Ratiometric fluorescent pH sensor for non-invasive monitoring

• US 20050090014A1
• Filed: 06/30/2003
• Published: 04/28/2005
• Est. Priority Date: 12/17/2002
• Status: Active Grant

• Current Assignee: University of Maryland
• Original Assignee: University of Maryland
• Inventors: Rao, Govind, Kostov, Iordan V., Harms, Peter, Kermis, Haley R.
• Granted Patent: US 7,390,462 B2
• Time in Patent Office: Days
• US Class Current: 436/166
• 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 fluorescent dye having the structure of

2. The fluorescent dye of claim 1, wherein said fluorescent dye is 6-methacryloyl-8-hydroxy-1,3-pyrene disulfonic acid (MA-HPDS).

3. A ratiometric fluorescent pH sensor comprising:

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

a polymer matrix, said fluorescent dye immobilized therein, said polymer matrix comprising a front surface and a back surface;

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

an adhesive means.

4. The ratiometric fluorescent pH sensor of claim 3,

wherein said fluorescent dye is 6-methacryloyl-8-hydroxy-1,3-pyrene disulfonic acid (MA-HPDS);

and

wherein said polymer matrix is a p(ethylene glycol) hydrogel.

5. The ratiometric fluorescent pH sensor of claim 4, wherein said p(ethylene glycol) immobilizes said MA-HPDS via covalent binding therewith.

6. The ratiometric fluorescent pH sensor of claim 4, further comprising a comonomer, a cross-linking agent or a combination thereof.

7. The ratiometric fluorescent pH sensor of claim 6, wherein the comonomer is a siloxane-based monomer.

8. The ratiometric fluorescent pH sensor of claim 6, wherein the cross-linking agent is trimethylolpropane triacrylate or ethylene glycol dimethacrylate.

9. The ratiometric fluorescent pH sensor of claim 3,

wherein said fluorescent dye is 8-hydroxy-1,3,6-pyrene trisulphonic acid trisodium salt (HPTS);

and

wherein said polymer matrix is p(ethylene glycol) diacrylate monomer polymerized around an anionic exchange resin.

10. The ratiometric fluorescent pH sensor of claim 9, wherein said anionic exchange resin immobilizes HPTS via ionic binding therewith.

11. The ratiometric fluorescent pH sensor of claim 3,

wherein said fluorescent dye is 8-hydroxy-1,3,6-pyrene trisulphonic acid trisodium salt (HPTS);

and

wherein said polymer matrix is a poly(2-hydroxyethyl methacrylate)-co-(methacryloyl ethyltrimethyl ammonium chloride) (pHEMA-METMA) copolymer hydrogel.

12. The ratiometric fluorescent pH sensor of claim 11, wherein said pHEMA-METMA immobilizes HPTS via ionic binding therewith.

13. The ratiometric fluorescent pH sensor of claim 3, wherein the optically isolating means is a microfiltration membrane.

14. The ratiometric fluorescent pH sensor of claim 3, wherein the adhesive means is an adhesive tape, a hydrophobic adhesive substrate or a transfer adhesive.

15. The ratiometric fluorescent pH sensor of claim 14, wherein said adhesive tape is a polyethylene tape.

16. The ratiometric fluorescent pH sensor of claim 14, wherein said hydrophobic adhesive substrate is a silicon rubber substrate or an acrylate-functional adhesive.

17. The ratiometric fluorescent pH sensor of claim 3, wherein the fluorescent dye comprising the sensor exhibits dual excitation maxima wavelengths and an emission wavelength maxima.

18. The ratiometric fluorescent pH sensor of claim 3, 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.

19. The ratiometric fluorescent pH sensor of claim 3, wherein the sensor is sterilized.

20. The ratiometric fluorescent pH sensor of claim 3, wherein the sensor comprises a patch.

21. The ratiometric fluorescent pH sensor of claim 20, wherein assembly of said sensor patch comprises the steps of:

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 said polymer matrix with the optically isolating means.

22. 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 3 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.

23. The system of claim 22, further comprising means for closed-loop feedback control of the system.

24. The system of claim 22, wherein said delivery means is a light emitting diode.

25. The system of claim 24, wherein said light emitting diode emits light in the ultraviolet wavelength range or in the blue wavelength range.

26. The system of claim 22, wherein said detecting means is a photodiode.

27. A method of continuous monitoring of pH of a growth media during a fermentation process comprising the steps of:

placing the growth media or a sample therefrom into the reaction vessel of the system of claim 22;

exciting the fluorescent pH sensor with the first of said pH-sensitive excitation maxima wavelengths;

measuring the intensity of fluorescent light emitted from the sensor at said emission maximum wavelength upon said first excitation;

exciting the fluorescent pH sensor with the second of said pH-sensitive excitation maxima wavelengths;

measuring the intensity of fluorescent light emitted from the sensor at said emission maximum wavelength upon said second excitation;

calculating a ratio of said emission intensity at the first excitation maxima wavelength to said emission intensity at the second excitation maxima wavelength; and

correlating said ratio to the pH of the growth media thereby monitoring pH during fermentation.