Optical membrane films for polycation detection

• US 6,156,274 A
• Filed: 02/26/1998
• Issued: 12/05/2000
• Est. Priority Date: 02/27/1997
• Status: Expired due to Fees

• Current Assignee: St John's University
• Original Assignee: St John's University
• Inventors: Wang, Enju
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): Handy, Dwayne K
• Application Number:
  US09/032,503
• Time in Patent Office: 1,013 Days
• Field of Search: 422/82.05, 422/82.06, 422/82.07, 422/82.08, 422/82.09, 422/82.11, 436/172, 436/800, 549/223, 549/224
• US Class Current: 422/82.06
• CPC Class Codes: C09B 11/08 Phthaleins; Phenolphthaleins; Fluorescein
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6809 involving fluorescent derivatizing reagents reacting non-specifically with all amino acids
• CPC Class Codes: G01N 33/86 involving blood coagulating time or factors, or their receptors

Abstract

Lipophilic fluorescein-based molecules have been synthesized and incorporated into thin plasticized polymeric membrane films as chromoionophores for the optical sensing and detection of polycationic protamine. The membrane response is based on the extraction of protamine into the film due to the interaction between the anionic fluorescein and the protamine polycation which results in a cation exchange between the protamine and proton, and thus, induces an absorbance spectra change of the polymeric film. The response speed is controlled by protamine diffusion through the stagnant diffusion layer adjacent to the film surface as well as within the bulk of the polymer film. When limited exposure time and non-stirring detection modes are used in a buffer solution, absorbance of a film changes as a function of the protamine concentration in the range of about 2 to 60 μg/ml (0.44 to 13.3 μM). The sensing film shows good selectivity over most common small cations, it can be used in the determination of protamine in diluted serum or whole blood. No response is observed when a protamine complexing reagent such as heparin is present, thus the instant invention is useful as an indicator for the protamine-heparin titration.

Claims

1. A membrane film for optical sensing of a molecular species comprising:

a lipophilic ester of a compound having a formula;

##STR3## where X¹ is selected from the group consisting of;

a hydrogen atom, a chlorine atom, and a bromine atom, where X² is selected from the group consisting of;

a hydrogen atom, a chlorine atom, and a bromine atom, where X³ is selected from the group consisting of a hydrogen atom, a chlorine atom, and a bromine atom, where X⁴ is selected from the group consisting of a hydrogen atom, a chlorine atom, and a bromine atom and at least one of X¹, X², X³ and X⁴ is a hydrogen atom, and where R¹ is selected from the group consisting of;

an alkyl having from 14 to 30 carbon atoms, an alkenyl having from 14 to 30 carbon atoms, a haloalkyl having from 14 to 30 carbon atoms, and a haloalkenyl having from 14 to 30 carbon atoms wherein in response to the polycation said ester exhibits an optical response; and

an optically inert polymer in which said ester is mixed.

2. The film of claim 1, wherein the molecular species is a polycation.

3. The film of claim 2, wherein the polycation is selected from the group consisting of protamine, poly-L-arginine, and poly-L-lysine.

4. The film of claim 2, wherein the polycation is protamine.

5. The film of claim 1 wherein X¹ and X² are the same type of atom and X³ and X⁴ are the same type of atom.

6. The film of claim 1 wherein R is an alkyl having an even number of carbon atoms, the number of carbon atoms being greater than 15 and less than 25.

7. The film of claim 1 wherein said ester is maintained at a pH greater than 6.00 and less than 10.00 during exposure to the polycation.

8. The film of claim 6 wherein the pH is maintained between 7.00 and 8.50.

9. The film of claim 7 wherein the pH is maintained between 7.3 and 7.5.

10. The film of claim 1 wherein said ester is present from about 0.1 to about 10 weight percent relative to total film weight.

11. The film of claim 1 wherein said optically inert polymer is present from about 15 weight percent to about 45 weight percent based on total film weight.

12. The film of claim 11 wherein said polymer is a mixture of poly(vinyl chloride) and polyurethane.

13. The film of claim 12 wherein the film has a thickness of from about 0.5 to about 25μ

.

21. A process for optical titration of heparin comprising the steps of:

(a) immersing the film of claim 1 in a solution containing heparin, said solution being buffered to fixed pH;

(b) adding an increment of protamine to said solution;

(c) providing time for the film to respond to said increment of protamine; and

(d) monitoring film absorbance after adding said increment of protamine.

22. The process of claim 21 wherein said solution is buffered to a pH between about 7.0 and 8.4.

23. The process of claim 21 wherein said solution is buffered to a pH between about 3.0 and 4.7.

24. The process of claim 21 further comprising repeating of steps (a)-(d).

25. The process of claim 21 wherein said solution is blood serum.

26. The process of claim 21 wherein said solution is whole blood.

14. A membrane film for optical sensing of a molecular species comprising:

a lipophilic molecule having the formula;

##STR4## where X¹ is selected from the group consisting of;

a hydrogen atom, a chlorine atom, and a bromine atom, where X² is selected from a group consisting of;

a hydrogen atom, a chlorine atom, and a bromine atom, and at least one of X¹ and X² is a hydrogen atom, where R¹ is selected from the group consisting of an alkyl having from 10 to 30 carbon atoms, an alkenyl having from 10 to 30 carbon atoms, a haloalkyl having from 10 to 30 carbon atoms and a haloalkenyl having from 10 to 30 carbon atoms, where R² is selected from the group consisting of;

a hydrogen atom, an alkyl having from 10 to 30 carbon atoms, an alkenyl having from 10 to 30 carbon atoms, a haloalkyl having from 10 to 30 carbon atoms and a haloalkenyl having from 10 to 30 carbon atoms, wherein in response to the polycation said molecule exhibits an optical response; and

an optically inert polymer in which said molecule is mixed.

15. The film of claim 14, wherein the molecular species is a polycation.

16. The film of claim 15, wherein the polycation is selected from the group consisting of protamine, poly-L-arginine, and poly-L-lysine.

17. The film of claim 15, wherein the polycation is protamine.

18. The film of claim 14, wherein R¹ is an alkyl having an even number of carbon atoms, the number of carbon atoms being greater than 15 and less than 25.

19. The film of claim 14, wherein R² is an alkyl having an even number of carbon atoms, the number of carbon atoms being greater than 15 and less than 25.

20. The film of claim 14, wherein said molecule is maintained at a small pH greater than 3.0 and less than 4.7 during exposure to the polycation.