CA2089649A1

CA2089649A1 - Self-compensating focs and other chemical sensors

Info

Publication number: CA2089649A1
Application number: CA002089649A
Authority: CA
Inventors: Kisholoy Goswami; Stanley M. Klainer
Original assignee: Individual
Current assignee: FiberChem Inc
Priority date: 1990-08-30
Filing date: 1991-06-13
Publication date: 1992-03-01
Also published as: JPH06500390A; KR930702670A; US5094958A; WO1992004618A1; EP0545949A4; EP0545949A1

Abstract

A chemical sensor, such as a fiber optic chemical sensor, is self-calibrated by measuring two output values which behave differently in response to an analyte, and forming a ratio between the two measured output values to cancel out effects of variations in external factors such as temperature variations differences between coatings, light (illuminator) variations, fouling, bleaching, leaching or the like. An indicator material may be used which produces both fluorescence and phosphorence, both monomer and aggregate emission or absorption bands, emission or absorption bands with or without an isosbestic point, emission peaks at one wavelength at two different excitation bands, or emission peaks at two wavelengths for excitation at two wavelengths.

Description

2 ~ 9 PCT/US91/0421 SELF-COMPENSATING FOCS AND OTHER CHEMICAL SENSORS
BACXGROUND OF TH INVENTION
The invention relates generally to fiber optical chemical sensors (FOCS) and other chemical sensors and more particularly to the elimination of sensor measurement errors caused by instrumental fluctuations, variations in optical properties, changes in temperature and slow loss of sensing material due to leaching or bleaching.
A fiber optic transmits light by total internal reflection at the core/clad interface when the refractive index of the clad is less than the index sf the core.
Optical fibers have been used in a wide variety of sensors, known as 'ioptrod~s" or "fiber optic chemical sensors"
~FOCS), which can detect various chemical species or physical par~meters. Illustrative U.S. Patents include 4,582,809 to Block, et al.; 4,321,057 to Buckles; 4,399,099 to Buckles; 4,682,895 to Costello; 4,600,310 to Cramp, et al.; 4,509,370 to Hirschfeld; 4,542,987 to Hirschfeld;
4,~47,546 to ~irschfeld, et al; 4,634,856 to Kirkham;
4,846,548 to Klainer; 4,892,383 to Klainer, et ai.;
4,824,206 to Klainer, et al.; 4,913,519 to Klainer, et al.;
4,272,485 to L~bbers; 4,269,516 to L~bbers; 4,476,870 to Peterson, et al.; 4,200,110 to Petersen, et al.
In addition to fiber optic type chemical sensors in which the fiber optic is used to transmit excitation and detector signals to and from the active sensor, other chemical sensors have been developed in which the fiber optic has been illuminated.
Typically an analyte specific reaction chemistry (e.g., an organic dye, transition ~etals, and their complexes or charge transfer complexes) is immobilized on the FOCS or placed in the chemical sensor cell body. The absorption or emission intensity is modulated as these W092/04618 2 ~ ~ 9 ~ ~ 9 PCT/US91/042l2 indicator materials interact with the specific analytes, thus producing a detector signal which provides a measure of the analyte o~ interest. For example, Ru(bpy) 32~
(Rutheniumtrisbipyridyl) which fluoresces with a peak at 610 nm, when excited at 440 nm, exhibits oxygen sensitive luminescence. In the presence of oxygen, Ru(~py) 321 luminescence is quenched and this quenching depends on the concentration of oxygen.
However, FOCS and other chemical sensors often exhibit poor performance because of degradation or other variations of the sensor. Sensor problems generally result from temperature variations~ differences between coatings, light (illuminator) intensity changes and flicker, loss of indicator materi~l and fouling of the ~ensor. For fluorescent sensors a particular problem is bleaching of the dye. Any of these variations, or others of a similar nature, cause changes in the sensor output which do not depend on the analyte, and thus produce an inaccurate measurem~nt. For example, with reference to the oxygen sensor describ~d above, if the Ru~bpy)32 undergoes degradation, or even ~f some Ru(bpy)32~ leaks out of the sensor, then the emission intensity of Ru(bpy)32~ will decrease, producing an erroneous oxygen reading. Thus it would be highly advantageous to have a FOCS or other chemical sensor in which the output is solely a function of the analyte or parameter of interest, and in which other variations in the senscr are compensated for so that they do not affect the sensor output. In particular, the objective is to use the FOCS or other sensor itself to provide the input ~or the necessary corrections.
SUP~ARY OF THE INVENTION
According to one aspect of the invention, there is provided a method of self-compensating a chemical sensor for W092/04618 2 ~ 8 ~ P~T/U~91/0~21' ~ariations in external factors when detecting an analyte of int~rest, comprising: forming the sensor with an indicator material which produces two di~tinct, differently varying spectral signals in response to the analyte, with both signals being affected by the variations in external actors; axciting the indicator material to produce the two spectral signals; forming a ratio o~ the two spectral signals;-determining analyte changes from the ratio.
According to another aspect o~ the invention, there is provided an apparatus for s lf-calibratinq the measurement o~ changes in an analyte in the presence of variations in external factors, comprising: a chemical sensor having an indicator material which produces two distinct, differently varying spectral signals in response to the analyte, with both si~nals being affected by the variations in external factors: excitations means operatively connected to the chemical sensor to excite the indicator ~aterial to produce the two p~ctral signals;
detection ~ea~s operatively ccnnected to the chemical sensor to detect the two spectral siynals; ~ans for forming a ratio of the two spectral ~ignals; ~eans for determining changes in the analyte from the ratio.
The invention may provide a self-compensating FOCS or other chemical sensor, preferably in which the output is solely a function of the analyte or parameter of interest, and in which other variations in tha sensor are compensated for so that they do not affect sensor output. The FOCS or other chemical sensor which is conveniently no~ affected by temperature variations, difference~ between coatings, light (illuminator) variations, fouling, bleaching, leaching or the like.
The in~ention may also provide a FOCS or other chemical sensor which is independent o~ sensor or indicator W092/0~ PCT/US91/0421~
. 2 ~

