DETECTION SYSTEM AND METHOD FOR HIGH SENSITIVITY FLUORESCENT ASSAYS

• US 20110312105A1
• Filed: 09/01/2011
• Published: 12/22/2011
• Est. Priority Date: 03/03/2009
• Status: Active Grant

• Current Assignee: Access Medical Systems, Ltd.
• Original Assignee: Access Medical Systems, Ltd.
• Inventors: Tan, Hong, Zuk, Robert F., Tan, Yushan, Cao, Erhua, Xia, Min, Chen, Jun
• Granted Patent: US 8,309,369 B2
• Time in Patent Office: Days
• US Class Current: 436/501
• CPC Class Codes: C08B 37/006 Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 2201/06113 Coherent sources; lasers
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Abstract

This invention relates to a detection system for measuring a fluorescent signal in a fluorescent assay. The system comprises a probe having a small sensing surface bound with a fluorescent label, and a light source and a detector both mounted at the proximal side of the sensing surface of the substrate. The invention also relates to a method for detecting an analyte in a liquid sample using a probe tip having a small surface area (≦5 mm) and a high molecular weight polymer (≧1 MD) having multiple binding molecules and multiple fluorescent labels. The binding reaction is accelerated by flowing the reaction solutions laterally and moving the probe tip up and down in the reaction vessels. The invention furthers relates to a fluorescent labeling composition comprising a cross-linked Ficoll molecule having a plurality of binding molecules and a plurality of fluorescent labels.

Claims

1. A detection system for measuring a fluorescent signal in a fluorescent assay, comprising:

a probe having an aspect ratio of length to width at least 5 to 1, the probe having a distal end and a proximal end, the proximal end having a sensing surface bound with a fluorescent label;

a light source for emitting excitation light directly to the probe'"'"'s sensing surface;

a collecting lens pointed toward the sensing surface; and

an optical detector for detecting the emission fluorescent light;

where the collecting lens collects and directs the emission fluorescent light to the optical detector.

2. The system according to claim 1, wherein the probe tip surface has a diameter equal or less than 5 mm.

3. The system according to claim 1, wherein the probe is transparent.

4. The system according to claim 3, wherein the distal end of the probe is tapered and/or roughened to reduce undesired reflection.

5. The system according to claim 3, wherein the light source is aligned so that the light beam projected onto the tip surface at an incident angle greater than the numerical aperture angle of the probe.

6. The system according to claim 1, wherein the probe is a non-transparent monolithic rod.

7. The system according to claim 1, wherein the sensing surface is coated with a film selected from aluminum, gold, or silver, the film has a thickness from about 50 nm to about 500 μ

m.

8. The system according to claim 1, further comprising a vessel containing a solution, wherein the proximal end of the probe is immersed in the solution, and the light source and the collecting lens are mounted on the bottom side of the vessel.

9. A method of detecting an analyte in a liquid sample, comprising the steps of:

obtaining a probe having a first antibody immobilized on the tip of the probe, wherein the diameter of the tip surface is ≦

5 mm;

dipping the probe tip into a sample vessel containing a liquid sample having an analyte;

moving the probe tip up and down in the sample vessel and flowing the reagent solution laterally in the sample vessel to bind the analyte with the first antibody;

dipping the probe tip into a reagent vessel containing a reagent solution comprising a polymer having a molecular weight of at least 1 million Daltons and conjugated with at least 5 second antibody molecules and at least 25 fluorescent labels;

moving the probe tip up and down and flowing the reagent solution laterally in the reagent vessel to form an immunocomplex among the analyte, the first antibody, and the second antibody on the probe tip;

dipping the probe tip into a washing vessel containing a wash solution, and

detecting the immunocomplex formed by detecting the fluorescent signal on the probe tip;

wherein the first antibody and the second antibody are antibodies against the analyte.

10. The method according to claim 9, wherein the tip surface is ≦

about 2 mm.

11. A method of detecting an analyte in a liquid sample, comprising the steps of:

obtaining a probe having a first antibody immobilized on the tip of the probe, wherein the diameter of the tip surface is ≦

5 mm;

dipping the probe tip into a sample vessel containing (i) a liquid sample having an analyte and (ii) a reagent solution comprising a polymer having a molecular weight of at least 1 million Daltons and conjugated with at least 5 second antibody molecules and at least 25 fluorescent labels;

moving the probe tip up and down and flowing the reagent solution laterally in the sample vessel to form an immunocomplex of the analyte, the first antibody, and the second antibody on the probe tip;

dipping the probe tip into a washing vessel containing a wash solution; and

detecting the immunocomplex formed by detecting the fluorescent signal on the probe tip;

wherein the first antibody and the second antibody are antibodies against the analyte.

12. The method according to claim 11, wherein the tip surface is ≦

about 2 mm.

13. A method of detecting an analyte in a liquid sample, comprising the steps of:

(a) obtaining a probe having a first antibody immobilized on the tip of the probe, wherein the diameter of the tip surface is ≦

5 mm;

(b) dipping the probe tip into a sample vessel containing a sample solution having an analyte, moving the probe tip up and down and flowing the sample solution laterally in the sample vessel;

(c) dipping the probe tip into a reagent vessel containing a reagent solution comprising a second antibody molecules conjugated with a first member of a binding pair, moving the probe tip up and down and flowing the reagent solution laterally in the reagent vessel;

(d) dipping the probe tip into an amplification vessel containing an amplification solution comprising a polymer having a molecular weight of at least 1 million Daltons and conjugated with at least 5 molecules of second member of the binding pair and at least 25 fluorescent labels, moving the probe tip up and down and flowing the amplification solution laterally in the amplification vessel to form an immunocomplex of the analyte, the first antibody, the second antibody, and the first and the second members of the binding pair on the probe tip;

(e) dipping the probe tip into a second washing vessel containing a second wash solution; and

(f) detecting the immunocomplex formed by detecting the fluorescent signal on the probe tip;

wherein the first antibody and the second antibody are antibodies against the analyte.

14. The method according to claim 13, wherein the tip surface is ≦

about 2 mm.

15. The method according to claim 13, wherein the first member of the binding pair is biotin and the second member of the binding pair is streptavidin.

16. A composition comprising:

a crosslinked Ficoll having a molecular weight of at least 1 million Daltons,

at least 5 binding molecules, and

at least 25 fluorescent dye molecules,

wherein the binding molecules and the fluorescent dye molecules are attached to the cross-linked Ficoll.

17. The composition according to claim 16, wherein the fluorescent dye molecules are attached indirectly to the cross-linked Ficoll through the binding molecules.

18. The composition according to claim 16, wherein the binding molecules are antibody molecules or streptavidin.

19. The composition according to claim 16, wherein the fluorescent dye molecules are arylsulfonate cyanines.

20. The composition according to claim 19, wherein the arylsulfonate cyanine is Cy5.