Polyallylamine conjugates and applications thereof for biological signal amplification

• US 20050106649A1
• Filed: 10/15/2004
• Published: 05/19/2005
• Est. Priority Date: 11/13/2003
• Status: Active Grant

• Current Assignee: Industrial Technology Research Institute
• Original Assignee: Co-Wealth Medical Science & Biotechnology Inc., Industrial Technology Research Institute
• Inventors: Yin, Li-Te, Chiou, Su-Fung, Tsao, Chao-Yun, Tsao, Jia-Huey, Pan, Chung-We
• Granted Patent: US 7,811,571 B2
• Time in Patent Office: Days
• US Class Current: 435/8
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding

Abstract

Polyallylamine conjugates and applications thereof for biological signal amplification are provided by utilizing the essential amino group of polyallylamine to covalently bind with capture agents and signal molecules having the functional groups selected from a group consisting of —NHS, —CO, —S═O₂ and —C═O—C═O. The resulting conjugates having more than one signaling entities can be further implemented for biological expression with enhancing effect on biological signal intensity, such that the sensitivity of detection for the variation between biological interactions is largely increased.

Claims

1. A polyallylamine conjugate, comprising polyallylamine, signal molecules, and capture agents, wherein each signal molecule and capture agent is attached to the polyallylamine via a —

NH₂ side chain of the polyallylamine.

2. The polyallylamine conjugate according to claim 1, wherein said signal molecules comprise enzyme, fluorescent molecules, luminescent molecules or other biologically signaling substances.

3. The polyallylamine conjugate according to claim 1, wherein said capture agent comprise protein, antigen, antibody, nucleic acid or other biomolecules with binding specificity.

4. The polyallylamine conjugate according to claim 1, wherein said capture agent can be further modified by compounds having functional groups selected from a group consisting of —

SH, —

CO, —

NH₂ and —

N═

C═

S and mixed with proper cross-linking agent or catalyst to assist the binding of capture agent with the —

NH₂ functional groups of polyallylamine.

5. The polyallylamine conjugate according to claim 4, wherein said cross-linking agent is a compound selected from a group consisting of N-succinimidyl 3-(2-pyridyldithio)propionate, glutaraldehyde, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), maleimide, iodoacetamide, protected thiol and sulfosuccinimidyl-6-(3′

-[2-pyridyldithio]propionamido)hexanoate (sulfo-LC-SPDP).

6. A method for preparing polyallylamine conjugates, comprising the steps of:

(a) binding polyallylamine with signal molecules having functional groups selected from a group consisting of —

NHS, —

CO, —

S═

O₂ and —

C═

O—

C═

O;

(b) purifying the resulting product from step (a);

(c) binding the purified product from step (b) with capture agents; and

(d) purifying the resulting product from step (c) to obtain a polyallylamine conjugate having polyallylamine, signal molecules and capture agents.

7. The method according to claim 6, wherein said signal molecules comprise enzyme, fluorescent molecules, luminescent molecules or other biologically signaling entities.

8. The method according to claim 6, wherein said signal molecules bind with the —

NH₂ functional groups of polyallylamine.

9. The method according to claim 6, wherein said capture agent comprises protein, antigen, antibody, nucleic acid or other biomolecules with binding specificity.

10. The method according to claim 6, wherein said capture agent binds with the —

NH₂ groups of polyallylamine directly or indirectly.

11. The method according to claim 6, wherein said capture agent can be further modified by compounds having functional groups selected from a group consisting of —

SH, —

CO, —

NH₂ and —

N═

C═

S and mixed with proper cross-linking agent or catalyst to assist the binding of capture agent with the —

NH₂ groups of polyallylamine.

12. The method according to claim 11, wherein said cross-linking agent is a compound selected from a group consisting of N-succinimidyl 3-(2-pyridyldithio)propionate, glutaraldehyde, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), maleimide, iodoacetamide, protected thiol and sulfosuccinimidyl-6-(3′

-[2-pyridyldithio]propionamido)hexanoate (sulfo-LC-SPDP).

13. The method according to claim 6, wherein the purification method in step (b) and step (d) comprise column chromatography, centrifugation, or precipitation.

14. A method for amplifying biological signals, comprising the steps of:

(a) binding polyallylamine with signal molecules having functional groups selected from a group consisting of —

NHS, —

CO, —

S═

O₂ and —

C═

O—

C═

O;

(b) purifying the resulting product from step (a);

(c) binding the purified product from step (b) with capture agents;

(d) purifying the resulting product from step (c) to obtain a polyallylamine conjugate containing polyallylamine, signal molecules and capture agents; and

(e) binding the resulting polyallylamine conjugate from step (d) with target molecules, then the biological signals are amplified.

15. The method according to claim 14, wherein said signal molecules comprise enzyme, fluorescent molecules, luminescent molecules or other biologically signaling entities.

16. The method according to claim 14, wherein said signal molecules bind with the —

NH₂ functional groups of polyallylamine.

17. The method according to claim 14, wherein said capture agent comprises protein, antigen, antibody, nucleic acid or other biomolecules with binding specificity.

18. The method according to claim 14, wherein said capture agent binds with the amino functional groups of polyallylamine directly or indirectly.

19. The method according to claim 14, wherein said capture agent can be further modified by compounds having functional groups selected from a group consisting of —

SH, —

CO, —

NH₂ and —

N═

C═

S and mixed with proper cross-linking agent or catalyst to assist the binding of capture agent with the —

NH₂ groups of polyallylamine.

20. The method according to claim 19, wherein said cross-linking agent is a compound selected from a group consisting of N-succinimidyl 3-(2-pyridyldithio)propionate, glutaraldehyde, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), maleimide, iodoacetamide, protected thiol, and sulfosuccinimidyl-6-(3′

-[2-pyridyldithio]propionamido)hexanoate (sulfo-LC-SPDP).

21. The method according to claim 14, wherein the purification method in step (b) and step (d) comprises column chromatography, centrifugation, or precipitation.

22. The method according to claim 14, wherein in step (e), the capture agents of the polyallylamine conjugate can identify target molecules and combine with them, thus the biological signals are amplified.

23. The method according to claim 14, wherein the target molecules in step (e) can be biological molecules immobilized on a biochip or contained in an assay specimen.

24. The method according to claim 23, wherein said biological molecules comprise protein, antigen, antibody, nucleic acid or other biomolecules with binding specificity.