Measurement of the cell activity and cell quantity

• US 20030203365A1
• Filed: 04/25/2002
• Published: 10/30/2003
• Est. Priority Date: 04/25/2002
• Status: Active Grant

• Current Assignee: Semibio Holdings Limited
• Original Assignee: Semibio Technology, Inc.
• Inventors: Cao, Bo
• Granted Patent: US 7,141,369 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: Y10T 428/315 Surface modified glass [e.g., tempered, strengthened, etc.]

Abstract

This invention uses a receptor molecule such as an antibody etc immobilized to a chip for a solid phase, and a sensor such as a pH electrode or an oxygen electrode or a glucose electrode for a search device. After immobilizing specific living cells by a bio-specific recognition reaction (a special immunity bond reaction) and washing the solid phase chip, one can measure the change of pH or oxygen consumption or glucose consumption from the metabolism of the specific living cells in substrate solution. This makes it possible to measure the activity and quantity of living cells specifically, accurately and quickly.

Claims

1. A method for measuring the activity or quantity of living cells and organelles comprising

a) attaching a receptor molecule to a chip, providing a solid phase

b) reacting a substrate solution containing living cells or organelles with the solid phase, immobilizing specific cells or organelles

c) measuring the metabolism of the cells or organelles with a detector.

2. The method, as in claim 1, wherein the chip is coated with polyions in a polyion charged binding reaction.

3. The method, as in claim 2 wherein the chip is coated with polyions selected from the group consisting of polylysine, polyallylamine, polyvinylpyridine, dextrans modified with amino groups, chitosan, polycarboxylic acids and polysulphonic acids.

4. The method, as in claim 3 wherein the receptor molecule is selected from one of the group consisting of an antibody reacting with various proteins exposed on the surface of cells, a ligand molecule reacting with various receptors exposed on the surface of cells, and a receptor reacting with various cell adhesion factors exposed on the surface of cells.

5. The method, as in claim 4 wherein the receptor molecule is complexed to a biotinylated compound complexed to an avidin-containing compound.

6. The method, as in claim 1 wherein said measurement is conducted instantaneously.

7. The method, as in claim 1, wherein said dectector is selected from the group consisting of a pH electrode, an oxygen electrode, and a glucose electrode.

8. The method, as in claim 1, wherein the metabolism is measured by measuring one of the group consisting of the change in pH, the change in oxygen consumption and the change in glucose consumption.

9. The method, as in claim 1 wherein the substrate solution further comprises NaCo₃ and carbonic anhydrase.

10. The method, as in claim 7, wherein the detector part of the electrode is adjacent to one end of the solid phase chip or inserted into the inner portion of the solid phase chip.

11. The method, as in claim 7, wherein the substrate solution is moved to the detection part of the electrode at the point of measurement.

12. The method, as in claim 1, wherein the measurement is conducted at a temperature of 32-38 degrees C., and thermostatically controlled by thermostatical control apparatus.

13. The method, as in claim 1, wherein the chip is constructed from a material selected from the group consisting of polyester, polyamide, polycarbonate, and cellulose polymers.

14. The method, as in claim 1, wherein the chip is composed of a short capillary with a narrow diameter.

15. The method, as in claim 14, wherein the chip is composed of glass.

16. The method, as in claim 15, wherein said glass is quartz glass that does not absorb carbon dioxide.

17. The method, as in claim 1, used to measure the activity of Human CD4 lymphoctyes or human neutrophils.

18. A method for immobilizing specific living cells on a solid phase chip, comprising

a. attaching a receptor molecule to a chip, providing a solid phase

b. reacting a substrate solution containing living cells with the immobilized solid phase chip.

19. The method, as in claim 18 wherein the receptor molecule is selected from one of the group consisting of an antibody reacting with various proteins exposed on the surface of cells, a ligand molecule reacting with various receptors exposed on the surface of cells, and a receptor reacting with various cell adhesion factors exposed on the surface of cells.

20. The method, as in claim 19, wherein the receptor molecules are complexed to biotinylated compounds complexed to compounds containing avidin.

