Protein chips, method producing it and detection system of the protein chips, and operating method of the detection system

• US 20030228631A1
• Filed: 06/18/2003
• Published: 12/11/2003
• Est. Priority Date: 01/04/2001
• Status: Abandoned Application

• Current Assignee: Shanghai Health Digit Limited
• Original Assignee: Shanghai Health Digit Limited
• Inventors: Hu, Gengxi
• Application Number:
  US10/464,127
• Publication Number:
  US 20030228631A1
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

The invention is related to protein chips, the methods producing it, the use thereof, wherein the protein chip can operate multiple index detection in parallel. The protein chip comprises a solid carrier and proteins being detectable multiple index in parallel, the various of proteins are fixed on the carrier in order by automatic sample system. The parts that does not drop samples on the chip are blocked by blocking solution, lyophilized and stored. The invention is also related to the detection system of the protein chip above, comprising protein chip, mixture of various labelled proteins with peroxidase, diluent, chemical composition that can perform chemoluminescence reaction, detergent and standard. The principle is: contacting the solid carrier combined with various proteins A that can be specially binding substance in vivo with sample solutions containing target proteins B, forming stable complex A-B, further reaction with the corresponding labelled AC-labelled specially binding protein to form stable A-B-C-labelled, in which the label is a chemoluminescence marker that can give detectable signal. The protein chip can significantly improve detection accuracy, and can advantageously give high reliability and detection sensibility. The protein chips are broadly useful in areas of molecular biology, bio-medicine, human proteinic study, especially clinical diagnosis.

Claims

1. A protein chip that comprises an organic substrate and proteins immobilized on the substrate, wherein said proteins can specifically bind with multiple tumor markers.

2. The protein chip of claim 1, wherein said organic substrate can be cellulose acetate membrane, nitrocellulose membrane, nylon membrane and polypropylene membrane.

3. The protein chip of claim 2, wherein the optimum organic substrate is nitrocellulose membrane.

4. The protein chip of claim 1, wherein the density of said proteins immobilized on the substrate is not lower than 25 dots per cm².

5. The protein chip of claim 1, wherein the amount of said proteins immobilized on the substrate ranges from 0.1 ng/dot to 10 ng/dot.

6. A method for preparing protein chip comprising the steps of:

(1) dissolving the various proteins to be immobilized with coating solution at certain concentration;

(2) immobilizing various proteins on the organic substrate by an automatic spotting system in a density of 25-200 dot/cm² wherein the amount of the said protein ranges from 0.1 to 10 ng/dot; and

(3) leaving for a period of time and blocking the chip with blocking solution;

drying and storing at 4°

C.

7. The preparation method of claim 6, wherein said blocking solution is TBS (0.7-0.9% NaCl, 1.0-1.5% Tris) containing sucrose, BSA, Tween20, tyrosine and proclin.

8. The preparation method of claim 7, wherein said blocking solution comprises 0.1-0.5% Tween, 0.02-0.3% tyrosine, 1-9% sucrose, 1-9% BSA and 0.1-1% proclin.

9. The preparation method of claim 8, wherein said optimum ingredient of blocking solution is TBS containing 0.2% Tween, 0.1% tyrosine, 4% sucrose, 5% BSA and 0.5% proclin.

10. The preparation method of claim 6, wherein said coating solution is NaHCO₃—

Na₂CO₃ buffer at pH9.0-10.0.

11. The preparation method of claim 6, wherein said organic substrate can be cellulose acetate membrane, nitrocellulose membrane, nylon membrane and polypropylene membrane.

12. The preparation method of claim 11, wherein said preferred organic substrate is nitrocellulose membrane.

13. The preparation method of claim 6, wherein said the proteins can be antigens, antibodies, receptors and ligands.

14. The preparation method of claim 13, wherein said proteins can specifically bind with disease-related proteins.

15. The preparation method of claim 14, wherein said proteins can specifically bind with tumor markers.

16. A protein chip detection system for parallel detection of multiple markers, using chemiluminescence reaction to detect the amount of multiple biomolecules in body fluids, wherein said system comprising:

(1) a protein chip for parallel detection of multiple markers;

(2) a mixture of various proteins labeled with peroxidase

(3) a diluent for diluting sample body fluids;

(4) a chemical composition that can generate and enhance chemiluminescence reaction;

(5) a washing solution; and

(6) a series of standards of protein to be detected.

17. The detection system of claim 16, wherein said peroxidase is horseradish peroxidase.

18. The detection system of claim 16, wherein said mixture of various proteins is labeled with peroxidase, which can specifically bind with target proteins in body fluids and are mixed at a certain concentration.

19. The detection system of claim 16, wherein said washing solution comprises 0.7-0.9% NaCl, 1.0-1.5% Tris, 0.05-0.2% Tween 20 and pH ranges from 6.0 to 9.0.

20. The detection system of claim 19, wherein said the optimum ingredient of washing solution comprises 0.9% NaCl, 1.21% Tris, 0.1% Tween20 at pH 7.50.

21. The detection system of claim 16, wherein said diluent comprises 0.7-0.9% NaCl, 1.0-1.5% Tris, 0.05-0.2% Tween20 and 0.05-0.2% tyrosine.

22. The detection system of claim 21, wherein said optimum ingredient of diluent comprises 0.9% NaCl, 1.21% Tris, 0.1% Tween20 and 0.1% tyrosine.

23. A detection method by using the protein chip detection system of claim 16 comprising the steps of:

(1) contacting the sample solutions with the solid substrate immobilized with various proteins(B) that can specifically bind with corresponding target proteins(A) in sample solutions, forming stable complex B-A;

(2) complex (B-A) further reacting with the corresponding protein(C-labeled), which can specifically bind with A, to form stable A-B-C-labeled complex;

the label is a chemiluminescent marker; and

(3) adding chemical composition which can lead to chemiluminescence reaction and generate light signal; and

detecting light signals.

24. The detection method of claim 23, wherein said label is horseradish peroxidase.

25. The detection method of claim 23, wherein said mixture of various proteins, labeled with peroxidase, can specifically bind with target proteins in body fluids and are mixed at a certain concentration.

26. The detection method of claim 24, wherein said peroxidase is horseradish peroxidase.

27. The detection method of claim 25, wherein said peroxidase is horseradish peroxidase.