CA2111838A1 - Fiber optic sensor, apparatus, and methods for detecting an organic analyte in a fluid or vapor sample - Google Patents

Abstract

The present invention provides fiber optic sensors, apparatus, methods of optical detection, and methods of sensor manufacture for detection of organic analytes having a fixed polarity. The sensor requires an optical fiber strand; an immobilized polarity-sensitive dye; and an immobilized polymeric material which not only contains the polarity-sensitive dye but also absorbs and partitions the organic analyte of interest.

Description

W093/21513 21 118 3 8 PCT/US93/0~W8 FIBER OPTIC SENSOR, APPARATUS, AND METHODS FOR
DETECTING AN ORGANIC ANALYTE IN A FLUID OR VAPOR SAMPLE
CROSS-REFERENCES
This application is a Continuation-ln-Part of application Serial No. 645,787 flled January 25. 1991. now pendlng.

RESEARCH SUPPORT
- 5 The research for the present invention was supported by a grant from the Northeast Hazardous Substances Research Center. an Environmental Protectlon Agency Research ~enter for ~ederal Regions 1 and 2, through the TuRs Center for Envlronmental Management.

~ . .
FIELD OF TH~: INVENTION
The present inventlon is concerned with optical sensors and optlcal sensing apparatus utilizing colorimetric or fluorometric techniques as qualitatlve and quantltative detection systems; and is partlcularly directed to flber optlc sensors utllizing polarlty sensitive solvachromic dyes and polymeric materials capable of absorbing and partitioning organlc analytes for optical determinations.

BACKGROUND OF THE INVENTION
The sclence and Instrumentation of spectroscopy as developed over the last centuly has become increasingly expanded and specialized as the various methods and applications of analysis came into existence. Today, spectroscopy has been divided into individual and distinctly different methods and instrumentation systems for:
ultraviolet and visible spectrophotometry; fluorescence and phosphorescence spectrophotometry; flame emission and atomic absorption spectrometry; atomic emission spectroscopy; infrared spectrophotometry; raman spectroscopy: nuclear magnetic resonance spectroscopy; electron spin resonance spectroscopy; refractometry and lnterferometry; and various others. Of these. the optlcal sensors alld optlcal sensing detectlon systems utilizing the ultraviolet and visible absorptlon methods and the fluorescence and phosphorescence excitation and emission systems are perhaps the best known and commonly utilized.

ml~ SHEEr WOg3/21513 ,~ 838 PCT/US93/~3448 In particular. the use of optical flbers and optical flber strallds in combinatlon wlth light ellergy absorbing dyes for medical.
envlronmental. and chemical analytical determinations has undergone rapld development especially wtthtn the last decade. Tl1e 5 use of optlcal flbers for such purposes and techniques generally is descrlbed by the following publications: Milanovlch et ah. "Novel Optical Flber Technlques for Medical Application," Proceedlngs of the SPIE 28th Annual Internatlonal Technlcal Symposium on Optics and Electro-Optlcs, Volume 294. 1980; Seltz, W.R., "Chemlcal Sensors 10 Based on Immoblllzed Indicators and Flber Optlcs," In C.R.C Crltiçal Reviews In Arl~al~cal Chen~lstry, Vol. 19, 1988, pp 135-173; Wolfbels.
O.S., "Flber Optical Fluorosensors in Analytlcal Chemlstry," in Molecula~ Lumlnescence Spectroscopv. Methods and Applications (S.G. Schulman, edltor), Wiley & Sons. New York (1988); Angel. S.M..
Spectroscopy~ 38 (1987); Walt et al.. "Chemical Sensors and Microinstrumentation," A~ Svmposlum Serles, Volume 403, 1989. p 252; and Wolf;bels, CRC Press, 1991.
The optlcal flber strands employed for analytical determlnatlolls typlcally are glass or plastlc extended rods having a small cross-20 sectlonal dlameter. When llght energy Is pro~ected Into one end of theflber strand (conventlonally termed the "proximal end"), the angles at which the vartous llght energy rays strike the surface are greater tha the critical angle; and such rays are "piped" through the strand's length by successive Internal reflections and eventually exit from the 25 opposlte end of the strand (conventionally termed the "distal end").
Typlcally, bundles of these strands are used collectively as optical flbers In a variety of dif~erent appllcations.
For maklng an optlcal flber Illto a sensor, one or more l~ght ener~ ~bsorblng dyes are attached to the distal end of the optical 30 flber. The sensor can then be used for both In-vltro and/or In-vivo applicat~ons. As used herein, llght energy Is photoenergy and Is deflned as electromagnetlc radlatioll of any wavelength. Accordingly.
the terms "llght ener0r~ and "photoet~ergv" include Infrared, vls~ble, and ultravlolet wavelengths convellt~ollally employed In most opt~cal W0 93/21513 .~ 1 .83~ P~r/US93/03448 nstruments and apparatus; the term also includes the other spectral regions of x-ray and microwave wavelel1gths (although these are - generally not used in conJunction wlth optical fibers).
Tvpically. Iight from an appropriate energy source is used to 5 Illuminate what is chosen to be the proximal end of an optical fiber or - a flber bundle. The light propagates along the length of the optical flber; and a portion of this propagated llght energy exists the distal end of the optical flber and is absorbed by one or more light energv absorbing dyes. As conventlonally known. the llght energy absorbing 10 dye may or may not be Immobitized: may or may not be directly attached to the optical flber itself: may or may not be suspended in a fluid sample containing one or more analytes of tnterest to be detected: and may or may not be retainable for subsequent use in a second optical determlnation.
Once the llght energy has been absorbed by the dye. some light energy of varying wavelengths and intenslty typically returns through the distal end of the optical flber and is then conveyed through either the same flber or a collection flber or flbers to a detection system where the emerging light energy is observed and measured. The 20 interactions bet~,veen the Incomlng llght energy conveyed by the optical flber and the properties of the llght absorbing dye - both in the presence of a fluid sample containing one or more analytes of interest and ln the absence of any analytes whatsoever - provide an optical basis for both qualitative and quantitative spectral determinations.
25 Merely illustratlng the use of some presently known optical flber sensors in a varietv of dlfferent conditions. apparatus. dyes, and apptlcatlons are U.S. Patent Nos. 4.822.746; 4.144.452: 4.495.293:
and Re. 31.879.
~ Moreover. In view of the microcircultry and enhanced telev~siol1 30 technology presently a~rallable, a varlerv of ilght tmage processing and analytical systems have now come illtO existence ln order to enhance~
anatyze. and mathematicatly process the tlght energles Introduced to and emcrglng from the absorbing dyes ill such optical analytical techniques. Typicallv. these svstellls provide components for image WO 93/21513 PCI'/US93/03448 ''' 2111X38 capture; data acquisition: data processing and analysis; and visual presentation to the user. Commercially avallable systems include the 9X-7 image processlng and analysis system sold by 9uante.Y. Inc (Sunnydale. CA); and the IM Spectrofluorescence imaging system 5 oifered by SPEX Industrles. Inc. (Edlson. NJ). Each of these systems may be combined wlth microscopes. cameras, and/or televlsion monltors fior automatlc processing of all llght energy determinat~ons.
Of the many dlfferent classes of llght absorbing dyes which may be employed with slngle optlcal flber strands and with bundles of 10 optlcal fibers for dlfferent analytlcal purposes are those composltions whlch emlt light energy after flrst absorb~ng energy and are termed "fluorophores"; and those composltlons whlch absorb llght energv and Internally convert the absorbed light energy into heat or kinetic energy rather than emit It as llght and are termed "chromophores" or 15 "absorbers". Fluorophores and fluorescent dctectlon methods employing optical flbers are recognized as being markedly different and dtstingutshable irom llght energy absorbance and absorptlon spectroscopy.
Fluorescence is a physical phenomenon based upon the abilitv 20 of some molecules to absorb light energy (photons) at speclfled wavelengths and then emit llght energy of a longer wavelength and at a lower energy. Such emisslons are called fluorescence lf the emissio is relatlvely long-lived. typlcally 10-1 ~ to 10-~ seconds. Substances able to nuoresce share and display a number of common 25 characterlstics: they absorb light energy at one wavelength or frequency; reach an excited el1ergy state; and subsequently emit light at another light frequency and ellergy level. The absorption and fluorescence emisslon spectra are thus indivldual for each fluor~phore: and are often graphlcall~ represented as two separate 30 curves which are sllghtly overla~ g.
All fluorophores demonstr.lte tlle Stokes' shift - that Is. the emltted llght Is always at a lon~er ~ elellgth (and at a lower energv leveU relatlve to the wavelengtl~ 1 e l~ergy level) of the excltlng llght absorbed by the substance. Mor~ r. the same fluorescence emlssio WO g3t21513 ~2 1 1 1 8 3 8 Pcr/US93/03448 s spectrum is generally observed ir~espective of the wavelength of the excltlng light and. accordingly. the wavelength and energy of the exciting light may be varied w~thln limits: but the light emitted by the tluorophore will always provide the same emission spectrum as 5 emerging llght. Finally. fluorescence may be measured as the quantum yleld of light emltted. The fluorescence quantum yield is the ratlo of the number of photons emltted in comparison to the number of photons inltlally absorbed by the fluorophore. For more detaiîed informatlon regardlng each of these characterlstlcs, the follow~ng 10 references are recommended: Lakowicz. J.R.. Prlnclples of Fluorescen ,c,e Spectros~. Plenum Press. Ncw York. 1983: Frelfelder.
D.. Physical Blochemlstry. sccond ctlltlon, W.H. ~rccman and Company. New York. 1982; Molccular Lumlncscence Spectroscopy Methods and Applicatlons: Part 1" (S.G. Schulman. edltor) ln Chem,~,al ~alvsls. vol. 77. Wlley 8c Sons. Inc.. 1985; The Theorv of Lumlnescence. Stcpanov and Crlbkovskll. Illffc Books. Ltd.. I,ondon.
; 1968.
In comparison. substances whlch absorb llght energy and do not fluorcsce usually convert the llght enerLv Into heat or kinetlc 20 encrgy. Thc abillty to Intcrnally convert tbe absorbed light energy Idcntlflcs the dye as a "chromophore." Dyes whlch absorb llght energ~
as chromophores do so at Indlvldual wavelengths of energy and are characterized by a dlstinctlve molar absorptlon coefficlent for llght energy at that wavelength. Chemical analyses employlng flber optic 25 strands and absorptlon spectroscopy using vlslble and ultravlolet llght wavclengths In comblnatlon with the absorptlon coefficient allow for the determination of concentratlon for speclflc analytes of Interest by spectral measurement. The most common usc of absorbance measurçment vla opncal flbers Is to determlne conccntratlon which is 30 calculàted In accordance wlth Beers' law: accordlngly, at a slngle absorbance wavelcngth; the greater the quantity of the composltlon whlch absorbs light encrgy at a giv,ell photo wavelength. the greater thc optlcal dcnslty for thc sample. Ill this way. thc total quantlty of light absorbed directly correlatcs w~th the quantity of the compositio WO 93/21513 Pcr/US93/03448 n the sample.
Many of the recent improvements employlng optical fiber sensors in both qualitative and quantitatlve analytical determinations concern the desirability of depositing and / or 5 immobilizing various llght absorbing dyes at the dlstal end of the optlcal flber using a glven technlque or apparatus. In this manner. a varlety of dlfferent optical flber chemical sensors and methods have been reported for speclflc analytlcal determinations and appllcations such as pH measurement. oxygen detection. and carbon dioxlde 10 analyses. These development are represented and exempllfied by the following publications: ~reeman et al..Ana~ hem. 53:9~ (1983);
Llpp~tsch et ~1-. Anal. Chem. Acta. ~ I . ( 1988~: Wolfbels et al.. Anal.
Che~. 60:2028 (1988); Jordan _ al.. Anal. Chen~. 59:437 (1987):
Lubbers et aL. Sens. Actuators. 1983; Munkholm et aL. Talanta 35:109 (1988): Munkholm et al.. ~a~hem~. 58:1427 (1986); Seitz.
W.R.. Ana~. Chem. 56:116A-34A (1984): Peterson et al.. ~. Chem.
52:864 (1980): Saarl et al.. Anal. Chem. 54:821 (1982); Saari et al..
Anal. Chem. 55:667 (1983); Zhujun et al.. hU~hÇ~Acta. 160:47 (1984); and Schwab et al.. Anal. Chem~. 56:2199 (1984).
Concurrent wlth developments in flber optic technology have been the dramatlc and devastatlng changes ln our envlronment. C)ver the last several decades there has been increasing awareness and concern over organic contaminatiol1 from hazardous waste sttes and underground storage tanks. This contamlnatlon threatens the quallty of groundwater at aquifers, thereby polluting the only drlnking water source ln many communltles. These concerns have generated a massive effort of sampllng and analvsis at an ever-lncreaslng number of monltorlng wells. Exlsting mollitoring technology Ias descrlbed in Koehn, J.W. and G.H. Stanko. EI)viroln Sç~ 2:1262-1263 tl988)1`relies typlcally on expensive. labor-lntenslve, dlscrete sample methods that lntroduce uncertaillt~es il~ the sampllng and handling procedures. Often there is a long de l.1~ between sample collectlon and communlcatlon of results caused I)~ e illablllb of conventlonal methods to provtde in situ real-t~ e Illollitorlng. Moreover, extensive WO 93/21S13 Pcr/us93/o3448 ~li1'~38 documentation ~s required due to chain-of-custody concerns. The application and generation of a low-cost rellable monitoring system employing flber optlc sensors and flberoptic detection apparatus would reduce the need for frequent samples and provide timely continuous informatlon of water quallty.

