein by reference in full, specifically and respectively are incorporated herein by reference as each independent publication, patent, patented claim or other list of references.This paper should not be construed as quoting of list of references and admits that these are prior aries of the present invention.

Although this paper shows and has described the preferred embodiments of the invention, it will be apparent to one skilled in the art that what these embodiments just provided as an example.Those skilled in the art can expect a large amount of variations, change and alternative, and do not deviate from the present invention.Should be appreciated that the various replacement schemes that to use embodiment of the present invention described herein in the embodiment of this invention.Following claim is intended to limit scope of the present invention, therefore covers method and structure and equivalent thereof in these claim scopes.

Claims (80)

1. single-particle analyzer system comprises:

(a) can gather a plurality of samples and sampling container and first automatically and inquire after the sampling system that the space fluid is communicated with; With

(b) can detect the analyzer of single-particle, comprise:

(i) be used to send the electromagnetic radiation source of electromagnetic radiation;

(ii) be set to accept first of electromagnetic radiation that described electromagnetic radiation source sends and inquire after the space;

(iii) be set to accept second of electromagnetic radiation that described electromagnetic radiation source sends and inquire after the space; Wherein said second inquires after space and described first inquires after the space fluid and is communicated with, and wherein inquires after space and second first and inquire after and have motive power between the space, causes particle can inquire after in first space and second between the space and moves;

(iv) inquire after first electromagnetic radiation detector that the space can be operatively connected, be used to measure first electromagnetic signature of particle with first;

(v) inquire after second electromagnetic radiation detector that the space can be operatively connected, be used for measuring at least one of second electromagnetic signature of particle and first electromagnetic signature of particle with second.

2. the analyzer system of claim 1 further comprises with second and inquires after the sample retrieval system that the space fluid is communicated with, and this sample retrieval system can reclaim all described samples basically.

3. the analyzer system of claim 2 further comprises sample preparation system.

4. the analyzer system of claim 3, wherein said sample preparation system carries out specimen preparation, that described specimen preparation is selected from is centrifugal, filtration, chromatography, lysis, change pH, add damping fluid, add reagent, heating or cooling, irradiation, interpolation label, label in conjunction with, separate unconjugated label and their combination.

5. the analyzer system of claim 1 further comprises the data analysis system of analyzing described first and second electromagnetic signatures and reporting described analysis result.

6. the analyzer system of claim 1, wherein said electromagnetic radiation source is the continuous wave electromagnetic radiation source.

7. the analyzer system of claim 6, wherein said continuous wave electromagnetic radiation source is selected from light emitting diode and continuous wave laser.

8. the analyzer system of claim 1, the carry-over rate of wherein said sampling system is less than about 0.02%.

9. the analyzer system of claim 1 wherein first and second is inquired after the space and is had the volume of about 0.02pL to about 300pL separately.

10. the analyzer system of claim 1 wherein first is inquired after at least one of inquiring after in the space in space and second and is had the volume of about 0.05pL to about 50pL.

11. the analyzer system of claim 1 wherein first is inquired after space and second and is inquired after in the space at least one and have the volume of about 0.1pL to about 25pL.

12. the analyzer system of claim 1, wherein first and second at least one the volumes of inquiring after in the space are adjustable.

13. the analyzer system of claim 1 further comprises at least and first inquires after space and second and inquire after the 3rd electromagnetic radiation detector that one of space can be operatively connected, and is used for measuring at least one of first electromagnetic signature of particle and second electromagnetic signature of particle.

14. the analyzer system of claim 1, wherein said motive power comprises pressure.

15. the analyzer system of claim 13, wherein said pressure is provided by the source that is selected from pump, vacuum source, hydro-extractor and their combination.

16. the analyzer system of claim 14, wherein fluid is communicated with pipeline or the passage that comprises in the microfluidic devices, and wherein said pressure is provided by one or more pumps.

17. the analyzer system of claim 5, wherein said analysis comprise the existence of determining particle, the concentration that does not exist and choose wantonly, and based on described existence, do not exist and/or concentration is determined possible diagnosis, prognosis, therapeutic state or recommended therapy.