variations. Preferably, the invention is self-compensating or int~rnally calibrated FOCS or other chemical sensor, and the methodology for self-compensating or internally calibrating a FOCS or other chemical sensor. The invention may be based on using two difference spectral values from a single sensing reagent, and forming a ratio of the two measurements which removes variations in the output caused by the sensor. There are several possible embodiments, wher~by, the ratiometric techniques can be used for sensor measurements:
a. The sensing reagent produces both ~luorescence and phosphorescence emissions, only one of which is analyte speci~ic.
b. At higher concentra~ions, the sensing reagent shows monomer and "aggregate" emission (or absorption) bands and these two bands have different quenching rates for a given analyte.
c. The interaction of an analyte makes two (or more) distinct absorption bands or emission bands with one or more clearly defined isosbestic points. One of the isosbestic points is considered as the reference and its constant intensity is ratioed with the variable intensity of a growing or d~caying peak. Alternatively, the absorbance or the emission intensities of the two maxima can be ratioed, as in the next embodiment.
d. Interaction of an analyte results in two absorption or emission bands without isosbestic points. The peak intensities will be ratioed to obtain maximum stability with respect to calibration.

W092/04618 2 ~ PCT/US91/04212 e. For a given emission wavelength, two excitation regions will develop because of the interaction of an analyte with the sensing reagents. The emission peak inten ikies in the two excitation ~ands will bP ratioed.
f. A dye is excited in two different wavelengths, the emission intensities are measured and the two intenslties are ratioed. This is applicable when the quantum yields of emission depend on wavelength.
BRIEF DESCRIPTION OF THE DRAWINGS
In the accompany drawings:
Figures lA, B, C, D, are schematic views of 3 types of FOCS and one type of non-fiber optic sensor which may be ~ade self-compensating ~e~sor6.
Figure 2 i~ a graph of intensity vs. wavelength showing separate fluorescence and phosphorescence emissions of an i~dicator material, biacetyl, in the presence and ab~ence of oxygen.
Figure 3 illustrates the "~onomerl' and "aggregate"
emission method using pyrene, in the prese~ce and absence of oxygen.
Figure 4 illustrates the isosbestic point method, and also the two absorption band method, using congo red in the presence o~ chlorine.
Figure 5 illustrates the two excitation/single emission methGd, using hydroxypyrenetrisulfonate as a function of pH.
Figure 6 illustrates the two excitation/two emission method, usin~ a mixture of crystal violet and polyacrylic acid as a function of pH.
Figure 7 is a sch~matic view of a self-calibrating sensor apparatu~.