SU~Y OF THE INVENTION
The present inventlon provides optical flber artlcles, apparatus.
and methods able to be employed in the fleld for low cost and reliable systems for monitoring the environment in a timely and contlnuous manner. One aspect of the present Inventlon thus provides a flber opttc sensor for dctecttng an organlc analyte of Interest In a fluld sample. sald flber opttc sensor comprlsing:
an optlcal flber strand able to convey llght energy of a predetermlned wavelength, sald optlcal flber strand having a proximal end. a dlstal end. and a strand length;
at least one polarlty-sensltive dye immobilized at the distal end of sald optlcal flber strand, sald polarlty-sensltlve dye being able to absorb ligbt energy of a predetermlned wavelength: and at least one polymerlc material immoblL~ed at the dlstal end of sald optical flber strand such that sald Immoblltzed polarlty-sensitlve dye Is contained wlthin sald polymeric materlal, through whlch at least a portlon of such organic analyte as is presented by the fluid sample becomes absorbed and partitloned by sald immobilized polymerlc material and a measurable change in the spectral properties of contained polarlty-sensitive dye is produced.
A second aspect of the present invention provldes a flber optlc sensor apparatus for detecttng an organic analyte ln a fluld sample.
said apparatus comprlslng:
at least one flber optlc sensor comprised of an optlcal flber strand able to collvey llght energy of a predetermlned wavelength. said o,~tical flber strand havtng a proximal end. a dlstal end. and a strand length:
at least one polarlty-sens~ti-e dve Immoblll~ed at the dlstal end WO 93/21513 PCr/USg3/03448 33~ 8 of sa~d optical flber strand. said polarity-sensltlve dye being able tO
absorb light energy of a predetermined wavelength: and at least one polymertc material immoblllzed at the distal e~ld of sald optlcal flber strand such that sald immoblllzed polarlty-sensitive 5 dye Is con~ained withln said polymerlc materlal, through whtch at least a portlon of such organic analyte as Is presented by the fluid sample becomes absorbed and partltioned by sald immobilized polymerlc materlal and a measurable change In the spectral properties of said contained polarlty-sensltlve dye Is produced:
means for introducing llght energy of a predetermlned wavelength to the proximal end of sald flber opt~c sensor: and means for detecting llght energy emltted by said contalned polar1ty-sensitive dye.
Moreover, a thlrd aspect of the present Invention provides a method for detectlng an organic analyte of Interest In a fluid sample.
said method comprlslng the steps of:
contacting the fluld sample comprlslng the organlc analyte of interest wlth a flber optic sensor comprlsed of an optical flber strand able to convey llght energy of a determinable wavelen~th, sald optlcal flber strand having a proximal end, a dlstal end, and a strand length, at least one polarlty-sensltlve dye, Immobillzed at the distal end of said optlcal fiber strand, sald polarlty-sensitive dve being able to absorb light energy of a determinable wavelength~
and at least one polymeric material Immobillzed at the distal end of sald optical flber strand such that said Immobilized polarlty-sensltive dye is contalned within sald polymeric _ materlal, through whlch at least a portlon of such organlc analyte as Is presented by the fluld sample becomes absorbed and partltloned by sald Immobilized polymeric materlal and a measurable change ln the spectral propertles of sald contained polarlty-sensltive dye Is produced:
Introduclng llght energy of a predetermlned wavelength to the WO 93/21513 Pcr/uss3/o3448 9 ~ 3 8 proximal end of said fiber optic strand whereby said llght ener~y is conveyed to sald distal end of said strand and said contained polarity-sensitive dye absorbs at least a portioll of sald llght energy: and detecting light energy emitted by said contalned polarlty-sensitive dye at said distal end of said flber optic sensor, said detected llght energy belng a measure of the organlc analyte In the fluld sample.
Yet a fourth aspect of the present invention provides a method for making a flber optic sensor able to detect an organlc analyte of interest in a fluld sample. sald method comprlslng the steps of:
obtalning an optical flber strand able to convey light energy of a predetermined wavehngth. sald optlcal fiber strand vnth at least on polymerizable materlal to form a reactlon mixture: havlng a proximal end. a distal end. and a strand length; -~
admixing at least one polarlty-sensltive dye able to absorb exciting light energy of a predetermined wavelength with at least one polymerlzable material to form a reactlon mixture; and ` polymerizing sald reactioll mixture at the distal end of said optical flber strand such that sald polarlty-sensltlve dye Is contalned wlthln said immobillzed polymerlc materlal, through whlch at least a portion of such organic analyte as Is presented by the nuid sample becomes absorbed and partitloned by said immobilized polymerlc ~ -materlal and a measurable change in the spectral propertles of sald contained polarity-sensltlve dye is produced.

