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WO2005119265A1 - Detection de proteines - Google Patents

Detection de proteines Download PDF

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Publication number
WO2005119265A1
WO2005119265A1 PCT/GB2005/002153 GB2005002153W WO2005119265A1 WO 2005119265 A1 WO2005119265 A1 WO 2005119265A1 GB 2005002153 W GB2005002153 W GB 2005002153W WO 2005119265 A1 WO2005119265 A1 WO 2005119265A1
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Prior art keywords
protein
sample
analyte
ligands
ligand
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PCT/GB2005/002153
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English (en)
Inventor
David Klenerman
Haitao Li
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Cambridge University Technical Services Ltd CUTS
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Cambridge University Technical Services Ltd CUTS
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Publication of WO2005119265A1 publication Critical patent/WO2005119265A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the present invention relates generally to methods and materials for detecting or quantifying protein analytes in a solution.
  • the present inventors have used two colour fluorescence coincidence detection to directly count .individual protein analytes (both free in solution, and present as structural components e.g. in viruses). This allowed quantitative measurement of protein analytes at the femtomolar level in complex media such as serum, the relationship being linear over three orders of magnitude .
  • Fig. 1A One embodiment of the invention is illustrated in Fig. 1A.
  • the protein analyte is labelled with red-excited and blue- excited antibodies. Coincidence bursts of fluorescence are detected only in cases where a target molecule labeled with both a red and blue-excited antibody diffuses into the probe volume. In contrast only single color events (red or blue) will be detected for molecules labeled only with antibodies of a single color or unbound antibodies. At the low concentrations used there is a low probability of a red and blue excited antibody entering the probe volume at the same time. This constitutes a statistical background of coincident events below which it is not possible to detect the target.
  • the background from impurities is also significantly reduced, by two orders of magnitude relative to a single color experiment due to the low probability of an impurity fluorescing when red-excited and blue excited simultaneously, owing to their wide spectral separation.
  • the method of the invention enables the measurement to be performed in the presence of complex media such as serum.
  • the sample preparation methods of the invention are simple, since detection takes place in solution and requires no prior separation.
  • the invention thus provides general methods to detect and quantitate proteins, and to characterize macromolecular complexes, with applications in ultra-sensitive proteomics and clinical diagnostics .
  • a method for detecting a protein analyte in a sample comprising the steps of: (i) providing a sample solution, (ii) providing at least two ligands capable of binding specifically to the protein analyte, each ligand being labelled with a different fluorophore, which fluorophores are excitable by spectrally separated wavelengths of light, (iii) combining the sample solution and the ligands to form a detection sample such that protein analyte if present is labelled with the at least two ligands, and wherein each ligand is present at less than 1 nM, (iv) illuminating the sample solution with at least two coherent light beams having wavelengths capable of exciting the fluorophores, which beams coincide to form a coincident probe volume within the sample solution,
  • step (v) detecting the number of coincident fluorescent events within the coincident probe volume over a period of time, (vi) optionally using the result from step (v) to quantify the protein analyte in the sample solution.
  • each ligand is present at less than 500, less than 100 or most preferably less than 50 pM.
  • V eff the effective focus volume
  • the method permits molecule by molecule direct and quantitative counting of ligand-protein complexes in the coincident probe volume, and hence permits the quantification of the protein analyte in the sample solution. Since the number of protein analytes is counted directly, the measurement is quantitative with a large dynamic range.
  • step (vi) may comprise comparing the result from step (v) with a calibration curve generated using a known range of concentrations of analyte in the presence of ligands at the concentration given in step (iii) .
  • step (vi) may comprise repeating steps (iii) to (v) with a series of lower ligand concentrations and comparing the results from step (v) with respective calibration curves generated using a known range of concentrations of analyte in each of said ligand concentrations .
  • protein analyte an analyte it is desired to detect, which contains, consists essentially of, or consists of a protein (including lipoproteins and glycoproteins e.g. an enzyme; hormone; toxin; receptor or structural protein) .
  • the invention may be practised on any such analyte for which specific ligands can be made available.
  • the protein analyte may be an individual protein, or may be part of a target complex or other structure, for example a virus or bacterium containing one or more proteins (e.g. envelope proteins) .
  • Other targets may be pathological protein aggregates e.g. prions, amyloid, particularly small size nucleation aggregates, and so on.
  • the ligands can bind to the same or different proteins or epitopes of the single target - provided only that the single target molecule or complex is able to bind the two or more ligands.
  • the target provides more than one of the same epitope or protein
  • the ligands will be specific for different parts of the target so that they do not sterically interfere and compete for binding sites. This can also improve the specificity of the method by reducing the likelihood that a non-target analyte will bind both of the ligands.
  • the analyte complex may be constituted by two interacting entities e.g. two proteins, each labeled with one of the ligands. This embodiment may be used e.g. in proteome research .
  • the present invention is capable of sensitive detection without the need for any separation or amplification steps.
  • the ligands may be added directly to the sample solution to form the detection sample.