18. an analyzer system comprises:

(a) sampling container and first is inquired after the sampling system that fluid is communicated with between the space;

(b) comprise that described first inquires after the single-particle analyzer that the space is inquired after in space and second, wherein said second inquires after space and first inquires after the space fluid and is communicated with, and inquire after space and second first and inquire after and have motive power between the space, cause particle to inquire after space and second and inquire after between the space and move first;

(c) inquire after the detecting device that the space can be operatively connected with described first and/or described second, be used for when there is detectable feature in particle, detecting this feature;

(d) sample retrieval system can be from sampling receptacle to inquiring after spatial movement and return sampling receptacle by this system's sample, and do not contact other parts of this analyzer, and do not contact the cleaning buffer in this analyzer basically; With

(e) data-analyzing machine, it receives the input of self-detector, and analyze the existence of particle or do not exist, and based on described existence or do not exist and report the result.

19. the analyzer system of claim 18 further comprises sample preparation system.

20. a single-particle analyzer system comprises:

(a) can gather a plurality of samples and sampling container and first automatically and inquire after the sampling system that the fluid between the space is communicated with; With

(b) can detect monomolecular analyzer, comprise:

(i) be used to send the electromagnetic radiation source of electromagnetic radiation;

(ii) be set to receive described first of electromagnetic radiation that described electromagnetic radiation source sends and inquire after the space; With

(iii) inquire after first electromagnetic radiation detector that the space can be operatively connected, be used to measure first electromagnetic signature of particle with first.

21. analyzer system, comprise analyzer, when in this analyzer, adding first sample and second sample, described first sample of adding analyzer and the volume of described second sample are less than 5 μ l, and when the concentration of analyte flew mole less than 50 in described first and second samples, this analyzer can detect between first sample and second sample analyte concentration difference less than 20%.

22. a single-particle analyzer comprises:

At least one is used to send the continuous wave electromagnetic radiation source of electromagnetic radiation;

Be set to receive first of electromagnetic radiation that described electromagnetic radiation source sends and inquire after the space, this first volume of inquiring after the space is about 0.02pL about 300pL extremely;

Be set to receive second of electromagnetic radiation that described electromagnetic radiation source sends and inquire after the space, this second volume of inquiring after the space is that about 0.02pL is to about 300pL, wherein second inquire after space and first and inquire after the space fluid and be communicated with, and inquire after space and second first and inquire after and have electromotive force between the space, cause and use electric power can make particle inquire after space and second to small part to inquire after between the space and move first;

With first inquire after first electromagnetic radiation detector that the space can be operatively connected, be used to measure first electromagnetic signature of particle; With

With second inquire after second electromagnetic radiation detector that the space can be operatively connected, be used for measuring at least one of second electromagnetic signature of particle and first electromagnetic signature of particle.

23. according to the analyzer of claim 22, further comprise at least and first inquire after space and second and inquire after the 3rd electromagnetic radiation detector that one of space can be operatively connected, be used for measuring at least one of first electromagnetic signature of particle and second electromagnetic signature of particle.

24. according to the analyzer of claim 22, wherein said continuous wave electromagnetic radiation source is selected from light emitting diode and continuous wave laser.

25., wherein at least the first inquire after the volume of inquiring after one of space in space and second and be about 0.1pL about 25pL extremely according to the analyzer of claim 22.

26. according to the analyzer of claim 22, wherein at least the first and second volumes of inquiring after one of space are adjustable.

27., wherein at least the first inquire after space and second and inquire after one of space and limit by one of the cross-sectional area of the electromagnetic radiation beam that receives from electromagnetic radiation source and sensing range of one of at least the first electromagnetic radiation detector and second electromagnetic radiation detector at least according to the analyzer of claim 22.

28. the analyzer of claim 27, wherein said sensing range is determined by the gap width in adjacent with one of first electromagnetic radiation detector and the second electromagnetic radiation detector at least spatial filter.

29. according to the analyzer of claim 22, wherein at least the first and second inquire after one of space and limited by housing at least in part, this housing comprises and is selected from glass, quartz, melts the solid material that coagulates quartzy, plastics or its combination in any.

30., wherein at least the first inquire after space and second and inquire after one of space and limit by fluid boundary at least in part according to the analyzer of claim 22.

31. according to the analyzer of claim 22, wherein one of at least the first electromagnetic radiation detector and second electromagnetic radiation detector are selected from CCD camera, video input module camera, streak camera, bolograph, photodiode, photodiode array, avalanche photodide detecting device, photomultiplier detector and combination in any thereof.

32. according to the analyzer of claim 22, further comprise one of pump, vacuum source and hydro-extractor at least, be used to promote particle to inquire after space and second and inquire after moving between the space first.