.~ .
BRIEF DESCRIPTION OF THE ~CURES
The present inventlon may be more easlly and completely understood when taken In ColljllllCtiOIl wlth the accompanylng draw ng. In wh~ch:
Flg. 1 is a perspectlve view of a sillgle, optical flber strand;
Flgs. 2A and 2B are overlle.~ iews of the proximal and dlstal ends of the slngle optlcal flber str;~l~d of Flg. I;
Flgs. 3A and 3B are persp~ iews of alternattve embodlments fot the dlstal end ~ )tical flber strand:

WO g3/21513 ` Pcr/us93/03448 21~ 3 8 10 Fig. 4 is a cross-sectional view of the sensor collfiguratloll for an organic vapor sensor;
F`ig. 5 is a block diagrarn of a field-portable fluorometer:
Fig. 6 is a graph lllustrating the excitation and emission spectra of a sensor exposed to benzene;
Flg. 7 is a graph illustratlng the sensor responses to a BTEX
serles and gasollne:
Flg. 8 is a graph Illustratil1g the serlsor's response tO xvlene over tlme;
- 10 Flg. 9 Is a graph showing the sensor`s cal~bration curves of xylene and gasoltne at vary~ng concentratlon;
,.A ~ . I;'lg. 10 Is a graph Illustrating the sensor's temperature dependence of basellne slgnal;
Flg. I I is a graph Illustrating the sensorts temperature dependence of xykne caitbration 250C. 30~C. and 350C:
Flg. 12 Is a graph lllustrating field data analyses at four dlfferent wells contaminated wlth )et fuel;
Flg. I3 ts a graph Illustrating the emisslon spectrum of an acrylodan/parafllm sensor exposed to toluene vapors;
Flg. 14 ts a graph ~llusattng the emission spectrum of a donsyl/parafllm sensor exposed to toluene;
Flg. lS is a graph Illustrating the response of an anthracene-9-carboxyaldehyde carbohydrazone/dimethyl and methyl vinyl silo.xalle sensor to toluene;
Flg. 16 is a graph illustrat~ng the excltation spectrum of octadecylrhodamine in the copolymer dimethyl and methyl vinyl sllox~ne for the sensor on exposure to gasoline at d~fferent time intervals:
_- ~FIg. 17 Is a graph Illustratlllg the emlsslon spectrum of octadecyl rhodamlne (ODR) In the copolymer dlmethyl and methvl vinyl slloxane (DMMV) for the sellsor oll exposure to gasoline at dlfferent t~me l~tervals:
Flg. 18 Is a graph Illustratillg the response proflle of ODR/DM
MV slloxane In a sensor exposed to toluel)e:

, WO 93/215t3 ~ 3 8 PC~/US93/03448 Figs. l9A-19C are graphs showing the response values of ODR/DM MV siloxane in a sensor e~cposed to var~ous volumes of gasoline in air:
Fig. 20 is a graph illustrating the calibration curve of a 5 ODR/DM MV slloxane sensor;
Flg. 21 is a graph Illustrating slc)pe callbration measured after 90 mtnutes of four different ORD/DM MV siloxane sensors: and Flg. 22 is a graph illustrating the optimal dye concentration for maximum fluorescence in an ORD/DM MV siloxane sensor.

i DETAILED DESCRIPTION O~ THE INVENTION
.

- The present Invention is a marked lmprovement in aber optic sensors. apparatus. and methods for performtng qualltative and quantitative optical measurements and determinatlons of organtc analytes. The physlcal construction of thls singular and unique fiber 15 optic sensor and the manner of Its manufacture are the most critical - -and demanding aspects of the sub~ect matter as a whole whlch is the prescnt Inventlon. The apparatus. the methods for making optlcal .
determinatlons. and the systems of qualltative and quantitative detection subsequently described are based and rely upon the 20 existence and use of the properly constructed flber optic sensors as the essential article.
Although the unlque flber optic sensor and the alternative construction and methods employing this sensor as described hereinafter may bear a superflclal similarity to conventionally known 25 optlcal flber strands. sensors, and tluorometrlc or colorimetric optical systems for making analytical determillations~ It will be recognized and apprec~ated that the subJect matter as a whole whlch is the pres~nt Inventlon provldes multl~le belleflts and ma~or advantages not previously known or avallable t1eretofore. Among these beneflts - 30 and advantages are the followlng:
1. A fully constructed fii)cr ol)tical senso~ comprising an Indlvldually clad. optlcal flber stral~ tlich has at least one Immobillzed polarlty-sensltlve sol~ llrolllic dyc and at least one WO 93/21513 Pcr/us93/o3448 immobilized polymeric material at the distal end. The immobilized polarity-sensitive dye Is able to absorb light energy at a determillable wavelength: and the immobilized polymeric material encloses and encompasses the immobilized polarlty-sensltive dye such that at least 5 a portlon of the organic analyte becomes absorbed and partltioned bv the lmmobllized polymeric materlal concomltant with making reactive contact with the lmmobllized polarlty-sensitlve dye itself. This unique mode of constructlon and organization permits the use of many dlfferent dyes to measure a variety of different organlc analytes.
10 the crttical requirement for the tmmobilized dye being only that tt be polarlty-sensitlve. Simllarly. the use of an Immobillzed polymeric material whose prlmaly functlon ls to absorb and partltlon at least a portlon of such organic analyte of lnterest as Is present is a distinctive and requlsite feature of the flber optlc sensor. This sensor 15 construction ls uniquely slmple and reproduclble as a chemlcal detector; and allows retiabk, accurate. and preclse determinations of various organlc analytes whlch were not conveniently deeectable before.

2. The present flber optlc sensor. apparatus, and 20 methodology for detection allows for several d~fferent mechanisms of Interactlon - a sltuatlon which is completely different and dlvergent from those systems conventionally employed for detection of organic analytes. The crltical and essential interactlon ~regardless of mechanism) occurs between the immobillzed polarity-sensitive dye. ~`
25 the sensor microenvlronment provided by the immobilized polymerlc materlal. and the presence or absence of the organic analyte of interest. Before the organic analyte is introduced. the spectral properties and the degree to which the immobitized polarity sensitlve dye ~bsorbs and releases llght energy of a g~ven wavelength Is dlrectly, 30 lnfluenced by the surroundlng Immobillzed polymeric material in which the dye Is contalned and dlspersed. However. after the organic analy~e of lnterest Is introduced to the sensor and the polymeric materlal has absorbed and at least ~)artially partitloned the organic analyte. the spectral propertles at)d the degree to whlch the :

W093/21513 ~ X3~ Pcr/Usg3/03448 immobilized polaritv-sensitive dve absorbs and releases light energ~ of a given wavelength is now influellced by a combined resulting effect provided bv the surrounding polvmeric material as altered and modified bv the absorbed and partitioned organic analyte thell presellt 5 within the local polymeric microenvironment. Thus. it is the merged resule of the polymeric materlal's indivldual propertles in combinatio with the addltional tnfluences exerted by the absorbed and partitioned organic analyte in-situ wlthin the polymeric material that causes the polarity-sensitlve dye contained wlthln the local mlcroenvlronment to 10 alter Its llght energy absorbing and releasing properties in measurable degree. Consequently. it is the change in the microenvironment gcnerated by the presence of the organic analyte within the polymeric material that causes mean~ngful and discernable dlfferences in spectral properties of the immobilized dye: and thus the presence or 15 absence of the organlc analyte of interest can be detected in a sens1tive and reproduclble manner by the change in llght energy absorbing and releasing properties for the immobllized dye. This form of Interaction and spectral change is truly unique in the art.

