Ratiometric fluorescent pH sensor for non-invasive monitoring

• US 7,390,462 B2
• Filed: 06/30/2003
• Issued: 06/24/2008
• Est. Priority Date: 12/17/2002
• Status: Expired due to Fees

• Current Assignee: University of Maryland
• Original Assignee: University of Maryland Baltimore County (University of Maryland)
• Inventors: Kostov, Iordan V., Harms, Peter, Kermis, Haley R., Rao, Govind
• Primary Examiner(s): Warden; Jill
• Assistant Examiner(s): Moss; Keri A
• Application Number:
  US10/609,720
• Publication Number:
  US 20050090014A1
• Time in Patent Office: 1,821 Days
• Field of Search: 422/82.05, 422/82.08, 422/55, 422/86, 436/68, 436/163, 436/172
• US Class Current: 422/82.08
• CPC Class Codes: C09B 57/001 Pyrene dyes
• 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 31/221 for investigating pH value

Abstract

The present invention provides ratiometric fluorescent pH sensors for non-invasive, continuous monitoring of pH in such applications as fermentation processes. The ratiometric fluorescent pH sensors comprise a fluorescent dye that exhibits a shift in excitation wavelength with a corresponding shift in pH in the local environment of said fluorescent dye. Ratiometric measurements of the emission intensities at dual excitation maxima correlate to pH. Also provided is a fluorescent dye 6-methacryloyl-8-hydroxy-1,3-pyrene disulfonic acid (MA-HPDS). Further provided are systems and methods to non-invasively and continuously monitor pH.

Claims

1. A ratiometric fluorescent pH sensor comprising:

a fluorescent dye immobilized within a polymer matrix comprising,

6-methacryloyl-8-hydroxy-1,3-pyrene disulfonic acid (MA-HPDS) covalently bound within a p(ethylene glycol) hydrogel matrix;

or

8-hydroxy,1,3,6-pyrene trisulphonic acid trisodium salt (HPTS) ionically bound within a p(ethylene glycol) diacrylate monomer that is polymerized around an anionic exchange resin;

said fluorescent dye exhibiting a shift in excitation wavelength with a corresponding shift in pH in the local environment of said fluorescent dye and

said polymer matrix comprising a front surface and a back surface;

a siloxane-based comonomer;

embedded in the polymer matrix,

a means of optically isolating said polymer matrix comprising said fluorescent dye; and

an adhesive means on the front surface of the polymer matrix.

2. The ratiometric fluorescent pH sensor of claim 1, further comprising a cross-linking agent.

3. The ratiometric fluorescent pH sensor of claim 2, wherein the cross-linking agent is triethylolpropane triacrylate or ethylene glycol dimethacrylate.

4. The ratiometric fluorescent pH sensor of claim 1, wherein the optically isolating means is a microfiltration membrane.

5. The ratiometric fluorescent pH sensor of claim 1, wherein the adhesive means is an adhesive tape, a hydrophobic adhesive substrate or a transfer adhesive.

6. The ratiometric fluorescent pH sensor of claim 5, wherein said adhesive tape is polyethylene tape or said hydrophobic adhesive substrate is a silicon rubber substrate or an acrylate-functional adhesive.

7. The ratiometric fluorescent pH sensor of claim 1, wherein the fluorescent dye comprising the sensor responds to a shift in pH within a range of about pH 6.0 to about pH 9.0.

8. The ratiomeric fluorescent pH sensor of claim 1, wherein the sensor is sterilized.

9. The ratiometric fluorescent pH sensor of claims 1, wherein the sensor comprises a patch, assembly of said sensor patch comprising

placing the adhesive means on a flat surface;

adhering the front surface of said polymer matrix comprising said fluorescent dye to the adhesive means; and

covering the back surface of sai polymer matrix with the optically isolating means.