33. an analytical approach comprises and uses the single-particle analyzer system to measure the existence of particle in the sample that obtains or do not exist from individuality, this single-particle analyzer system comprises:

(a) can gather a plurality of samples and sampling container and first automatically and inquire after the sampling system that the fluid between the space is communicated with;

(b) can detect the analyzer of single-particle, this analyzer comprises:

(i) be used to send the electromagnetic radiation source of electromagnetic radiation;

(ii) be set to receive first of electromagnetic radiation that described electromagnetic radiation source sends and inquire after the space;

(iii) be set to receive second of electromagnetic radiation that described electromagnetic radiation source sends and inquire after the space; Wherein second inquire after space and first and inquire after the space fluid and be communicated with, and inquire after space and second first and inquire after and have motive power between the space, cause particle to inquire after space and second and inquire after between the space and move first;

(iv) inquire after first electromagnetic radiation detector that the space can be operatively connected, be used to measure first electromagnetic signature of particle with first;

(v) inquire after second electromagnetic radiation detector that the space can be operatively connected, be used for measuring at least one of second electromagnetic signature of particle and first electromagnetic signature of particle with second.

34. the method for claim 33, wherein said analyzer further comprise the data analysis system of analyzing described first and second electromagnetic signatures and reporting described analysis result.

35. the method for claim 34 further comprises based on described analysis result and determines diagnosis, prognosis, therapeutic state and/or methods of treatment.

36. the method for claim 33, wherein said analyzer system further comprise with second inquire after that the space fluid is communicated with, can reclaim the sample retrieval system of whole described samples basically.

37. the method for claim 33, wherein said analyzer system further comprises sample preparation system.

38. the method for claim 33, wherein said electromagnetic radiation source are the continuous wave electromagnetic radiation sources.

39. the method for claim 33, wherein said first and second inquire after the space has the volume of about 0.02pL to about 300pL separately.

40. the method for claim 33 wherein at least the first is inquired after space and second and is inquired after one of space and have about 0.05pL to the volume of about 50pL.

41. the method for claim 33 wherein at least the first is inquired after space and second and is inquired after one of space and have about 0.1pL to the volume of about 25pL.

42. the method for claim 33, wherein at least the first and second volumes of inquiring after one of space are adjustable.

43. the method for claim 33, wherein said motive power comprises pressure.

44. the method for claim 43, wherein said pressure is provided by the source that is selected from pump, vacuum source, hydro-extractor and their combination.

45. the method for claim 33, wherein said individuality is an animal or plant.

46. the method for claim 45, wherein said individuality is an animal.

47. the method for claim 46, wherein said individuality is a mammal.

48. the method for claim 47, wherein said individuality is the people.

49. the method for claim 33 comprises the multiple particle in the sample is analyzed.

50. the method for claim 49, every kind of detected in wherein said multiple particle particle comprises label, and every kind of wherein detected particle is distinguished with other particle mutually according to being selected from label identity, label intensity, mobility or its combined feature.

51. the method for claim 33, wherein said sample is selected from blood, serum, blood plasma, bronchoalveolar lavage fluid, urine, cerebrospinal fluid, hydrothorax, synovia, ascites, amniotic fluid, gastric juice, lymph liquid, interstitial fluid, tissue homogenate, cell extract, saliva, phlegm, ight soil, physiology secretion, tears, mucus, sweat, milk, seminal fluid, seminal fluid liquid, vaginal fluid, from ulcer and other surperficial rash, the liquid of blister and abscess and comprise normal, the extract of the tissue in pernicious and suspection is organized in, any other composition that maybe may contain the health of particle.

52. the method for claim 51, wherein said sample is selected from blood, blood plasma or serum.

53. the method for claim 52 further comprises the particle in the described sample of mark, wherein analyzes described sample and comprises the existence of the particle that detects described mark or do not exist.

54. the method for claim 53 further comprises and remove unconjugated label from described sample.

55. the method for claim 33 further comprises obtaining described sample from described individuality.

56. the method for claim 53, wherein said particle is selected from protein, nucleic acid, nanosphere, microballoon, dendrimer, chromosome, carbohydrates, virus, bacterium, cell and combination in any thereof.

57. the method for claim 53, wherein said particle is selected from protein, nucleic acid, virus, fungi, bacterium and combination in any thereof.

58. the method for claim 53, wherein said particle are selected from amino acid, nucleotide, lipid, sugar, granule toxin, peptide toxin, venom, medicine and combination in any thereof.

59. the method for claim 52, wherein said sample are the blood serum samples that contacts with fluorescently-labeled intended particle specific antibody; And wherein said analysis comprises the existence of the particle of certification mark, does not exist and/or concentration.

