Patent Application
20060105174
This invention relates to an optical sensor for measuring pH with improved stability and sensitivity. A pH sensitive fluorescent material is conjugated to a water-soluble polymer chain attached to a support. The water-soluble polymer chain extends away from the support when contacted with an aqueous sample resulting in improved sensitivity for pH measurement.
Fluorescent indicators have been used as an alternative to potentiometric techniques to measure pH. 8-Hydroxyl-1,3,6-pyrenetrisulphonate (HPTS) has been considered one of the best potential indicators for pH determination because of its excellent photo-stability, high quantum yield, dual excitation, large Stokes' shift and long fluorescence emission. An essential feature of this indicator is that the acidic (associated, HPTS) and basic (dissociated, PTS−) forms have different excitation wavelengths at 406 and 460 nm, with an isosbestic point at 418 nm, but exhibit a similar fluorescence emission maximum at 515 nm. The dual excitation and single emission make HPTS suitable for ratiometric detection of pH. The fluorescence intensity at 406 nm for the acid form decreases but the intensity at 460 nm for the base form increases as the pH is raised accompanying the conversion of the acidic into basic forms of HPTS. However, when HPTS is directly physically or covalently immobilized in polymer membranes, researchers have found that the fluorescence excitation intensities of both acid and base forms increased along with increasing pH, and that there was no isosbestic point accompanying the conversion between acid and base form. This change results in a lowered sensitivity for ratiometric pH measurements.
Accordingly, it is an object of the invention to prepare an optical sensor with high stability and enhanced sensitivity which can be used to determine pH in a sample.
A further object of the invention is to provide an optical sensor which can be used to determine pH in environmental studies of natural waters, sediments, and soils. These and other objects of the invention are achieved by providing an optical sensor including a copolymer having a water soluble portion conjugated to a pyrenetrisulphonate wherein one end of the copolymer is covalently bound to a support. When the sensor is contacted with aqueous solution, the water-soluble portion causes the copolymer to extend or stretch out thereby facilitating contact with the sample. In this manner the immobilized pyrenetrisulphonate exhibits fluorescence properties similar to free pyrenetrisulphonate in solution.
The invention also provides for a method of making an optical sensor comprising conjugating a pyrenetrisulphonyl chloride and a water soluble material to form a conjugate, and copolymerizing the conjugate and an acrylamide wherein a copolymer is formed and wherein one end of the copolymer is attached to a support.
The invention further provides a method of determining the pH of a sample comprising providing an optical sensor foil comprising a transparent support material and a copolymer having a water-soluble porting conjugated to pyrenetrisulphonate wherein the copolymer has one end attached to the transparent support material to form a sensor foil, contacting the sample with the optical sensor foil, obtaining the fluorescence intensity of the sensor foil in the sample, and determining the pH of the sample from a fluorescence intensity ratio.
The relative fluorescence spectra of immobilized HPTS and pH response in this new membrane are similar to those of free HPTS, but actually show higher overall sensitivity to pH compared with free HPTS. The rugged physical properties of this new sensor membrane are also such as to make it particularly desirable for practical use in a variety of applications such as environmental studies of natural waters, sediments, and soil. The use of optodes to measure pH is described in Hulth, S., Aller R., Engstrom P. and Selander E., Limno. Oceanogr., 47 (1), 2002, pp. 212-220 which is incorporated by reference herein.
This invention also relates to a thin-film pH standard apparatus comprising a transparent support material, a film of a pH standard solution, and an optical sensor material having a sensor surface facing the standard solution.
The sensor of the invention is based on a new immobilization method of HPTS onto a polymer membrane. The traditional immobilization method directly binds or entraps HPTS in a membrane, so the vibration of immobilized HPTS molecules is largely limited. Restricted vibration results in changes of HPTS excitation bands and thus pH response characteristics. In a method according to the invention, a water-soluble single polymer chain is conjugated to HPTS. This soluble polymer chain is then covalently bound to the surface of a rugged insoluble polymer membrane through a co-polymerization procedure. When this kind of membrane is placed into aqueous solution, the water-soluble polymer chains stretch out, and the immobilized HPTS exhibits characteristics very similar to free HPTS.
A schematic illustration of preparation of an optical sensor in accordance with the invention is shown in
Preparation of pH Sensor Foil
Preparation of HPTS Conjugate
1. Preparation of HPTS-Sulfonyl Chloride.
Mix 100 mg HPTS with 1 gram PCl5 in a 4-mL vial and allow the mixture to stand for 1 hour at room temperature. Transfer the mixture into an agate mortar and grind for 5 minutes under a hood. Extract the mixture twice with 10 mL acetone. Combine the extract solutions and filter.
