W. Rudolf Seitz

A fluorescence sensor for quantifying pH in the range from 6.5 to 8.5

A sensor for quantifying pH values in the physiological range has been prepared by immobilizing the trisodium salt of 8-hydroxyl-1,3,6-pyridine trisulfonic acid (HOPSA) on an anion-exchange membrane. Because electronically excited HOPSA undergoes rapid deprotonation, both acid and base forms of HOPSA lead to fluorescence from the excited state of OPSA−. However, the acid and base forms of HOPSA can be selectively detected by appropriate choice of excitation wavelengths. The ratio of fluorescence intensities resulting from excitation at 470 and 405 nm can be used to quantify pH values between 6 and 9. The ratio is unaffected by variables such as temperature and ionic strength which affect abslute intensities. At coverages below 15 μg cm−2, the ratio varies only slightly with the amount of HOPSA immobilized on the membrane. Membranes treated with HOPSA can be stored for extended periods of time without changing characteristics. However, they undergo slow photodegradation which will limit their useful lifetimes.

A fluorescent sensor for aluminum(III), magnesium(II), zinc(II) and cadmium(II) based on electrostatically immobilized quinolin-8-ol sulfonate

Abstract An optical sensor responding to Al(III), Mg(II), Zn(II) and Cd(II) is prepared by immobilizing quinolin-8-ol-5-sulfonate (QS) on an ion-exchange resin and attaching the resin to the end of a trifurcated fiber-optic bundle. Immobilization leads to weak fluorescence from QS and causes shifts in the fluorescence spectra of the QS/metal complexes. Detection limits for the metal ions studied are all below 1 × 10−6 M. Response to metal ion concentration is nonlinear. The shape of the response fits a model that assumes a 1:1 metal/QS chelate is formed. Forrnation constants for immobilized QS complexes calculated from the model are similar to those observed for dissolved QS. Immobilized and dissolved QS behave similarly with respect to pH and interferences.

Membrane for in situ optical detection of organic nitro compounds based on fluorescence quenching

Abstract Fluorescent membrane formulations for detecting organic nitro compounds by fluorescence quenching were evaluated. The most sensitive membrane is prepared by solvent casting from cyclohexanone to incorporate pyrenebutyric acid into cellulose triacetate plasticized with isodecyl diphenylphosphate. The response follows the Stern-Volmer law for 2,4,6-trinitrotoluene (TNT) and 2,4-dinitrotoluene (DNT). The membrane also responds to hexahydro-1,3,5-tri- nitro-1,3,5-triazine (RDX). For a given set of conditions, the primary factor determining sensitivity is the extent to which each nitro compound partitions into the membrane. Detection limits are ca. 2 mg l−1 for DNT and TNT and 10 mg l−1 for RDX. Nitrogen purging prior to the measurement enhances the sensitivity and eliminates interference from oxygen. The membrane is designed to be used for remote optical in situ screening of groundwater for contamination by explosives.

Polymer-Coated, Tapered Cylindrical ATR Elements for Sensitive Detection of Organic Solutes in Water

Commercially available tapered chalcogenide fibers have been coated with poly(vinyl chloride) (PVC) plasticized with 47% (w/w) chloroparaffin containing 60% Cl by weight. The coating procedure involves applying a drop of a solution containing PVC and the plasticizer in tetrahydrofuran along the fiber while allowing the solvent to evaporate. The coated fibers were exposed to 0.15% (v/v) benzene in water (1479-cm−1 band), 0.40% (v/v) chloroform in water (1216-cm−1), and 0.10% (v/v) nitrobenzene in 1.5% (w/v) methanol/water (1348-cm−1). All three organic solutes gave readily detectable signals with the coated fibers but were not observable when the aqueous solution was sampled with the use of an uncoated, tapered fiber. Detection limits for benzene, chloroform, and nitrobenzene were calculated to be 0.02%, 0.11%, and 0.006% by volume, respectively. These data show that the advantage of using a polymer coating to concentrate the analyte and reduce the water background may be combined with the advantages of using a tapered optical fiber to yield a sensitive method for detecting nonpolar organic solutes in water.

A fiber optic sensor for water in organic solvents based on polymer swelling

The sensing element is a bead of commercially available anion exchange resin formed by introducing a quaternary ammonium group onto porous crosslinked polystyrene. The diameter of the bead is 20-40% larger in water than in organic solvents investigated in this study including acetone, ethanol, methyl ethyl ketone, 3-pentanone, 4-methyl-2-pentanone and 3-heptanone. The size of the bead varies continuously with the activity of water. In the sensor bead size changes are coupled to movement of a reflecting diaphragm. Movement of the diaphragm changes the intensity of light reflected into an optical fiber. Light intensity is measured as a function of water activity. The results demonstrate that it is possible to sense reversibly and continuously the activity of water in an organic solvent using the phenomenon of the polymer swelling. Problems encountered with cracking of the polymer bead with use and swelling of the diaphragm in the sensor can be addressed by modifying the polymer formulation and changing the sensor design.