  • the ligands used in the present invention may be labelled directly with the fluorophore, or may optionally be labelled via a secondary ligand - this can increase the intensity of labelling, hence enhance the signal to noise, and also makes the method very general and easily applicable.
  • Preferred ligands are antibodies. Each antibody may be monoclonal or polyclonal. It may also be derived from a monoclonal antibody by expressing all or part of the nucleic acid encoding therefore in a suitable host cell such as to produce a polypeptide comprising all or part of the antigen binding site of the original antibody. Such antibodies and derivatives can be raised using any techniques commonly used in the immunology art (see e.g. Roitt et al in '"Immunology 5th edition" - Pub. 1997 by Moseby International Ltd, London) .
  • antibody as used herein should be construed as covering any specific binding substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or synthetic. Chimaeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of Chimaeric antibodies are described in EP-A- 0120694 and EP-A-0125023.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VI and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S.
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804) .
  • Other ligands include aptamers .
  • the present invention may include the step of providing ligands such as antibodies and optionally labelling them.
  • ligands In preferred embodiments only 2 ligands are used. However, it will be appreciated that the invention is also applicable to the use of greater numbers of ligands if that is desired e.g. 3 ligands, each distinctively labelled.
  • Suitable fluophores which can be used to label the ligands (e.g. via functional groups), can be selected from those known in the art, and widely available commercially.
  • the fluorophores are excitable by spectrally separated wavelengths of light - thus preferred combinations of fluorophores will preferably include a red or blue excited fluorophores, preferably both.
  • Example fluorophores used herein include Alexa Fluor® 488 N- hydroxylsuccinimide ester, Alexa Fluor® 647 N-hydroxyl- succinimide ester from Molecular Probes Europe BV (Leiden, The Netherlands) .
  • Other suppliers are well known to those skilled in the art e.g. Amersham Pharmacia.
  • the inventors used 488 and 632 nm excitation- 144 nm separations. These sources are readily available and may be used with a dichroic mirror that reflects both wavelengths while transmiting fluorescence .
  • Detection may occur within a fixed probe volume of the detection sample, the system being based on diffusion of labelled analyte into this probe volume.
  • the detection limits of the invention may be further improved by relative movement between the detection sample and the illumination device (s) e.g. by use of a flow cell or use of a laser scanner 6 .
  • the encounter rate of the labelled analyte with the probe volume is increased.
  • step (v) may be performed wherein the detection sample is passed or flowed through a coincident probe volume defined by the coincidence of the beams .
  • step (v) may be performed wherein a coincident probe volume defined by the coincidence of the beams is scanned within the detection sample.
  • the invention is used in combination with a microfluidic device for sample preparation and flow, using the methods of the invention for in situ detection.
  • the bin time used will be determined by the length of time it takes for a molecule on average to diffuse through the probe volume. In examples used herein this was of the order of 1 ms, so the signal was generally integrated over a slightly longer time - 5 ms .
  • the length of time used for counting may be dictated by the concentration of labelled analyte and required accuracy.
  • a threshold setting may be used in order to distinguish true events from background as discussed above, with a high threshold setting generally requiring a longer count time to make an accurate measurement.
  • calibration experiments performed with samples of known concentration under the same conditions as the detection sample may be used to ensure consistency and permit quantification.
  • the detecting of coincident fluorescent events can be performed using conventional equipment e.g. using 2 different detector devices appropriate to the respective emissions of the fluorophores .
  • samples may include CSF, saliva, sweat, urine, blood, faeces etc.
  • Another application is the assessment of pathogens or pollutants in soils, water, plants and other matrices.
  • the invention provides a method as discussed above where the sample is a biosample of an organism, tissue, cell or body fluid. It further provides a method of diagnosis or prognosis, in an individual, of a disease which is associated with a protein analyte, which method comprises taking a biopsy sample from the individual and detecting or quantifying the analyte using a method as described above.
  • the invention further provides a method of assessing infection or contamination of an environment, wherein the infection or contamination is associated with a protein analyte, which method comprises taking a sample from the environment and assessing the analyte using a method as described above.
  • the methods of the invention may also be employed to allow the detection and identification and also the measurement of protein-protein, protein-DNA and protein-RNA interactions.
  • the methods of the invention may be employed to study the proteome e.g. measuring the amount of a specific protein from a collection of cells.
  • Another embodiment permits the study of the interactions of low abundance proteins and proteins that are expressed at low levels, whereby labelled, interacting, proteins can be detected as coincident events . This may useful where there is interest in understanding the interaction between proteins .
  • kits or other components for use in carrying out the method of the present invention. These may include any one or more of the following:
  • At least two ligands capable of binding specifically to a protein analyte each ligand being labelled with a different fluorophore, which fluorophores are excitable by spectrally separated wavelengths of light
  • Sodium hydrogen carbonate (NaHC0 3 ) , hydroxylamine, protein G, bovine serum albumin (BSA) , and rabbit immunoglobulin G (IgG, 95%) were all purchased from Sigma-Aldrich Company (Dorset, UK) .
  • Alexa Fluor® 488 N-hydroxylsuccinimide ester, Alexa Fluor® 647 N-hydroxyl-succinimide ester was both purchased from Molecular Probes Europe BV (Leiden, The Netherlands) .
  • PBS (10 mM phosphate, 150 M NaCl, 2 mM NaN 3 , pH 7.2), NaHC0 3 (0.1 M, pH ⁇ 8.3), and hydroxylamine-HCl (1.5 M, pH 8.5) buffers were all prepared using ultra-pure MilliQ water (resistance > 18 M ⁇ .cm).