WO 92/04618 PCr/US91/0421' 2 ~

pETAILED DESCRI~rION OF THE PREFERRED EMBODIMENTS
Figures lA-D show a variety of sensor desig~s. The invention applies to any of these sensor designs or to other sensor designs which ~all within the purview of fiber optic chemical sensors and non-fiber optic chemical sensors, such as reservoir cell s~nsors with either internal or external sources and detectors.
Figure lA shows side-coated FOCS 1. FOCS 1 is made of a fiber with core 2, clad 3 (with re~ractive index less than the refractive index of core 2) surrounding core 2, and (optional) buffer (protective layer) 4 surrounding clad 3.
A portion 5 of the fiber is reduced ko a bare core (core 2 with clad 3 and bu~fer 4 removed), and side-coating 6 is applied to core 2 along exposed portion 5. Side-coating 6 is the reactive coating. The light enters FOCS 1 at end 7.
End 8 is the distal end (termination of FOCS 1).
Figure lB shows tip-coated FOCS 9. FOCS 9 is made of core 2, surrounding clad 3, (optional) buffer 4 around clad 3, and tip-coating 10. Tip-coating 10 can be the same material as side-coating 60 Figure lC shows a reservolr FOCS 11. Fiber optic 12 is held in fiber optic mount 13 which is placed in reservoir cell body 14. Spacers 15 control volume 16 and optical path 17 of cell body 14. ~embrane 18 holds sensing solution 19 in cell body 14. Retaining ring 20 makes membrane 18 liquid tight. Sensing solution 19 may be a solution of the same material as side-coating 6 and/or tip coating 10.
Figure lD shows a reservoir chemical sensor 21 which does not utilize optical fibers. S~nsor 21 is formed of a cell body 22 having a port 25 fo~med therein. Port 25 is covered by a semi-pexmeable membrane 18 which is held in place by retaining rings 200 A source 23 and a detector 24 are mounted in opposed ends of cell body 22, by attachment , .

2 ~
-!:

means or adapters 26, 27, respectively. sensor 21 is filled with sensing solution l9.
In FOCS l, 9 and ll, and sensor 21, light enters the sensor and excites the sensing chemistry t6, lO and l9) which then interacts with the tar~et (chemical/biological) of interest. This interaction results in a spectral change (intensity, wavelength shift, change of lifetime) which can supply both qualitative and quantitative information about the sample being measured.
In a preferred embodiment of the invention, as shown in Figure 2, the indicator material of the sensor is a dye which produces both fluorescence and phosphorescence emissions, only one of which is analyte specific. Any luminescent species having analyte specific fluorescence and nonspecific phosphorescence or vice versa can be used. An example is biacetyl lu~inescence quenching by oxygen~
Biacetyl (or Diacetyl or 2, 3-Butanedion~) has fluorescence peaks 30, 31 at 450 nm and 490 ~m and phosphorescence peaks 32, 33 at 518 nm and 570 nm with corresponding excitation at 420 nm. The phosphorescence peaks at 518 and 570 nm are very sensitive to oxyyen while the fluorescence pe~ks at 450 and 490 ~m are oxygen insensitive. Emission spectra curve 34 of biacetyl (in cyclohexane solution~ is produced in the presence of 20% o~ygen while emission spectra curve 35 is produced in the absence of oxygen. Therefore, by ratioing the peak intensities of any of the phosphorescence peaks to any of the ~luore~cence peaks, a measurement slgnal is obtained which is independent of bleaching, leaching, degradation, temperature and other external factors. This is because the non-specific peak reacts to all the external factors, while the specific peak responds to both the external factors as well as the analyte of interest. By forming a ratio the effect of external factors is WO9~/046~ PCT/~S91/04212 rS~