21. The method, as in claim 20, wherein said biotinylated compound comprises biotinylated bovine serum albumin.

22. The method, as in claim 19, wherein the chip is coated with polyions in a polyion charged binding reaction.

23. The method, as in claim 22, wherein said chip is coated with successively alternating layers of polyanions and polycations

24. The method, as in claim 23 wherein said polyions are selected from the group consisting of polylysine, polyallylamine, polyvinylpyridine, dextrans modified with amino groups chitosan, polycarboxyllic acids, and polysulphonic acids.

25. The method, as in claim 23, wherein the top layer of polyions comprise polylysine.

26. The method, as in claim 22, wherein the chip is coated by immersion in a solvent containing 1-100 mg/mls of polyions.

27. The method, as in claim 26, wherein the solvent is selected from the group of cyclic amides.

28. The method, as in claim 24, wherein an activated biotin ester reacts with the polylysine directly at room temperature.

29. A method for determining the quality of a a receptor-molecule solid phase chip comprising

a. Attaching a receptor molecule to a chip, thereby providing a receptor-molecule solid phase chip

b. reacting said receptor-molecule solid phase chip with fluorescence-labelled chemical agents.

c. detecting the fluorescence emission with a UV transilluminator or charge coupled device camera.

30. The method, as in claim 29. wherein the receptor molecule is selected from one of the group consisting of an antibody reacting with various proteins exposed on the surface of cells, a ligand molecule reacting with various receptors exposed on the surface of cells, and a receptor reacting with various cell adhesion factors exposed on the surface of cells.

31. A method, as in claim 30, wherein the fluorescence-labelled chemical agents are selected from the group consisting of fluorescence-labelled polyanions, fluorescence-labelled polycations, fluorescence-labelled streptavidin, fluorescence-labelled biotin and fluorescence-labeled antibodies.

32. A method for observing the morphology of cells or organelles comprising

a) attaching a receptor molecule to a chip, providing a solid phase

b) reacting a substrate solution containing living cells or organelles with the solid phase

c) dying the resulting chip-immobilized cells with a stain

d) observing the morphology of specific cells or organelles with a device.

33. The method, as in claim 32 wherein the receptor molecule is selected from one of the group consisting of an antibody reacting with various proteins exposed on the surface of cells, a ligand molecule reacting with various receptors exposed on the surface of cells, and a receptor reacting with various cell adhesion factors exposed on the surface of cells.

34. The method, as in claim 33, wherein said receptor molecules comprise biotinylated compounds or compounds containing avidin.

35. The method, as in claim 32, wherein the chip is immersed in a cyclic amine containing solvent containing 1-100 mgs/ml of polylyine.

36. A method, as in claim 32 wherein said device is a light microscope.

37. A method, as in claim 32 wherein said device is a charge coupled device.

38. A method, as in claim 32, wherein said specific cells in step d) comprise tumor cells.

39. A chip for measuring the activity or quantity of living cells and organelles wherein said chip is attached to a receptor molecule, said receptor molecule

selected from

one of the group consisting of an antibody reacting with various proteins exposed on the surface of cells, a ligand molecule reacting with various receptors exposed on the surface of cells, and a receptor reacting with various cell adhesion factors exposed on the surface of cells. wherein the receptor molecule is complexed to a biotinylated compound complexed to an avidin containing compound.

40. The chip, as in claim 39, wherein said chip is coated with

polyions selected from the group consisting of polylysine, plyallylamine, polyvinylpyridine, dextrans modified with amino groups, chitosan, polycarboxylic acids and polysulphonic acids before attaching a receptor molecule.

41. A chip, as in claim 40, wherein said chip is a narrow glass capillary free of dust.

43. An apparatus, as in claim 40, wherein said chip is coated with polyions selected from the group consisting of polylysine, polyallylamine, polyvinylpyridine, dextrans modified with amino groups, chitosan, polycarboxylic acids and polysulphonic acids before attaching a receptor molecule.

42. An apparatus for measuring the activity and quantity of cells, comprising

(a) a chip for measuring the activity or quantity of living cells and organelles said chip attached to a receptor molecule, wherein said receptor molecules is selected from

one of the group consisting of an antibody reacting with various proteins exposed on the surface of cells, a ligand molecule reacting with various receptors exposed on the surface of cells, and a receptor reacting with various cell adhesion factors exposed on the surface of cells, wherein the receptor molecule is complexed to a biotinylated compound complexed to an avidin containing compound,

(b) a pumping means to pump sample through the chip, and

(c) a detection device.