  • rabbit IgG was dissolved in the 0.1 M NaHC0 3 buffer (pH 8.3) to obtain a concentration of 5 mg/mL, then 1 mL of the freshly prepared IgG solution was added to a vial of 1 rug Alexa Fluor® 488 N- hydroxylsuccinimide ester. After the dye was thoroughly dissolved and mixed with the protein, the resulting solution was allowed for gentle magnetic stirring for 1 hr at room temperature. After which the labeling reaction was terminated by addition of 100 ⁇ l of the 1.5 M hydroxylamine-HCl.
  • the resulting mixture was then loaded on a purification column using Bio-Rad BioGel P-30 Fine size exclusion purification resin, and the PBS buffer was used as the eluting buffer.
  • the first yellowish fluorescent band was collected which was the labeled rabbit IgG.
  • Alexa 647 labeled rabbit IgG was prepared by the same procedure, except the IgG solution was added to a vial of Alexa Fluor® 647 N-hydroxylsuccinimide ester.
  • the average labeling, detected by measuring the UV absorbance at 280 and 650 nm was 8 fluorophores per IgG molecule .
  • HSV-1 Herpes Simplex Virus Type 1
  • HFEMdelUL22Z 10 Herpes Simplex Virus Type 1
  • Tissue culture medium from infected cells was clarified by centrifugation at 2000 x g for 10 minutes, and virus particles were then pelleted from the supernatant by centrifugation at 18,000 rp for 2 h in a Beckman type 19 rotor at 4 °C. The pellets were resuspended in a small volume of PBS and sonicated before being layered on 30ml 15-30% Ficoll gradients in PBS.
  • the gradients were centrifuged at 12,500 rpm for 90 minutes in a Beckman SW28 rotor at 4 °C, and the visible band at the centre of the gradient was harvested, diluted with PBS and pelleted by centrifugation at 21,000rpm in an SW28 rotor. The final pellet was resupended in PBS, and aliquots were stored at -70 °C.
  • Virus particle numbers were estimated by comparison with latex particles of known concentration using negatively stained preparations as described by Watson et al. 12 LP2 is a mouse monoclonal antibody, which recognizes the HSV envelope glycoprotein, gD.
  • IgG was purified from the tissue culture supernatant from hybridoma cells producing this antibody by immunoaffinity chromatography on a Protein A sepharose column and eluting the IgG with 0. IM glycine pH3. IgG concentrations were determined by measuring the optical density at OD 280. This antibody was labeled by Alexa 647 or Alexa 488 by the same labeling procedure as above. The average labeling numbers are 12 and 14 respectively.
  • APD avalanche photodiode
  • Red fluorescence was also filtered by long-pass and band-pass filters (565ALP and 695AF55, Omega Optical Filters) before being focused onto a second APD (SPCM AQR-141, EG&G, Canada) . Dark count rates for the two APDs were found to be below 100 counts per second. Outputs from the APDs were coupled to two PC implemented multi-channel scalar cards (MCS-Plus, EG&G, Canada) , the synchronous start output of one MCS card being used to trigger the second.
  • MCS-Plus multi-channel scalar cards
  • HSV-1 particles were diluted to 5 pM in PBS then 25 pM Alexa 488 labeled LP2- IgG and 25 pM Alexa 647 labeled LP2-IgG were added. This sample was further diluted for low concentration coincidence counting experiment. All experiments were carried on at room temperature. The excitation laser powers were the same as protein G experiment.
  • Protein G has up to three available binding sites for IgG with a sub- nanomolar dissociation constant. 14
  • Fig. IB Typical data obtained with both labeled antibodies and protein G at 50 pM concentration is shown in Fig. IB, where coincident events are marked with asterisks. Not all the events are coincident due to two reasons. Firstly single labeled protein G and free antibodies are also present. Secondly the overlap between the red and blue excited probe volumes is imperfect, 30% in these experiments, so some molecules diffuse through a region that is just excited by the red or blue laser only. 4 Note the good signal to noise, typically 50:1, on both channels for individual protein- antibody complexes .
  • Fig. 2C the concentration of IgG used was the same as protein G.
  • the number of coincident events is directly proportional to protein G concentration over three orders of magnitude.
  • the number of coincident events counted is significantly higher than the statistical background so that the sensitivity is only limited by the encounter rate of the protein G-IgG complex with the probe volume. In this case, relying only on diffusion, the limit is about 50 fM.
  • Example 2 detection of protein analytes in a virus
  • HSV Herpes Simplex Virus
  • the virus particle is approximately 120 nm in diameter, comprising an icosahedral nucleocapsid surrounded by a lipid bilayer embedded with multiple virus-specific membrane glycoproteins, including glycoprotein D (gD).
  • gD glycoprotein D
  • fluorophore labeled antibodies We found in this case that the binding was irreversible over the three hour measurement time. Experiments with antibodies only gave no detected coincidence events and hence a zero statistical background. This is because multiple antibodies bind to the virus, so a higher fluorescence threshold was used for coincidence detection, rejecting two differently labeled antibodies in the probe volume at the same time.
  • analyte samples of known concentrations e.g. as in protein G in Example 1
  • the analyte solutions are diluted, ensuring that sufficient time is permitted for equilibrium had been reached before measurement for these experiments. Different dilutions are assessed until the coincident events detected have reached the background level.
  • the calibration curve is then be repeated with a lower antibody concentration e.g. 10 pM, 1 pM , 100 fM to give a series of calibration curves at different fixed antibody concentrations .
  • the unknown sample is then run firstly at the highest fixed antibody concentration to determine if the number of coincident events is above background, in which case this number is determined and the amount of target read off the calibration curve. If the number was no higher than background then the experiment is repeated at the next lower antibody concentration until the coincident events are above background and this concentration can be determined.
  • the number of coincident events may be directly converted to a bound-analyte concentration by reference to the overlap probe volume and detection efficiency of coincident events which may be measured in separate calibration experiments.
  • the true concentration may then be determined from the dissociation constant or off-rate for the ligand using conventional thermodynamic equations.