compensated so that the variat.ions in the fluorescence to phosphorescence ratio are due only to the analyte (external effects X oxygen effect/external effects - oxygen effect).
Thus one of the luminescence p,eaks acts as a~ internal 5 standard. The technique can be applied to pH, CO2 and other sensors as well as 2 In the second embodiment, shown in Figure 3, the sensing dye exhibits two emission (or absorption) bands depending on the concentration of the sensing dye or on the 10 concentration of a non-sensing entity (e.g., a polymer).
These two bands have different rates of quenching toward a particular analyte. The intensities at the peak wavelengths are ratioed. For example, when a 103 M solution of pyrene in toluene is excited in the ultraviolet, two emissions 15 appear as shown in the emission spectra curve 40, at 20~
oxy~en concentration, and emis~ion ~pectra curve 41 with no oxygen present. The peaks in ~he 360-420 nm region (band 45~ belong to th~ mon~mer emission, whereas, the 440-640 nm band ~band 46) is the excited dimer or excimer band. As :~20 Figure 3 shows, in the presence of 20% oxygen (emission spectra curve 40), the excimer band decreases more than the monomer band. Ratioing the intansities at 390 nm (peak 43) and 480 nm (peak 44~, at any oxygen concentration, gives sensor performance which is independent o~ many variables 25 which affect FOC5 performance.
In certain situations, the interaction of an analyte with the sensing reagent leads to growth of a new absorption or emission band with the simultaneous decay of the original band. The existence of two distinct species is indicated by the appearance of clearly defined isosbestic points. In a ; third e~bodiment, an isosbestic point is used as the internal standard. Figure 4 shows the interaction of congo red with chlorine. Absorption spectra curves 51, 52, 53, WO 92/04S18 2 ~ 9 PCT/US91/04212 , ~, .

54, 55, 56 of congo red and its complex with chlorine result from no chlorine, and 50, lO0, 200, 400 and 800 ppb chlorine, respectively. Congo red shows an intense abs~rption band in the visible wavelength ranye with a peak 57 at 500 nm. As chlorine is added, the absorbance at 500 nm diminishes while another absorption band grows with peak 58 at 650 nm. As Figure 4 shows, the absorbance at 560 nm and 800 nm remains unchanged. These are called the isosbestic points (59, 60, respectively). For an accurate determination of chlorine, the absorbances at 500 nm and 560 nm, or 650 nm and 560 nm are ratioed. The isosbestic point at 800 nm can also be used. The choice of 560 nm is more accurate because detQctor response at 500 nm or 650 nm is much closer to 560 nm. The absorbances at 560 nm and 800 nm will change only because of external effects. At 500 nm and 650 nm the absorbances are affect~d equ~lly by the ~xternal ~` effPcts as well as the presence o~ chlorine. The ratio is, therefore, only sensitive to chlorine concentration.
When the interaction of an analyte produces two absorption or emission bands wi~hout an isosbestic point, or alternatively even when there is ~n isosbestic point, the intensities of the two max.ima can be ratioed. This is ; illustrated using peaks 57, 58 in Figure 4, which can be ratioed. This fourkh embodiment is similar to that shown in Figure 3.
In ~ fifth e~bodiment, shown in Figure 5I the emission spectra of the sensor mater.ial provides two different excitation bands or regions which behave differently as a function of the analyte. Emission spectra curves 36, 37 for hydroxypyrenetrisulfonate (HPTS) at a pH
of 5.0 and 7.0, respectively (pH = hydrogen ion concentration) are shown in Figure 5. The curves 36, 37 are plots of the emission intensity of HPTS at 570 nm as a W092/04618 2 ~ ~ ~ g ~ PCT/US91/04212 --:10--function of the excitation wavelength over the range 380-500 nm. The emission spectra has two distinct excitation regions ox bands, a first region 38 between 380-410 nm and a second region 39 between 420-480 nm, which behave very differently as a function of pH. Region 38 varies only slightly in emission intensity as a function of p~ while region 39 varies greatly. Thus, the sensor can be self-calibrated by taking a ratio of intensity peaks ~8, ~9 within the two excitation bands 38, 39, respectively.
In a sixth embodiment, shown in Figure 6, the sensor material is excited at two different wavelengths and the Pmission intensities produced by the two excitations behave differently as a function of the analyte. Self-compensation is performed by ratioing the peak emission intensities at - 15 the two excitation wavelengths. Figure 6 shows both the absorption and emission spectra of crystal violet with polyacrylic acid ~in water solution) as a function of pH.
Curves 61, 62 are the absorption ~pectra at pH of 4.0 and 7.5, r~spectively. Curves 63, 64 are the corresponding emission spectra at a pH of 4.0 and 7.5, respectively. The absorption spectra has peak 65 at 508 nm and peak 66 at 590 nm while the emis~ion spectra has a peak 67 at 635 nm and a peak 68 at 740 ~m. Excltation of 508 nm leads to emission at 740 nm while excitation at 590 nm leads to emission at : 25 635 nmO It is the combination of crystal violet with polyacrylic acid which produces the double peak; crystal violet alone provides the 590/635 nm peak while the combination adds the 508/740 nm peakO Peak 67 behaves differently from peak 68 as a function of pH, i.e., it changes more, as shown in Figure 6. Therefore, a self-calibrated or compensated sensor output signal is produced by ratioing peaks 67, 68.