3. The flber optic sensor. apparatus, and me~hods for 20 detection may be employed with organic analytes which are volatile or non-volatile. The inventlon is of particular value for accurate determination of organlc analytes such as hydrocarbons. including those principally present in petroleum products. The sensor is most sensltive to lower- molecular welght hydrocarbons because these have 25 hlgh rates of dtffusion wtthin the polymeric material and thus allow a rapid rate of absorption and partition. This conse~uently permits such- lower molecular weight hydrocarbons to come lnto contact wlth the Immobllized polarlty-sensitive dve ill an unusually fast time peri~d'and thus provides the sel~sor wlth a rapld response tlme. Such 30 hydrocarbons also have relatively hîgh solubllltles withln the polymeric material whlch also provides hlghly sensitlve optlcal determlnattons when using the fiber OptiC sensor.

4. The sensor. apparatlls. alld methods of detection permit determlnatlons and measurement of organ~c analvtes in the gaseous WO 93/21513 Pcr/uS93/03448 ~11183~

or vaporized state as well as in the liquid state. The present flber optic sensor is a maJor improvement over laboratory based analytical methods such as gas chromatography in that the present sensor may be used practically ln the fleld or envlronment generally, thus 5 avoiding the ma)or delays currently assoclated with sampllng and translt time presently requlred; and elimlnates sources of addltlonal error due to sample handllng.
5. The present invent~on Is Intended to be operated In sltu, dwelllng at the point of analysls. Thls elimlnates the long recognized 10 problems ln obtaining a representatlve sample ex-sltu for analysis, Whlle some of the present avallable methods may also be used In the fleld. each of them requlres actually drawtng a sample from the source and then analyzing the limlted sample quantlty. In contrast, the present Inventlon allows a flber optic sensor to be Itself Inserted into 15 the source such as a weU contalnlng potable or contaminated water, a munlclpal reservoir, contamlnated soll. or the vapor space surrounding under- or above-ground storage tanks. Thus, the present Inventlon does not requlre removal of sample for analysls. To the contrary. the results are the direct evaluatlon and determinatlon of 20 the fluld composltlon as It occurs over tlme In the envlronment and at the naturally occurring source of the fluld.

6. The present flber optic sensor, apparatus, and method of optical determinations provide practical results in a matter of minutes or seconds and thus provide immedlate data. This real time 25 analysis and determlnatlon capability is presently unavallable by conventionally known apparatus and is a necesslty in practical terms for monitorlng a process or for following the effects of environmental hazards or controls. In additlon, the flber optic sensor permlts contiauous monltorlng if deslred. or Inonltorlng and direct analysls at 30 present time Intervals or wlthln a scheduled program of determlnatlons over tlme.

7. The present flber op~ t~l~sor, apparatus. and methodology pro~lde a more se~ allalysls and determination than Is pre-ently posslble by otll- r ~ oll~elltlonally known In-sltu WO 93/21513 2 ~ ~ 1 8 3 ~ Pcr/US93,03448 devices such as metal-oxide sensors which typically detect onl~
several hundred parts per millioll vapor volume concentratiom Tlle present invention achieves at least another order of magnitude ill sensitivtty generally: and with respect to known chemical sensors for - 5 detection of hydrocarbons, is unusually sensltlve because a dlscernlble response signal (with respect to background noise) is generated at markedly lower organ~c analyte concentrations.

8. The present flber optic sensor, apparatus. and method for detectton are completely automatic and require no human Intervention from the time of placing the sensor in the desired locatlon to the time of recordlng of the slgnal representlng the raw data Itself. Thls capablllty and advantage Is of ma~or importance because so mucp of the present and future needs for analytical determinations Is for remote envlronment monltorlng such as at storage tank sldes. in wells, and wtthin and along pipellnes. The present invention also permlts repeated use as a fleld screening device and technique whlch would detect the presence of organic analytes wh~ch are ma~or pollutants: and then would trlgger addltlonal sampling automatlcally for a more comprehenstve analy,sis at multiple sltes. Tbls automat~c sensing and monltorlng can be an essentiall~
continuous operation if deslred because the cost of contlnuous operatlon does not markedly increase with a large increase in the number of actual analyses. Alternatively. the monltoring may be performed on a regular or Irregular time schedule at one or more locattons. concurrently or in series.
Slnce the present invention is definable alternatively il~
multiple formats as a flber optic sensor~ an apparatus, a method for detectlon, and a method of mallufact-lre: and may be employed in a vartety of dlvergent purposes and appllcations to detect a large and d~verse range of organtc analytes of illterest. the subJect matter as a whole whtch is the present inventiotl will be presently described il1 multiple textual sections indlvldllallv a!ld collectively in order that the prospective user may more qllickl~ recognlze and appreciate their ma~or dlfferences and dlstlnctiolls ill comparlson to the flber optic WO93/21513 2~ 38 rcr/usg3/o~8 sensors. apparatus. and svstems conventionally known ~oda~,.

1. The Construction and Organizat~on of the Fiber Optic Sensor The singular flber optic sensor is compr~sed of three essential - 5 components: an optlcal flber strand: at least one polaritv-sensitive or -solvachromlc dye immobllized at the distal end of the optical fiber strand: and at bast one polymeric material Immobllized at the distal end of the optical flber strand such tbat the Immobllized polarit~
- sensitive solvachrom~c dye Is contained within (I.e.. dlspersed in and 10 enclosed by) the polymerlc materlal. Each component will be ~ndlvldually descrlbed ~n detail.

A. The Optical Flber Strand - ~ A preferred opttcal flber strand is Illustratcd by Figs. 1 and 2A
and 2B. As seen thereln. an Individual optical flber strand 10 is }5 comprised of a slngle optlcal nber 12 having a cyllndrlcal shaft 14 and two flber ends 16.18.~ each of whlch provldes a substantlally planar end surface. Thc tntended dlstal su rface 20 at the flber end 16 ~s Illustrated by Flg. 2A whlle the intended proximal surface 22 at the flber end 18 is lllustrated within F~g. 2B. It will be recognized and appreciated that the terms "proximal" and "dlstal" are relative and interchangeable untll the strand is ultimately posltioned in an apparatus. The opttcal flber 12 is composed typlcally of glass or plastic and Is a flexible entity able to convey l1ght energy introduced at elther of its ends 16.18. Such optical fibers 12 are conventionally known and commerctally available. Alternatively. the user may hlmself prepare optical flbers ~n accordance w~th the conventlonal practices and tcchniques reported bv the scient~c and industrial literature. For these reasons. the o,otical flber 12 Is deemed to be conventionally known and available as such.
It~will be~appreclated that Fi~s. 1-2 are Illustrations in which the features have been purposel~ ed and exaggerated bevond thelr normal scale in order to pro~ l(It l)oth clarlty and visualizatlon of . . .