2. Preparation of HPTS-Allylamine Conjugate.
Dissolve 30 μl of allylamine or other unsaturated alkyl or aromatic amine such as methallylamine, 4-aminostyrene, and vinylaniline etc. in 2 mL acetone. Gently drop the solvent mixture into the HPTS-sulfonyl chloride solution at 0° C. while stirring. Adjust the pH of the reaction solution to 8 using 2 M NaOH solution, and allow the mixture to react overnight at 0° C. while stirring. Remove the solvent under vacuum and save the residue for further reaction.
Preparation of Polyvinyl Alcohol Membrane with Vinyl Group on the Surface.
Add 10 mL of 4.2% polyvinyl alcohol, 1.0 mL of 2% allyl alcohol or other unsaturated alkyl alcohol or amine such as 3-butene-1-OH, methallylalcohol, allylamine etc., 1.2 mL of 5% glutaraldehyde and 1 mL of 4 M HCl into a 20-mL vial. Stir the mixture and then spread it on the surface of 300 cm2 support transparency film (polyester). Keep the film level for several hours to dry the polymer membrane.
Preparation of Optode.
Dissolve the residue obtained in step 2 in 60 mL water. Then add 226 mg N-isopropylacrylamide or acrylamide, 20 mg (NH4)2S2O8, and 20 μl of N,N,N′,N′,-tetramethyl-ethylenediamine (TEMED). Mount (suspend) the polyvinyl alcohol membrane obtained above in a thin plastic tank and add the polymerization mixture. Allow the co-polymerization to occur for 2 hours at room temperature. Remove the membrane and wash with water. Place the membrane into pH 9 water solution overnight to remove any remaining free HPTS totally. The resulting fluorescent sheet may be stored dry or in water at room temperature.
The fluorescence spectra of the immobilized HPTS in various seawater pH buffers are shown in
An embodiment of a schematic illustration of a thin-film pH standard apparatus in accordance with the invention is shown in
Suitable support materials include, but are not limited to, a polyester which is transparent, e.g., Mylar sheet. The pH standard solution is selected on the basis of the preferred pH reference scale for a given application. For example, some reference scales include but are not limited to the NIST (National Institute of Science & Technology) pH low ionic strength buffer scale, Hanssen seawater H+ activity scale and seawater total H+ concentration scale. A separate pH standard apparatus can also be prepared for each of a range of standard pH solution, for example, pH 6.0, 6.4, 6.8, 7.2, 7.6 and 8.0.
The thin-film pH standard apparatus of the invention is stable at room temperature and can be stored in a refrigerator for at least a year. The thin-film pH standard apparatus can be imaged directly at the same site or surface and condition where the pH is to be determined using an optical sensor and thus provides for in situ standardization of the optical sensor. The thin-film standard apparatus has been successfully used under water at the sea floor.
Preparation of Thin-Film pH Standard Apparatus
A 2×10 cm strip of a 125 μm thick of Mylar sheet is covered with a surface tension film of a buffer solution (Hanssen pH scale) applied with pH measurements in marine environments. A 2×10 cm sensor optode as prepared above is place over the buffer solution with sensing surface facing the solution to form a sandwich. The edges are sealed with Teflon tape or by using a heat sealer.
The thin-film pH standard apparatus can be fabricated either individually or in a serial array and can have any planar dimension or be cut to an arbitrary shape which is suitable for the measurement environment. For example, a series of pH standard thin film strips each having a rectangular size 0.5×2 cm or each having a circular diameter 0.5 cm can be placed in proximity to a target location for which pH is to be determined for simultaneous standardardization within the imaged plane.
Although preferred embodiments are specifically illustrated and described herein above, it will be appreciated that many modifications and variations of the present invention are possible in light of the above teachings and within the purview of the appended claims without departing from the spirit and intended scope of the invention.
This application is a continuation-in-part of U.S. Ser. No. 10/973,663 filed Oct. 25, 2004, which is incorporated by reference herein.
The invention also relates to a thin-film pH standard which can be used for pH calibration in the local environment. Partial funding for the development of this invention was provided by U.S. Government Grant NSF grant OCE0117062. The United States government may own certain rights to this invention.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10973663 | Oct 2004 | US |
| Child | 11257481 | Oct 2005 | US |