WO92/0~18 P~/US~l/042~2 ~'0 ~

The above mentioned examples illustrate how ratioing is used for self-compensating FOCS and other chemical sensor measurements. The corrections are intrinsic to the spectral response of the sensing chemis1ry. The self-correction is based on comparing signal intensities at two different wavelengths where the two signal intensities are affected in a different functional manner by the presence of the analyte. Since both signal values will be affected in the same way by the external ~actors, a ratio of the two signal values will cancel out the variation introduced by the external factors. The two wavelengths can be either input or output wa~elengths; i.e., the output signal (e.g., emission or absorption spectra) is measured by two different - wavelenyths or is measured at a single wavelength for two different excitation wav~lengths. The two compared signals must behave dif~rently with respect to the analyt~, eOg.
only one changes, or one increases while the other decr~ases, or they change by di~rent relative amounts.
For example, a signal that varies only slightly or not at all with the species of interest is ratioed with a signal that varies with the species of interest; both vary with the external factors whose effect is cancelled out. The resultant signal is only a function oP the analyte. Thus~
thQ invention re~uires a minimum of two distinct differently varying spectral features in response to an analyte.
Figure 7 i5 a schematic view of a self-aompensating sensor apparatus 70. Source (excitation means) 71 pr~avides an input signal into sensor 72. Sensor 72 may be any of the types illustrated in Figures lA-D, or any other type : 30 chemical sensor in which an optical signal is produced which has the requisite two distinct differently varying spectral features in response to a species of interest. Sensor 72 may produce any of the effects shown in Figures 2-6, or -1;2-other similar effects. The output of sensor 72 is measured at each of two wavelengths, as previously described, by measurement means 73, 74, respectively (which may be comhined in a single physical unit). ~he measured signals at the two wavelengths are input into ratio-forming means 75, which provides a self-calibrating output signal. Ratio-forming means 75 may be a computer or other calculating means to provide a sel~-calibrated output (and may be used for further signal processlng in addition to forming the ratio of the two signals). Source 71 may provide excitation at a single wavelength or at two (or more) wavelengths and the measurement of the sensor output at the two wavelengths may be mad~ substantially simultaneously or sequentially~
Changes and modifications in the specifically described embodi~ents can be carried out without departing ~rom th2 scope of the invention which is intended to ~e limited only by the scope of the appended claims.