WO 93/21513 2 1 1 1 8 3 ~ Pcr/us93/o3448 extreme detail. Typlcally. the conventional optical flber stral1d has a cross-section diameter of 10- l ,000 mlcrometers and is routinely .employed In lengths ranging between centimeters ~in the laboratorv) to kilometers (In fleld telecommunlcations). Moreover, although the 5 optical flber Is illustrated vla Flgs. 1-2 an extended cyltnder having substantially clrcular prox~mal and dlstal end surfaces, there is no requlrement or demand that thls speclflc conflguratlon be maintained. To the contrary, the optical flber may be polygonal or asymmetrlcally shaped along its length: provlde speclal patterns and 10 shapes at the proximal and/or dlstal faces: and need not present an end surface which ts substantlally planar. Nevertheless, for best results, It Is presently belleved that the substantlally cylindrical rod-llke optlcal flber strand having planar end surfaces Is most desirable.Each optlcal flber strand 12 is deslrably, but not necessarily.
15 indlvldually clad by cladding 26 axiaUy along Its length. This cladding 26 Is composed of any material wh~ch has a lower refractlve Index and prevents the transmlsston of light energy photons from the optlcal fiber 12 to the external envlronment. The claddlng materlal 26 may thus be composed of a vartety of radically different chemlcal 20 formulatlons Includlng varlous glasses, silicones, plastlcs, cloths.
platings, and shielding matter of dlverse chemlcal composltlon and formulatlon. The manner In whlch the optical flber 12 is clad is consequentlal and of no Importance to the present inventlon. Those methods of deposition, extruslon, palnting, and coverlng are 25 sclentifically and industrtally avallable; and any of these known processes may be chosen to meet the requlrements and convenlence of the user. Moreover, the quantlty of claddlng employed need be only that minlmal amount whlch effectively prevents llght energy conveyed by the~ optlcal flber 12 from escaplllg Il1to the general surroundlngs. It 30 ~nll be recognlzed and appreclated therefore that the depth of cladding 26 as appears wlthin Figs. I and 2 respectlvely Is greatly exaggerated and purposely thlckened ln order to show the general relationship:
and Is wlthout scale or preclse ratio between the claddlng 26 and the optlcal flbeF 12.

WO g3/21513 2 1 1 1 8 3 8 Pcr/US93/03448 It will also be recognlzed that the conflguratlon of the claddillg 26 as appears wlthln Flgs. 1 and 2 has been shaped as a round coatlng as a preferred embodlment only. Alternatlvely, It is often desirable that the cladding take shape in specific multi-sided and 5 regular geometric forms such as a round. oval. clrcular, or even irregular shape. The illustrated conflguration. however. is merely one ~, embodlment of the cladding 26 as it extends co-axially along the length of the optical flber strand 10. For purposes of added clarity also. Fog. 1 reveals the Indlvidually clad. optical flber strand 12 in 10 par,tial cross-sectlon vlews to demonstrate the relatlonshlp between the optical flber 12 and the claddlng 26 whlch Is coextenslve along Its length.
The user also has a variety of cholces at hls discretlon regarding the conflguratlon of the dlstal end 16 of the optlcal flber strand 12 as 15 Is demonstrated by ~Igs. 3A and 3B. As seen via Fig. 3A. the distal end 16 Is substantlally cyllndrlcal In shape and destrably presents a surface 20 whlch Is substantlally planar and smooth. As an alternatlve in Flg. 3B. the dlstal end 30. while malntalning its substantlally cylindrlcal shape. nevertheless provldes a very dlfferent 20 end surface for the optlcal flber 12. The surface 32 Includes a depresslon or well 34 which extends into the substance of the optical fiber 12 at a depth typlcally of several micrometers. Although the well 34 appears substantlally circular within Flg. 3B. oval or irregularly conflgured depresslons may also be employed as flts the needs or 25 convenlence of the user. Simllarly, the vold volume of the well 34 from tts greatest depth to the surface 32 may also be cons~derably varled.
It wlll be recognlzed and appreclated as well that the range and variet~f of dirncnslonal and conflguratlol1al dlvergence for the strand 30 end Is limlted only by the user's abillty to subsequently dlspose and Immobillze a polarlty-sensltlve dye composltlon/formulatlon on the Intended distal surface of the optlcal fiber 12. The alternatlve iUustrated by Flg. 3B urlU lncrease the quantlty of dye materlals deposlted and also permit a greater sllrface area of dye for reactlve WO 93/21513 Pcr/uss3/o3448 contact on the surface for speclfic us~d àssay applicatlons. Il~
some embodiments. the greatest possible surface area configurations of the distal end surface may be highly desirable; nevertheless. for most general assay purposes. both quantltatlve and qualltative. the 5 intended distal surface illustrated w1thin F'lg. 3A as a substantlally planar and smooth surface Is deemed to be sultable and deslrable.
For general constructlon of the optlc flber sensor and for most purposes and appllcations of the Improved optical detectlng system and procedures descrlbed herelnafter. It is destrable to employ the - 10 Indlvtdually clad. flber optlcal strand Illustrated by F`igs. 1. 2A. and 2B.

B. The Polarltv-Sensltlve or Solvachromlc Dye The second cr1tlcal requirement and feature of the present flber 15 optic sensor Is the presence of at least one polarity-sensitive or solvachromlc dye immobllized at the intended distal end of the optical flber strand. Solvachromlc dyes. regardless of spec~flc compositlon and formulatton. are Identifled and deflned in operatlonal terms as a llght energy absorblng substance whose absorption and/or emiss~on 20 spectra are sensltlve to and altered by the polarity of their surroundlng environment - includltlg gaseous. Ilquld. and/or solid molecules and lons whlch are temporarlly or permanently present in the Immedlately ad~acent spatlal volume. The term "solvachromlc" is derlved from the recognlzed and lollg establlshed characteristics of 25 many fluorophores whose fluorescence emission spectra are sensitive to the polarity of the solvents in which they are employed or found.
For example. if the emission spectrum of a fluorophore such as ANS
( 1 -anilino-8-naphthalenesulfonyl acid) ~s examined in different solvents of varytng polarlty. one fillds that the emlsslon spectrum 30 shifts to shorter wavelengths ~blue shifts) as the solvent polarity is decreased. Conversely. Increasillg solvent polarlty generally results In shifts of the emlsslon spectrulll of tlle fluorophore to longer wavelengths ~red sh~fts). Red sllif~x ?re often. but not always.
accompanled by a decrease in ttl~ l)tum yleld or total of photons WO g3/21513 ~ 3~ PCI`/US93/03448 emitted for the fluorophore being evaluated, This phenomenol1. the change in emission spectrum of manv fluorophores with respect to different solvents of varving polaritv. is well descrlbed bv the follow publications: Joseph R. Lakowicz, Princ~ples of Fluorescence Spectroscopv, Chapter 7, Plenum Press. New York. 1983. pp 187-25~:
Mataga etal., Bull. Chem. Soc. Jpr. ~:465-470 (1956); ~akh~shiev.
N.G.. Opt. Spectrosc.10:379-384 (1961), and Opt. Spectrosc. 12:309-313 (1962). and Opt. Sectrosc.13:24-29 ~1962); MacCregor, R.B. and G. Weber, Proc. N.Y. Acad. Sci. 366:140-154 (1981).
While the best known examples of solvachromic dyes are fluorophores. the membershlp of this class as a whole Includes both absorbers or chromophores as well as fluorescent molecules. The essentlal property common to each and every member of this class of dyes is that the chosen dye substance change its spectral properties when exposed to dlfferent solvents of varying polarlty. For fluorophores. this spectral change can include elther an emission intenslty change or a change in the wavelength of the emitted fluorescent llght. For an absorber or chromophore dye, the intensit~, of color may change or the absorptlon spectrum of the dye may shlft elther toward the red or the blue elld of the spectrum. To determine whether a chosen dye composltion is a member of the class defined as a solvachromic dye. the test is solely an empirical one, When the dve Is exposed to dlfferent organic solvents of varying polarlty, the dye changes lts color whlch is empirically observed as a spectral change.
P 25 Thus. an absorber dye demonstrates a spectral change through its color, elther by alterlng the Illte~lsity of the color or by the obse'rvatlon of an actual color change, Alternat~vely, a fluorescent d~ e demonstrates its sensttlvity to dlfferellt solvents of varylng polaritv threugh changes in elther Its absorbing excitlng light; or by a chan~e in wavelength of the emitted llght: or bv a change in the intensitv of the emltted llght.
By thls operatlonal definitioll alld the empirical test method through whlch any person of ordillar~ sklll In thls art may identifv a chosen dye substance as being a sol-acllromic dye, it w~ll be WO 93/21513 2 1 ~. 1 8 3 ~

