Method for detecting analyte(s) using magnetic colloidal particles

• US 20050048673A1
• Filed: 11/20/2002
• Published: 03/03/2005
• Est. Priority Date: 11/20/2001
• Status: Active Grant

• Current Assignee: Bibette Jerome, Diagnostica Stago (Stago International)
• Original Assignee: Bibette Jerome, Diagnostica Stago (Stago International)
• Inventors: Bibette, Jerome, Rousseau, Alain, Baudry, Jean
• Granted Patent: US 7,332,353 B2
• Time in Patent Office: Days
• US Class Current: 436/526
• CPC Class Codes: G01N 33/5434 using magnetic particle immunoreagent carriers which constitute new materials per se

Abstract

The invention concerns a method for detecting and/or quantifying at least an analyte in a liquid medium, characterized in that it consists in using magnetic colloidal particles functionalized at the surface with at least a ligand specific of the analyte to be detected and/or assayed and in that it comprises: contacting said particles with said medium to be analyzed; applying a magnetic field to said medium, at an intensity sufficient to cause said magnetic particles to be structured in the form of chains; maintaining said magnetic field for a sufficient time interval to enable coupling or combination of the analyte concerned with at least two specific ligands present respectively on two neighbouring particles of a chain; cancelling the magnetic field, and determining the presence and/or absence of said analyte and, as the case may be, its concentration via the presence and/or the absence of said permanent chains of magnetic particles.

Claims

1. A method for detecting and/or quantifying at least one analyte in a liquid medium, characterized in that it uses magnetic colloidal particles functionalized at the surface with at least one ligand specific for an analyte to be detected and/or assayed and in that it comprises:

1. bringing said particles into contact with said medium to be analyzed,

2. applying a magnetic field to said medium, at a strength sufficient to cause said magnetic particles to be structured in the form of chains,

3. maintaining said magnetic field for a period of time sufficient to enable coupling or combination of the analyte concerned with at least two specific ligands present respectively on two consecutive neighboring particles of a chain,

4. removing the magnetic field, and

5. determining the presence and/or absence of said analyte and, where appropriate, its concentration by the presence and/or absence of permanent chain(s) of magnetic colloidal particles.

2. The method as claimed in claim 1, characterized in that said particles are superparamagnetic colloidal particles.

3. The method as claimed in claim 2, characterized in that the superparamagnetic colloidal particles are obtained by coprecipitation of a polymer with an aqueous ferrofluid or by emulsification with a ferrofluid in organic phase.

4. The method as claimed in claim 1, characterized in that the magnetic colloidal particles are between 5 and 10 000 nm, and more preferably between 100 and 500 nm, in size.

5. The method as claimed in claim 1, characterized in that the specific ligands are immobilized at the surface of the particles by absorption interactions, covalent interactions and/or high affinity interactions.

6. The method as claimed in claim 1, characterized in that the immobilized ligand is one of the two partners of a high affinity binding couple.

7. The method as claimed in claim 6, characterized in that the ligand is chosen from one of the partners of the couples (poly)carbohydrates/electin, biotin or biotinylated compounds/avidin or streptavidin, protein receptor and its specific ligand, and haptene/antibody.

8. The method as claimed in claim 1, characterized in that the immobilized ligand is a compound chosen from peptides, proteins, including glycoproteins, lipoproteins and other similar or derived structures, in free or complex form, immunoglobins, nucleic acids such as DNA or RNA and their homologs, saccharides such as monosaccharides or bisaccharides, oligosaccharides and polysaccharides, lipids, hormones, receptors, metabolites or other biological substances.

9. The method as claimed in claim 1, characterized in that the analytes targeted are antigens, antibodies and nucleic acids and/or proteins.

10. The method as claimed in claim 1, characterized in that the medium to be analyzed is pretreated beforehand in order to make the analyte being sought reactive with respect to at least two molecules of one of its specific ligands.

11. The method as claimed in claim 10, characterized in that the pretreatment comprises functionalization of the analyte with one of the partners of a high affinity binding couple.

12. The method as claimed in claim 1, for detecting and/or quantifying (an) analyte(s) of the type antibodies to pathogens such as viruses, bacteria or protozoa and other infectious agents, antibodies against tumors, antibodies directed against allergens, or autoantibodies.

13. The method as claimed in claim 1, for detecting and/or quantifying antigens such as free antigens, for instance blood proteins and factors, such as clotting factors, metabolites, hormones or mediators, or antigens attached to supports, for instance surface antigens of microorganisms, membrane-bound receptors, blood group antigens and antigens of the major histocompatibility system.

14. The method as claimed in claim 1, characterized in that the magnetic colloidal particles also carry a secondary label.

15. The method as claimed in claim 14, characterized in that this label can be characterized visually, optically, mechanically and/or electrically.

16. The method as claimed in claim 14, characterized in that this label is chosen from color pigments and fluorescence agents or phosphorescence agents.

17. The method as claimed in claim 1, characterized in that several populations of magnetic colloidal particles functionalized, respectively, with at least one ligand specific for the various analytes to be detected are used.

18. The method as claimed in claim 1, characterized in that the magnetic field is continuous.

19. The method as claimed in claim 18, characterized in that the continuous magnetic field is from 5 to 500 mT, preferably 10 to 100 mT.

20. The method as claimed in claim 1, characterized in that the magnetic field is alternating.

21. The method as claimed in claim 1, characterized in that the characterization of the chains is carried out directly by visualization under a microscope and/or, where appropriate, indirectly by any optical, mechanical or electrical method.

22. The method as claimed in claim 21, characterized in that the characterization of the chains is carried out by photometry or turbidimetry.

23. The method as claimed in claim 21, characterized in that the characterization of the chains is carried out by image processing.

24. The method as claimed in claim 23, characterized in that a CCD camera is used as image-capturing device.

25. The method as claimed in claim 21, characterized in that a laser is used as light source.

27. The use of the method as claimed in claim 1, for detecting and/or quantifying at least one analyte in a microfluid system.

26. A kit of reagents for detecting an analyte in a liquid medium, characterized in that it comprises at least magnetic colloidal particles functionalized at the surface with at least one ligand specific for the targeted analyte.