Immunoassay labels based on chemiluminescence and bioluminescence

Reagents required for reactions that produce chemiluminescence (CL) or bioluminescence (BL) may be coupled to antibodies or antigens and used as labels for immunoassay. Because methods based on CL and BL have very low detection limits, they have the potential to replace assays that currently employ radioisotopes as labels. The feasibility of several BL and CL labels has been demonstrated. To date, isoluminol derivatives have been most widely studied. Several steroid assays involving isoluminol labels have been reported, and labelled compound has been detected at levels approaching 10(17) moles. Acridinium ester-labelled compounds have also been detected at this level. In addition to systems in which the label is a reactant required for light, CL and BL can be used to analyze the amount of product generated by enzyme labels. This approach has also yielded very low detection limits. Systems have been developed using enzyme labels that catalyze formation of ATP which is then assayed by the firefly reaction or that catalyze formation of peroxide which is determined by either luminol or peroxyoxalate CL.

Amylase substrate based on fluorescence energy transfer

Abstract A fluorigenic substrate for measuring α-amylase (E.C. 3.2.1.1) activity was prepared by double labeling soluble starch with 5-(4,6-dichlorotrizain-2-yl)aminofluorescein and Procion Red MX8B. Because the absorption spectrum of Procion Red MX8B overlaps the fluorescein emission spectrum, Procion Red efficiently quenches fluorescein emission when it is closer than the critical radius for fluorescence energy transfer. When amylase catalyzes cleavage of a starch molecule between a fluorescein and a Procion Red MX8B, the distance between the two labels increases and the degree of quenching decreases. The rate at which the fluorescence intensity increases is proportional to amylase activity. To maximize the sensitivity it is critical to maximize the amount of Procion Red MX 8B coupled to the starch and to use a high-precision spectrofluorimeter which can measure a small rate of increase in fluorescence above a large constant background.

Fiber-optic sensor for salt concentration based on polymer swelling coupled to optical displacement

Abstract A sensor that responds reversibly to salt concentration was prepared in which the sensing element is an ionic polymer, either sulfonated polystyrene or sulfonated dextran, that shrinks as the ionic strength is increased. The polymer is coupled to a reflector such that changes in polymer size cause a change in reflector position, which in turn causes a change in the intensity of light reflected back into an optical fiber. The range of salt concentrations that cause a change in polymer size depends on the charge density on the polymer. Larger intensity changes are observed as the degree of polymer cross-linking decreases. Intensity changes are larger for smaller beads. Plots of intensity vs. concentration vary from salt to salt but follow concentration more closely than ionic strength. The response is more sensitive with a two-fiber optical arrangement than with a single fiber.

A sensor for pH based on an optical reflective device coupled to the swelling of an aminated polystyrene membrane

We have developed a low cost pH sensor based on an optical reflective device (ORD) coupled to a toughened, lightly crosslinked aminated polystyrene membrane. Protonation of the amine group causes the polymer to swell. This is accompanied by a decrease in the turbidity of the membrane. As a result, reflected intensity measured by the ORD is a function of pH. The resulting pH sensor is inexpensive and has modest power requirements. The sensor is stable, provided the membrane is not allowed to dry out and crack. Changing ionic strength affects response both by changing the turbidity of the membrane due to refractive index effects and also by reducing the extent to which the protonated polymer swells. Increasing temperature leads to a larger change in turbidity.

Single fiber absorption measurements for remote detection of 2,4,6-trinitrotoluene

Abstract Single fiber optical measurements have been used to follow changes in the color of an initially clear poly(vinyl chloride) membrane as it reacts with aqueous 2,4,6-trinitrotoluene (TNT) to form a colored product that absorbs strongly at 500 nm. Attempts to make this measurement via the effect of the colored product on the emission spectrum of a fluorophore incorporated into the PVC membrane were unsuccessful. However, single fiber absorption measurements were successful. Refractive index matching to reduce stray light and a reflector behind the membrane to increase the reflected intensity were essential to keep the stray light levels small relative to the signal of interest. To compensate for drift, the reflected intensity at 500 nm is measured relative to the reflected intensity at 824 nm, a wavelength at which intensity is not affected by color formation in the membrane. The rate at which the ratio of reflected intensity at 824 nm to that at 500 nm increases is a function of TNT concentration. It is estimated that TNT levels as low as 0.10 mg 1 −1 can be determined by this technique.