, 1 recognlzed and appreclated that the terms ~solvachromic~ and ''polarity-sensitlve'l are directlv retated and often interchangeable. Tlle meaning of each of these terms. however. is not exactl-, alike To the contrary, the term ~'polarity-sensltive dye~ deflnes and identifles a dye 5 formulatlon which is not only sensitive to different solvents of varv~ng polarity. but also to any other organic entity. molecule. or substance which has a discernable - that is. a demonstrable or determinable -polarity. Thus. organic compos~tions. compounds, and formulations of varving polarity which are not solvents as such are clearly 10 encompassed and included by thls term in add~tion to those composltlons whi~h are classlcally deflned as "organlc solvents."
Thus. organic solvents constltute merely one group or famtly w~thin the membership as a whole for the class of organic analytes having a discernable polarlty. In thls manner. while tt Is most convenience tO
15 test and evaluate a chosen dye using a pluraltty of solvents of varying polarlty to empirlcally dcmonstrate that the chosen dye is spectrally tnfluenced and altered by the polarity of the surrounding envlronment. any other klnd or type of organic molecule may also be employed to demonstrate the spectral sensltlvity of the chosen dye -20 albeit under less convcnient and/or more rigorous test conditions.
To demonstrate thc range and diverslty of the membershlpcomprising the class as a whole which constitutes polar~ty-sensitive or solvachromic dyes. a non-exhaustive listing of representative examples if provided hereinafter bv Tables 1 and 2 respect~vely. Table 25 1 provldes a representative l~st of polaritv-sensltive fluorophores.
Correspondingly. Table 2 provides a rallge of illustrative examples which are polarlty-sensitive absorber or chromophoric dyes.

WO 93t21513 Pcr/Us93/03448 h~ ') 2 Table I
POLARITY-SENSITIVE F'LUOROPHORES

Phospholipid Fluorophores N-(7-nltrobenz-2-oxa-1.3-diazol-4-yl) dipalmlttcyl-L-a-5 phosphatidylethanolamine (NBD-PE) N-(5-fluoresceinthlocarbomoyl) dipalmitoyl-L-a-phosphatidylethanolamine triethylammonium salt (fluoresce in - PE ) N-t6-tetramethylrhodaminethiocarbamoyl) dipalmitoyl-L-a-phosphatldylethanolamlne trlethylammonium salt (TRITC DPPE) 10 N-(Lissamine rhodamine B sulfonyl) dtpalmitoyl-L-a-phosphatldylethanolamine trlethylammonium slat (rhodamine DPPE) N-~Texas Red sulfonyl) diolsoyl- L-a- phosphatidylethanolamine trtethylammonium salt 15 N-lTexas Red sulfonyl) dipalmltoyl-L a-phosphatidylethanolamine triethylammonium salt (Texas Red DPPE) 3-palmltovl-s-(1-pyrenedecanoyl)-L a phosphatidylcholine (10-py-PC) N (5-dimethylamlnonaphthalene- 1 -sulfonyl) dipalmitoyl-L-a-p~osphatidylethanolamine trlethylammonium salt 20 N-(l-pyrenesulfonyl) dlpalmitoyl-L-a-phosphatldylethanolamine triethylammonium salt N-(6-t5-dlmethylaminonaphthalene-1-sulfonyll amino) hexanoyldlpalmitoyl-L-a-phosphatldylethanolamine trlethylammonium salt 25 N-(biotinoyl) dipalmitoyl- L-a- phosphatidylethanolamine trlethylammonium salt i Ani~n~c Fluorophores cis-parinarlc acld 30 trans-parinaric acid p-((6-phenyl)-1.3.5-hexatrlenyl) beIlzoic acld (DPH carboxvlic acid) 3-(p-(6-phenyl)- 1 .3.5-hexatrienyl) ~llell~ lpropleonic acid (DPH
prop~onic acld) , ~

WO 93/21513 PCI~/US93/03448 3 g l-pyrenecarboxylic acid l-pyrenebutanolc acld (pyrenebutyric actd) l-pyreneonanolc acld I-pyrenedecanolc acld 5 l-pyrenedodecanolc acid l-pyrenehexadecanolc acld 1 1-( l-pyrenesulfonyl) amlno) undecanotc acld 2-(9-anthroyloxy) palmltic acld (2-AP) 2-(9-anthroyloxy) stearlc acld (2-AS) 10 3-(9-anthroyloxy) stearlc acld (3-AS) 6-(9-anthroyloxy) stearlc acld (6-AS~
7-(9-anthroyloxy) stearlc acid 17-AS) 9-(9-anthroylo~r) stearlc acid (9-AS) 1 0-(9-anthoyloxy) stearlc acld ( I 0-AS~
1 l-(9-anthroyloxy) undecanolc acid ( 1 1 -AU) 1 2-(9-anthroyloxy) stearlc acld ( 1 2-AS) 12-(9-anthroyloxy) olelc acld ( I 2-A0) 16-(9-anthroyloxy) palmltlc acld (16-AP) 9-anthraceneproplonlc acld 9-anthracenedodecanolc acld l-pe~ylenedodecanolc acld 6-(N-(7-nltrobenz-2-oxa- 1 .3-dlazol-4-yl) amino) haxanolc acld ( N BD
hexanolc acld) 12-(N-methyl-N-((7-nltrobenz-2-oxa- 1 .3-dlazol-4-yl) amlno) dodecanolc acid 1 2-(N-methyl-N-((7-nltrobenz-2-oxa- 1 .3-d~æol-4-yl) amino) octadecanoic acld 1 2-(N-(u-nltrobenz-2-oxa- 1 .3-dlazol-4-yl) amlno) dodecanolc acld 11 -(9-4a~bazole) undecanolc acld ( I I -CU) - 30 11-((5-dlmethylamlnonaphthalene-1-sulfon~l) amlno) undecano~c acid 5-(N-dodecanoyl) amlnofluorescein 5-1N-hexadecanoyl) amlnofluorescein 5-(N-octadecanoyl) amlnofluoresce~
5-(N-hexadecanoyl) amlnoeosln wo 93/21513 Pcr/us93/~.~448 `~,Jll`l838 I -anilinonaphthalene-8-sulfonic acid ( I ,8 -ANS ) 2-anlllnonaphthalene-6-sulfonic acid 12,6-ANS) 2-(p-toluldlnyl) naphthalene-6-sulfonic acid sodium salt (2.6-TNS) 2-(N-methylanilino) naphthalene-6-sulfonic acld sodium salt (2,6-MANS) bls-ans (I,l'-bl(4-anlllno) naphthalene-5.5'-disulfonic acid, dlpotasslum salt) I-pyrenesulfonlc acid, sodium salt 2-(N-octadecyl) aminonaphthalene-6-sulfonlc acld, sodlum salt Catlonlc Fluorophores 1,1 '-dlhexadecyloxacarbocyanlne, perchlorate ~DIOC 16(3)) 3,3'-dloctadecyloxacarboxyanine perchlorate ("DiO", DIOCI8(3)) 1,1'-dldodecyl-3,3,3',3'-tetramethylindocarbocyanine, perchlorate 15` (DIIC 12(3)) l,l'-dlhexadecyl-3,3,3'.3'-tetramethyollndocarbocyanine perchlorate (DIIC 16(3)) l,l'-dioctadecyl-3,3,3',3'-tetramethyllndocarbocya,nlne perchlorate (":DII", DIIC18(3)) 1,1'-dldocosanyl-3.3.3',3'-tetramethyllndocarbocyanlne perchlorate ( DIIC22 (3)) 1,1'-dioctadecyl3,3,3',3'-tetramethylindodlcarbocyanine perchlorate (DIIC 18(5)) 3,3'-dloctadecylthlacarbocyanille perchlorate (DISC~8(3)) octadecyl rhodamine B, chloride salt (R 18) rhodamlne 6G, octadecyl ester, clllor~de rhodamlne 101, octadecyl ester, clllorlde N-4- ~dldecylamlnosty~l)-N-nlett~lpyrldlnlum`lodlde (4-dl-10-ASP) 1-(4-trlmethylammonlumphenyl) 6 ~ enyl- 1 .3,5-hexatriene. p-toluenesulfonate (TMA-DPH) 6-palmltoyl-2-(((2-(trlmethyl) allllllolllt~ ) ethyl) methyl) amino) naphthalene. chlorlde (PATMA~
l -pyrenemethyltrlmethylammoll lll lll lodlde WO 93/21513 2 ~ 1 t ~ 3 8 Pcr~US93/0~8 I-pyrenebutyltrimethylammon~um bromide 3-(-anthracene) propyl trimethylammonium bromlde acridlne orange-10-dodecyl bromlde (dodecyl acrldlne orange) acrldine orange-lOnonyl bromide (nonyl acridlne orange Neutral Fl~hQ~
1 ,6-dlphenyl- 1 ,3.5-hexatrlene (DPH) 1 -phenyl-6-((4-trlfluoromethyl) phenyl)- 1 .3.5-hexatr~ne ~C F`3 - DPH) p~ladlum dlsodlum allzarlnmonosulfonate (Pd(QS)2) Nile Red or 9-dlethylamlno-SH-benzol l phenoxaztne-5-one 6-proplonyl-2-dimethylamlnotbaphthalene (prodan) 6-dodecanoyl-2-dlmethylamlnonaphthalene (laurodan) N-phenyl- 1 1 -naphthylam~ne l,10-bis-(1-pyrene) decane 1,3-bts-(1-pyrene) propane p-dlmethylamlnobenzylldenemalononltrlle N-(5-dlmethylamlnonaphthalene-1-sulfonyl) hexadecylamine N-(5-dlmethylaminonaphthalene-1-sulfonyl) dlhexadecylamlne 4-(N,N-dlhexadecyl) amlno-7-nitrobenz-2-oxa=1,3-diazole (NBD
dlhexadecylamlne) 4-(N,N-dtoctyl) amino-7-nltrobenz-2-oxa-1,3-diæole (NBD-dloctylamine) 4-(hexadecylamlno)-7-nltrobenz-2-oxa- 1 ,3-dlaxole (NBD
hexadecylamine) 1-pyrenecarboxaldehyde 1-pyrenenonanol 7-dlmethylamlno-4-pentadecylcoumarin cholesteryl anthracene-9-carboxylate l-pyr_~emethyl 36-hydroxyl-22,23-btsl1or-5-cholenate (PMC) l-pyrenemethyl 38-(cls-9-octadecet~oyloxy)-22,2S-bisnor-5-cholel1ate (PMC oleate) 25-(NBD-methylamlno)-27-norcl1olesterol INBD~MANC) 25-(NBD-methylamlno)-27-norcholestervl oleate (NBD-MANC oleate) 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-~-yl) amlno)-23,24-blsnor-5-, WO g3/21513 ~,~ Pcr/uss3/o344x 21~ 3~
cholen-38-ol 2 6 22-(N-17-nltrobenz-2-oxa- 1 .3-dlazol-4-yl) amino)-23-24-bisllor-5-cholen-38-yl linoleate N-(3-sulfopropyl)-4-(p-d~decylamillostyryl) pyridintum. inner salt 5 (DllOASP-PS) 3-(N.N-dimethyl- N-( I -pyrenemethyl) ammonlum~ propanesu If onate .
lnner salt 4-(N.N-dlmethyl-N-(l-pyrenemethyl) ammonium) butanesulfonate, Inner salt 10 N e-(5-dlmethylamlnonaphthalene-1-sulfonyl)-L-lyslne (dansyl Iysine) WO 93/21513 ~ 8 3 Bcr/US93/03448 Table 2 POLARITY-SENSITIVE CHROMOPHORES