10. A system for non-invasive monitoring of pH comprising:

a reaction vessel in which to monitor the pH;

the ratiometric fluorescent pH sensor of claim 1 wherein the front surface of said pH sensor adheres to the reaction vessel via said adhesive means;

a means for delivering a first pH-sensitive excitation maximum wavelength to the front surface of said fluorescent pH sensor;

a means for delivering a second pH-sensitive excitation maximum wavelength to the front surface of said fluorescent pH sensor;

a means for detecting maximum emission intensity of a wavelength emitted from said fluorescent pH sensor at both of the first and second pH-sensitive excitation maxima wavelengths; and

a means for evaluating a ratio of the first and second maximum emission intensities as pH.

11. The system of claim 10, further comprising means for closed-loop feedback control of the system.

12. The system of claim 10, wherein said delivery means is a diode emitting light within the ultraviolet wavelength range or within the blue wavelength range.

13. The system of claim 10, wherein said detecting means is a photodiode.

14. A ratiometric fluorescent pH sensor comprising:

a fluorescent dye, 8-hydroxy-1,3,6-pyrene trisulphonic acid trisodium salt (HPTS), exhibiting a shift in excitation wavelength with a corresponding shift in pH in the local environment of said fluorescent dye;

a poly(2-hydroxyethyl methacrylate)-co-(methacryloyl ethyltrimethyl ammonium chloride) (pHEMA-METMA) copolymer hydrogel matrix, said HPTS immobilized therein via ionic binding, said pHEMA-METMA copolymer hydrogel matrix comprising a front surface and a back surface;

a means of optically isolating said pHEMA-METMA copolymer hydrogel comprising said HPTS; and

an adhesive means on the front surface of the polymer matrix.

15. The ratiometric fluorescent pH sensor of claim 14, further comprising a comonomer, a cross-linking agent or a combination thereof.

16. The ratiometric fluorescent pH sensor of claim 14, wherein the comonomer is a siloxane-based monomer.

17. The ratiometric fluorescent pH sensor of claim 16, wherein the cross-linking agent is triethylolpropane triacrylate or ethylene glycol dimethacrylate.

18. The ratiometric fluorescent pH sensor of claim 14, wherein the optically isolating means is a microfiltration membrane.

19. The ratiometric fluorescent pH sensor of claim 14, wherein the adhesive means is an adhesive tape, a hydrophobic adhesive substrate or a transfer adhesive.

20. The ratiometric fluorescent pH sensor of claim 19, wherein said adhesive tape is polyethylene tape or said hydrophobic adhesive substrate is a silicon rubber substrate or an acrylate-functional adhesive.

21. The ratiometric fluorescent pH sensor of claim 14, wherein the fluorescent dye comprising the sensor responds to a shift in pH within a range of about pH 6.0 to about pH 9.0.

22. The ratiomeric fluorescent pH sensor of claim 14, wherein the sensor is sterilized.

23. The ratiometric fluorescent pH sensor of claims 14, wherein the sensor comprises a patch, assembly of said sensor patch comprising

placing the adhesive means on a flat surface;

adhering the front surface of said polymer matrix comprising said fluorescent dye to the adhesive means; and

covering the back surface of sai polymer matrix with the optically isolating means.

24. A system for non-invasive monitoring of pH comprising:

a reaction vessel in which to monitor the pH;

the ratiometric fluorescent pH sensor of claim 14 wherein the front surface of said pH sensor adheres to the reaction vessel via said adhesive means;

a means for delivering a first pH-sensitive excitation maximum wavelength to the front surface of said fluorescent pH sensor;

a means for delivering a second pH-sensitive excitation maximum wavelength to the front surface of said fluorescent pH sensor;

a means for detecting maximum emission intensity of a wavelength emitted from said fluorescent pH sensor at both of the first and second pH-sensitive excitation maxima wavelengths; and

a means for evaluating a ratio of the first and second maximum emission intensities as pH.

25. The system of claim 24, further comprising means for closed-loop feedback control of the system.

26. The system of claim 24, wherein said delivery means is a diode emitting light within the ultraviolet wavelength range or within the blue wavelength range.

27. The system of claim 24, wherein said detecting means is a photodiode.