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Abstract

L'invention concerne des méthodes et des nécessaires pour la détection d'un analyte protéique dans un échantillon, lesdites méthodes consistant : (i) à utiliser une solution échantillon, (ii) à utiliser au moins deux ligands (par exemple, des anticorps) pouvant se fixer spécifiquement à l'analyte protéique, chaque ligand étant marqué à l'aide d'un fluorophore différent, lesdits fluorophores pouvant être excités par des longueurs d'ondes de lumière spectralement séparées, (iii) à combiner la solution échantillon et les ligands afin de former un échantillon de détection de sorte que si l'analyte protéique est présent, il est marqué à l'aide desdits deux ligands, chaque ligand étant présent à une concentration inférieure à 1 nM,(iv) à éclairer la solution échantillon à l'aide d'au moins deux faisceaux lumineux cohérents présentant des longueurs d'ondes pouvant exciter les fluorophores, lesdits faisceaux coïncidant afin de former un volume sonde coïncidant dans la solution échantillon, (v) à détecter le nombre d'événements fluorescents coïncidants dans le volume sonde coïncidant sur une certaine durée. Le système de détection sensible peut être utilisé pour détecter des analytes à des concentrations très faibles, par exemple dans des liquides corporels.
PCT/GB2005/002153 2004-06-03 2005-06-01 Detection de proteines Ceased WO2005119265A1 (fr)

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GB0412410A GB0412410D0 (en) 2004-06-03 2004-06-03 Protein detection