Phospholipld Chromophores 5 2~3-dlphenylhexatrienyl) propanoyl-3-palmitoyl-L-a-phosphatidyl choline (DPH-PC) N-(6-(blotlnoyl) amino hexanoyl) dlpalmltoyl~L a-phosphatidylethanolamlne trlethyl ammonlum salt (blotin-X-DPPE) N-t(4-maleimldylmethyl) cyclohexane-1-carbonyll dipalmltoyl-l,-a-10 phosphat~dyl-ethanolamlne trlethylammonium salt (MMCC-DPPE) N-(~2-pyrldyldlthlo) proplonyl) dlpalmltoyl-L-a-phosphatldyl-ethanamlne trlethylammonium salt :
AnlQnic ~ 2mQ~orç~ .
15 1 5-phenylpentadecanolc acld 5-(N-hexadecanoyl) amino fluorescein d~acetate :
:

.

WO 93/21513 ~ X 3 8 Pcr/us93/o3448 C. The Plymeric Material The third and final required compol1ent comprisillg the fiber optic sensor is the existence of at least one polvmeric material Immobilized at the distal end of the optlcal flber strand such that tl~e 5 Immobilized polarlty-sçnsltive dve is contained wlthin. that is -dlspersed, enclosed. and/or encompassed by - this polymeric material.
There are two characterlstics and functions for the polymeric material as It relates to the sensor construction and performance. The flrst characteristic and functlon is the primary role of the polvmeric 10 materlal - captur~ng the organlc analyte of Interest to be detected.
Thls capture function and capablllty is performed by absorbing and partltlonlng the organlc analyte of interest wKhln the substance and thlckness of the polymeric material itself as It lles immobilized at the distal end of the optical flber strand. The absorption and partition 15 occurs between the ,vapor or liquld phase of the fluid sample and the plymeric materlal formlng one component of the sensor construction.
The ~ partltionlng of the organic analyte of interest may be simllar withln the fluid sample and in the plymeric material. that is the concentratlon of vapor in each of these two phases may be the same:
20 or more likely. one of the two will be enrlched ~n concentratlon of the organic analyte relattve to the other. Under ideal ctrcumstances. the polvmeric materlal layer will serve to concentrate the organic analvte of interest vla Its superlor solubility characteristics relative to the vapor or liquld phase. In preferred embodiments of the flber optic 25 sensor comprising the present invent~on. the polymeric material will concentrate the organic analyte. which in turn. increases the sensitivity and detectlon limit of the sensor as a unit.
The second function and characteristic of the polymeric mat~r~al. whlch wlll not be present to a similar degree in all 30 embod~ments of the flber optic sensor, is the spectral influence exerted by the polymeric material alld its ablllty to alter or modifv the spectral characterist~cs of the d,ve ~ depel~dent and separate from the spectral influences and conseq-lellces caused by the organic analyte of Interest. This second prol~ert,v and characterlstic will ofte WO 93/21513 Pcr/uss3/o3448 ~9 ~11838 valy ur~th the degree of polaritv or the non-polaritv of the polvmer material as individually chosen for use in constructing the specific e~bodiment. Polaritv as such. however. is not the sole propertv or mechanism bv which the dye s spectral properties are mediated or affected. Thus. the properties of the polymerlc materlal containing the tmmobllized polarity-sensltive dye at the dtstal end of the optical flber strand mav or mav not ttself alone intluence and alter the spectral characterlstics ofthe immobtlized solvachromic dye apart from and prior to Introduction of an organlc analyte in a fluld sample.
It wtll be noted. however. that the essentlaa value and ctrcumstance lies tn the polymeric materlal tnteractlng wtth the immobilized poiart~y-sensltive dye and thus provlding a background or baseline of dye Interact~on and of dye spectral properties against whtch all other or subsequent optical determlnations and measurements are made and compared. As a consequence of the polarity-sensitive dye being contained. dispersed. or otherwtse immobtlized within a parttcular polymeric material at one end of the opttcal flber strand. a background or baseltnc set of spectral properttes for the immobilized and contained dye is produced whtch are the result and consequence of only the tnteraction between the polymertc matertal and the polarity-sensltlve dye. It Is this baseltne or background set of spectral characteristlcs against which all optical determinations and changes ~n spectral prope~ties are subsequently made and measured for the detectiol~ of an organic analyte of interest.
Accordtngly. when the fullv constructed flber optlc sensor is then placed in contact ~,vith a fluid sample belleved to contain one or more-organic analytes having a~ ducible or fixed polarit~. the organic analytes become captured. absorbed, and partltioned by the pol~neric material and generates l~larked changes in the spectral properties of the Immobllized polarltv-sensitlve dye In the sensor.
Thus. directly as a result of the orgallic analyte's absorption and part~tloning by the polymerlc la~-~r. ~ s~ectral llght absorblng and light emltting characterlstlcs of ~ obilized dve become changed from its background or baseline ~.nl~l(l.lr(l provlded by the effect of the -WO g3/21513 Pcr/US93/O344g ':~'11183'8 polymeric material alone.
There is a large and diverse range of polymeric materials sultable for use when constructing the embodlments of the presellt flber opt~c sensor. Many of these polymeric materlals have been 5 previously syntheslzed. characterized chemically. and are often commerclally prepared. A representative, but non-exhaustive listing of polymerlc materlals sultable for use when constructlng the present flber optlc sensor Is presented by Table 3 below.