;

Claims

1. A method of self-compensating a chemical sensor for variations in external factors when detecting an analyte of interest, comprising:
forming the sensor with an indicator material which produces two distinct, differently varying spectral signals in response to the analyte, with both signals being affected by the variations in external factors exciting the indicator material to produce the two spectral signals;
forming a ratio of the two spectral signals;
determining analyte changes from the ratio.

2. The method of Claim 1 wherein the spectral signals are emission intensity at different wavelengths.

3. The method of Claim 1 wherein the spectral signals are absorbance intensity at different wavelengths.

4. The method of Claim 1 wherein the spectral signals are fluorescence and phosphorescence emissions.

5. The method of Claim 4 wherein only one of the fluorescence and phosphorescence emissions is analyte specific.

6. The method of Claim 1 wherein the spectral signals are two absorption or two emission bands which have different quenching rates for the analyte.

7. The method of Claim 6 wherein the spectral signals are monomer and aggregate emission bands or absorption bands.

8. The method of Claim 1 wherein the two spectral signals are two emissions or two absorption intensities, one of which is at an isosbestic point.

9. The method of Claim 1 wherein the two spectral signals are emission intensity at a single wavelength for two different excitation bands.

10. The method of Claim 1 wherein the two spectral signals are emission intensity at two emission wavelengths for excitation at two wavelengths.

11. The method of Claim 1 wherein one of the two spectral signals is substantially invariant in response to the analyte.

12. The method of self-compensating a chemical sensor for variations in external factors when detecting an analyte of interest, comprising:
forming the sensor with an indicator material which produces both fluorescence and phosphorescence emissions, only one of which is analyte specific;
exciting the indicator material to produce fluorescence and phosphorescence peaks;
forming a ratio of a fluorescence peak intensity to a phosphorescence peak intensity;
determining analyte changes from the ratio.

13. The method of Claim 12 comprising forming the indicator material of biacetyl.

14. The method of self-compensating a chemical sensor for variations in external factors when detecting an analyte of interest, comprising:
forming the sensor of an indicator material which is excitable at a first and second wavelength;
exciting the indicator material at the first and second wavelengths;
measuring emission intensity peaks of the indicator material when excited at the first and second wavelength;
forming a ratio of the emission intensity peaks when excited at the first and second wavelength;
determining analyte changes from the ratio.

15. The method of Claim 14 comprising forming the indicator material of a mixture of crystal violet and polyacrylic acid.

16. The method of self-compensating a chemical sensor for variations of external factors when detecting an analyte of interest, comprising:
forming the sensor with an indicator material which produces an emission spectra or an absorption spectra which has an isosbestic point;
exciting the indicator material to produce the emission or absorption spectra;
forming a ratio of a peak intensity of the emission or absorption spectra to the intensity at the isosbestic point;
determining analyte changes from the ratio.

17. The method of Claim 16 comprising forming the indicator material of congo red.

18. The method of self-compensating a chemical sensor for variations of external factors when detecting an analyte of interest, comprising:
forming the sensor with an indicator material which produces both monomer and aggregate emission or absorption bands which have different quenching rates for the analyte;
exciting the indicator material to produce the monomer and aggregate bands;
forming a ratio of a monomer band peak intensity to an aggregate band peak intensity;
determining analyte changes from the ratio.

19. The method of Claim 18 comprising forming the indicator material of a solution of pyrene in toluene.

20. The method of self-compensating a chemical sensor for variations in external factors when detecting an analyte of interest, comprising:
forming the sensor of an indicator material which produces an emission intensity at a single wavelength which has a first peak in a first excitation band and a second peak in a second excitation band;
exciting the indicator material at the first and second excitation bands:
measuring emission intensity peaks when excited at the first and second excitation bands;
forming a ratio of the emission intensity peaks when excited at the first and second excitation bands;
determining analyte changes from the ratio.