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2456063A (en) * 2007-12-19 2009-07-08 Singulex Inc Optical scanning analyser for single molecule detection
US9040305B2 (en) 2004-09-28 2015-05-26 Singulex, Inc. Method of analysis for determining a specific protein in blood samples using fluorescence spectrometry
US9063131B2 (en) 2004-09-28 2015-06-23 Singulex, Inc. Methods and compositions for highly sensitive detection of molecules
EP3156799A1 (fr) * 2006-04-04 2017-04-19 Singulex, Inc. Analyseur et procédé hautement sensible de détection d'analytes

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19757740A1 (de) * 1997-12-23 1999-07-08 Evotec Biosystems Ag Verfahren zum Nachweis von Assoziations-, Dissoziations-, Verknüpfungs- oder Spaltreaktionen sowie Konformationsänderungen mittels Koinzidenzanalyse
US6200818B1 (en) * 1997-12-23 2001-03-13 Evotec Biosystems Ag Method for detecting reactions by means of coincidence analysis
WO2002008732A1 (fr) * 2000-07-20 2002-01-31 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procede et dispositif d"analyse de coincidence par fluorescence a deux photons polychrome
US6388746B1 (en) * 1998-02-06 2002-05-14 Cellectricon Ab Method, apparatus and flow cell for high-sensitivity detection of fluorescent molecules
WO2004011903A2 (fr) * 2002-07-25 2004-02-05 The Regents Of The University Of California Suivi d'interactions moleculaires par analyse de la distribution des intervalles entre temps d'arrivee de photons

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19757740A1 (de) * 1997-12-23 1999-07-08 Evotec Biosystems Ag Verfahren zum Nachweis von Assoziations-, Dissoziations-, Verknüpfungs- oder Spaltreaktionen sowie Konformationsänderungen mittels Koinzidenzanalyse
US6200818B1 (en) * 1997-12-23 2001-03-13 Evotec Biosystems Ag Method for detecting reactions by means of coincidence analysis
US6388746B1 (en) * 1998-02-06 2002-05-14 Cellectricon Ab Method, apparatus and flow cell for high-sensitivity detection of fluorescent molecules
WO2002008732A1 (fr) * 2000-07-20 2002-01-31 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procede et dispositif d"analyse de coincidence par fluorescence a deux photons polychrome
WO2004011903A2 (fr) * 2002-07-25 2004-02-05 The Regents Of The University Of California Suivi d'interactions moleculaires par analyse de la distribution des intervalles entre temps d'arrivee de photons

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KAPANIDIS ACHILLEFS N ET AL: "Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 101, no. 24, 2 June 2004 (2004-06-02), pages 8936 - 8941, XP002341408, ISSN: 0027-8424 *
LI HAITAO ET AL: "Ultrasensitive coincidence fluorescence detection of single DNA molecules.", ANALYTICAL CHEMISTRY. 1 APR 2003, vol. 75, no. 7, 1 April 2003 (2003-04-01), pages 1664 - 1670, XP002341407, ISSN: 0003-2700 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9040305B2 (en) 2004-09-28 2015-05-26 Singulex, Inc. Method of analysis for determining a specific protein in blood samples using fluorescence spectrometry
US9823194B2 (en) 2004-09-28 2017-11-21 Singulex, Inc. Methods and compositions for highly sensitive detection of molecules
US9063131B2 (en) 2004-09-28 2015-06-23 Singulex, Inc. Methods and compositions for highly sensitive detection of molecules
EP3156799A1 (fr) * 2006-04-04 2017-04-19 Singulex, Inc. Analyseur et procédé hautement sensible de détection d'analytes
US8917392B2 (en) 2007-12-19 2014-12-23 Singulex, Inc. Scanning analyzer for single molecule detection and methods of use
US8634075B2 (en) 2007-12-19 2014-01-21 Singulex, Inc. Scanning analyzer for single molecule detection and methods of use
GB2456063A (en) * 2007-12-19 2009-07-08 Singulex Inc Optical scanning analyser for single molecule detection
US8462339B2 (en) 2007-12-19 2013-06-11 Singulex, Inc. Scanning analyzer for single molecule detection and methods of use
US8264684B2 (en) 2007-12-19 2012-09-11 Singulex, Inc. Scanning analyzer for single molecule detection and methods of use
US9239284B2 (en) 2007-12-19 2016-01-19 Singulex, Inc. Scanning analyzer for single molecule detection and methods of use
US7914734B2 (en) 2007-12-19 2011-03-29 Singulex, Inc. Scanning analyzer for single molecule detection and methods of use
GB2456063B (en) * 2007-12-19 2010-10-20 Singulex Inc Scanning analyzer for single molecule detection and methods of use
US10107752B2 (en) 2007-12-19 2018-10-23 Singulex, Inc. Scanning analyzer for single molecule detection and methods of use

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