60. the method for claim 59 further comprises based on the existence of the particle of mark, does not exist and/or concentration is determined diagnosis, prognosis, therapeutic state and/or methods of treatment.

61. the method for claim 60 further comprises to individuality and reports described diagnosis, prognosis, therapeutic state and/or methods of treatment.

62. the method for claim 60, wherein said biomarker is TREM-1.

63. the method for claim 62, wherein said method is finished in less than 1 hour.

64. the method for claim 60, wherein said definite diagnosis, prognosis, therapeutic state and/or methods of treatment based on the existence of one group of biomarker, do not exist and/or concentration.

65. the method for claim 59, wherein said method is finished in less than 2 hours.

66. analytical approach, comprise existence, do not exist and/or concentration is determined diagnosis, prognosis, therapeutic state and/or methods of treatment based on particle from the sample that individuality obtains, wherein use a kind of analyzer system that can detect monomolecular analyzer that comprises to determine described existence, do not exist and/or concentration, wherein said analyzer comprises that at least one inquires after the space.

67. the method for claim 66, wherein said analyzer comprises that at least two are inquired after the space.

Can detect monomolecular analyzer 68. the method for claim 66, wherein said analyzer system comprise, this analyzer comprises that at least one is used to send the continuous wave electromagnetic radiation source of radiation, and wherein at least one is inquired after the space and is set to receive described radiation.

69. one kind exists disease and the method for examination individuality in order to determine whether, comprise that the analysis of operational analysis device is from one or more disease markers in the sample of individuality, when first sample and second sample add in this analyzer, the volume that adds interior described first sample of analyzer and described second sample is less than 5 μ l, and when the concentration of one or more labels flew mole less than 50 in described first and second samples, the concentration that this analyzer can detect one or more labels between first sample and second sample was less than 20% difference.

70. the method for claim 69 further comprises described analysis result and known label value are compared.

71. the method for claim 69, wherein said individuality is the smoker, and described cancer is a lung cancer.

72. a method that detects particle comprises:

By electric power particle being moved to volume inquires after in the space and volume is extremely second inquiring after in the space of about 300pL of about 0.02pL for about 0.02pL to first of about 300pL;

To at least one continuous wave electromagnetic radiation source of sample application;

Inquire after when interacting in the space first when particle and continuous wave electromagnetic radiation, inquire after first electromagnetic signature of measuring particle in the space first; With

When particle and continuous wave electromagnetic radiation are inquired after when interacting in the space second, inquire after one of first electromagnetic signature of measuring particle in the space at least and second electromagnetic signature second.

73. the method for claim 72, wherein said particle are first particles, described method further comprises:

Making second particle move to first inquires after space, second and inquires after space, the 3rd and inquire after space and the 4th and inquire after in the space at least two; With

Inquire after space, second when second particle and continuous wave electromagnetic radiation first and inquire after space, the 3rd and inquire after when interacting in space and the 4th at least one of inquiring after in the space, measure one of first electromagnetic signature of second particle and second electromagnetic signature of second particle at least.

74. a computer-readable recording medium comprises the one group of instruction that is used to have user interface, comprises the multi-purpose computer of display unit, this group instruction comprises:

(a) be used to import with logic with value that two single-particle detecting device analytic samples of inquiring after the space are obtained; With

(b) be used for showing input value result's display routine with described display unit.

75. the computer-readable recording medium of claim 74, wherein said instruction further comprises the comparison program that is used for comparison input value and database; And wherein said display routine further comprises the logic that is used to show the comparison program result.

76. electric signal or the carrier wave between computing machine, propagated by the internet, comprise the one group of instruction that is used to have user interface, comprises the multi-purpose computer of display unit, this group instruction comprises computer-readable storage medium, comprising being used to have user interface, comprising one group of instruction of the multi-purpose computer of display unit, this group instruction comprises:

(a) be used to import with logic with value that two single-particle detecting device analytic samples of inquiring after the space are obtained; With

(b) be used for showing input value result's display routine with described display unit.

77. the signal of claim 76 or carrier, wherein this group instruction further comprises the comparison program that is used for comparison input value and database; And wherein said display routine further comprises the logic that is used to show the comparison program result.

78. a method of carrying out business activity comprises by entity and uses two detecting devices of inquiring after the space that have can detect single-particle to obtain the sample determination results, reports described result, and to the expense of this entity pays result report.

79. being clinical labororatories, the method for claim 78, wherein said entity improve correction (CLIA) laboratory.

80. the method for claim 78, wherein said entity are not the CLIA laboratories.

Images