21. The method of Claim 20 comprising forming the indicator material of hydroxypyrenetrisulfonate.

22. In a self-compensating chemical sensor for detecting an analyte in the presence of variations in external factors, comprising: an indicator material which produces two distinct, differently varying spectral signals in response to the analyte, with both signals being affected by the variations in external factors.

23. In the sensor of Claim 22, wherein the indicator material produces both fluorescence and phosphorescence emissions, only one of which is analyte specific.

24. In the sensor of Claim 23, wherein the indicator material is biacetyl.

25. In the sensor of Claim 22, wherein the indicator material produces a first emission intensity peak when excited at a first wavelength and a second emission intensity peak when excited at a second wavelength.

26. The sensor of Claim 25, wherein the indicator material is a mixture of crystal violet and polyacrylic acid.

27. In the sensor of Claim 221 wherein the indicator material produces an emission spectra or absorption spectra which has an isosbestic point.

28. In the sensor of Claim 27, wherein the indicator material is congo red.

29. In the sensor of Claim 22, wherein the indicator material produces both monomer and aggregate emission or absorption bands which have different quenching rates for the analyte.

30. In the sensor of Claim 29, wherein the indicator material is a solution of pyrene in toluene.

31. In the sensor of Claim 22, wherein the indicator material produces an emission intensity at a single wavelength which has a first peak in a first excitation band and a second peak in a second excitation band.

32. In the sensor of Claim 31 wherein the indicator material is hydroxypyrenetrisulfonate.

33. Apparatus for self-calibrating the measurement of changes in an analyte in the presence of variations in external factors, comprising:
a chemical sensor having an indicator material which produces two distinct, differently varying spectral signals in response to the analyte, with both signals being affected by the variations in external factors;
excitations means operatively connected to the chemical sensor to excite the indicator material to produce the two spectral signals;
detection means operatively connected to the chemical sensor to detect the two spectral signals;
means for forming a ratio of the two spectral signals;
means for determining changes in the analyte from the ratio.

34. Apparatus of Claim 33, wherein the chemical sensor is a fiber optic chemical sensor.

35. Apparatus of Claim 33 wherein the indicator material produces both fluorescence and phosphorescence emissions, only one of which is analyte specific, the excitation means excites both fluorescence and phosphorescence emissions, and the ratio forming means forms a ratio between fluorescence and phosphorescence peaks.

36. Apparatus of Claim 35, wherein the indicator material is biacetyl.

37. Apparatus of Claim 33, wherein the indicator material produces a first emission intensity peak when excited at a first wavelength and a second emission intensity peak when excited at a second wavelength, the excitation means excites the indicator material at the first and second wavelengths, and the ratio forming means forms a ratio between the emission intensity peaks at the first and second wavelength.

38. Apparatus of Claim 37, wherein the indicator material is a mixture of crystal violet and polyacrylic acid.

39. Apparatus of Claim 33, wherein the indicator material produces an emission spectra or absorption spectra which has an isosbestic point, the excitation means excites the indicator material to produce the emission or absorption spectra, and the ratio forming means forms a ratio of a peak intensity of the emission or absorption spectra to the intensity at the isosbestic point.

40. Apparatus of Claim 39, wherein the indicator material is congo read.

41. Apparatus of Claim 33, wherein the indicator material produces both monomer and aggregate emission or absorption bands which have different quenching rates for the analyte, the excitation means excites the indicator material to produce the monomer and aggregate bands, and the ratio forming means forms a ratio between a monomer band peak intensity and an aggregate band peak intensity.

42. Apparatus of Claim 41, wherein the indicator material is a solution of pyrene in toluene.

43. Apparatus of Claim 33, wherein the indicator material produces an emission intensity at a single wavelength which has a first peak in a first excitation band and a second peak in a second excitation band, the excitation means excites the indicator material at the first and second excitation bands, and the ratio forming means forms a ratio of emission intensity peaks when excited at the first and second excitation bands.

44. Apparatus of Claim 43, the indicator material is hydroxypyrenetrisulfonate.