WO 93/21513 ~2 1 1 1 8 3 8 Pcr/US93/03448 Table 3 POLYMERIC MATERIALS

Silicones and Silicon-Containing PQlvmers Monomeric and ollgomeric flulds (including sllahydrocarbons) 5 Polydimethylslloxanes - conventional flulds Polydlmethylsiloxanes, silanol and moisture cure prepolvmers Polydlrnethylslloxanes. vinyl termination Polydimethylslloxanes. functional terminatlon Polydimenthylsiloxanes. vinyl functional copolymers 10 Polydimethylsiloxanes. copolymers with functlonal groups T-structure polymers wlth functlonallty Organohydrosiloxane polymers and copolymers Polymethylalkylslloxanes -Fluoroalkylsiloxanes 15 Aromatic ~phenyl containing~ siloxanes Aromatlc polymers with functiollal groups Aromatic substltuted alkyl polyslloxanes Sliicone gums Non-siloxane-slloxane copolymers 20 Polysilanes Polysilazanes Polyalkoxysiloxanes-Polys~licatse (including sol-gel intermediates) T-resins and ladder polymers Silane-modifled polymers (including polymer~c coupling agents) 25 Other Polvmers polyethylene p~rpropylene polymethylmethacrylate polystyrene 30 polyhydroxyethylmethacrylate polyurcthanes polyvinylc~lorlde WO 93~21513 PCI`/US93/03448 polyvinylidene chloride nuorinated polyoleflns chloronuoropolyoleflns polysubstituted siloxanes 5 Parafllm ~ copolymers of the above llsted compounds . . .

WO 93/21S13 Pcr/uss3~o3448 33 '~ 1 11838 D. Mechanism of Flber Optlc Sensor O~ F~lnctiol1 The sensors descrlbed herein are not controlled in operatioll or functlon by any particular mechanism of actlom The spectral changes exhibited by the sensors which may be operative. wlll Illclude:
(1) polarity changes in the polymeric materlal generated by the organic analyte of tnterest whlch consequently can impart changes in the spectral properties of the dyes. as these dyes are sensitlve to polarity:
(2) concentration quenching wherein dyes can associate with one another and through this association dlmlnlsh thelr llght Intenslty.
the degree of associatlon belng Influenced by the presence or absence of the organlc analyte: ~3) changes orientational In nature. in whlch the polymer. In the presence of the organlc analyte. orients the dye In a parttcular way whlch creates an envlronment for changes spectral propertles; and~4) swelllng in whlch the distance between dye molecules changes as a functlon of the volume change In the polymerlc materlal caused by the Introductlon of the organlc analyte.

Il. Organlc Analytes Having a Dlscernible Polarlty The analytes whlch may be optlcally detected and measured using the present Invention Indtvldually and collectlvely share several charactertstlcs and propertles. The flrst and foremost property Is that the organlc analyte have a dlscernible polarity. The polarlty includes polart~r of bonds caused by two atoms ~oined by a covalent bond whlch share electrons unequally; and the polarlty of molecules whlch occurs if the center of negatlve charge does not coincide wlth the center of positive charge within the molecular structure and thus - constltutes a dipole.
The second commonly shared characteristic of the organic analyte~ havlng a dlscernible polarlty is that they may In fact be in any phystcal state - that Is in a gaseous. I~quld. or even in a fluld-solid state. It is required that the orgal~ic analyte be able to mlgrate wtthin or be carried by a fluld sam~)le: to be absorbed and at least partially partltloned by the polymeric l~aterlal immobiltzed at the tip ofthe sensor: and that the absorbed al~d partitloned analyte of -WO g3/21513 Pcr/usg3/o~

nterest present in the polvmerlc material layer meaningfully alter or modi~ the baseline set of spectral properties generated by the interaction of the Immobllized solvachromic dye with the polymer~c material which exlsts prior to introductioll of the analyte of interest.
Thus. so long as the organic analyte of interest has a dlscernlble polarity and ls In a moblle and transportable state wherein it can be conveyed. that organic analyte may be detected. identifled. and determined optlcally by the present inventlon.
The ma~ority of analytes sultable for detection by the present Invention are expected prlmarlly to be In the vapor or liquid physlcal states; and. moreover. that these be recognized conventlonally as organ1c solvents whlch are well known and employed in research and industry. Nevertheless, such organlc substances whlch appear as fluid sollds in the fleld or in-situ are also suitable for detection and measurement using thepresent invention.
The third common property shared arnong the membership of organlc analytes of discernible polarity is that they are primarlly but exclusively hydrocarbons. Such hydrocarbons are composed primarily of carbon and hydrogen atoms: but may also contain one or more heteroatoms selected from the group consisting of nttrogen.
oxygen. sulfur. and halogen atoms. These hydrocarbons. wlth or wlthout one or more heteroatoms. may be saturated or unsaturated;
may take shape as llnear. branched. ring, or polycycllc structures: and present format whlch Include aliphatlc and aryl hydrocarbon structures or combinattons of these. Moreover. It Is intended and expected that the hydrocarbon molecule as a whole. exclusive of any heteroatoms which may opt10nally be present. will comprlse from I to about 25 carbon atoms in total; and that with1n thls range of carbon atoms. o,ne or more degrees of satllratioll: llnear. branched, and rlng entlties, and multlple structural forll~at wtll be present.
Since one of the ma~or Intell~lecl a,oplicatlons and advantageous uses of the present Inventton w~ `itllill the environmental area.
with partlcular emphasls upon ,v~ ter sources and so~l and water contamlnation from Induslrl;~l ~o~lrces. the flber opt~c sensor ' ' ~

WO g3/21513 Pcr/us93/03448 35 '~ 838 and method of detectlon are particularly valuable for the detectioll of hydrocarbons principally present 1l1 petroleum products. These include petroleum aromatlcs. tlaphthalenes. parafflns. alld olefil~s whlch are present withln crude oil or derived as petroleum products.
The flber optlc sensor is particularly sensltive to and exceptionally able to detect lower molecular welght liquld hydrocarbons because such molecules have hlgh solublllty In and h~gh diffuslvitles wlthln the chosen polymerlc materlal - thus permltting rapid absorptlon and partltion by the plymerlc material and a measurable change In the spectral propertles of the immobillzed polarlty-sensltive dye within a reasonably fast response time. In comparlson. for organlc analytes which are normally gaseous (such as .
methane.'ethane. and ethylene) the sensor Is expected to have lower sensltivlty In response because of the lower solublllty of these analyte,s withln the polymeric materlals generally expected to be employed within the fully constructed sensor. Hlgh molecular weight liquid hydrocarbons would also be expected to take a somewhat longer time~ to,be detected in comparlson to low molecular welght hydrocarbons because of lower dlffuslvltles In the polymerlc materlal.
Regardless of the partlcular molecular welght of the entlty whlch Is to be detected using the present Inventlon, any organic analyte which can penetrate and be captured by the polymeric materlal of the sensor (and thus be absorbed and partitloned during its migratlon) Is suitab!e for detection using the present inventiom The dlfferences among the varlous hydrocarbons and other organic compounds would be only in the magnitude of thelr individual effects upo~the polari~r-sensltive dye: and the time requlred for the sensor to respond spectrally to the presence of the organlc analytes withtn the fluid sample.
~o demonstrate. a representatlve but preferred range of hydrocarbons sultablè for detectlon by the present lnventlon are in the llsting of Table 4 provtded below.

: .

:

WO g3/21513 Pcr/uS93/03448 ~. lil8'~8 36 Table 4 HYDROCARBONS FROM PETROLEUM SOURCES
SUITABLE FOR DETECTION

5 Aromat1cs such as benzene, toluene. the ~ylenes, ethyl benzene, naphthalene, anthracene. phenanthrene. plus thelr hydrocarbon derivatives;

Naphthenes (sat~trated cyclics) such as cyclohexane, tetralin. and thelr hydrocarbon derivatlves;

10 Prafflns (branched and stralght chain) su,ch as propane; normal and isobutane; all paraffinic isomers of C5. C6, C7, C8, C9, and C10;

Olefins such as propylene: the butylenes; all oleflnic isomers of C5, C6. C7, C8. C9, and C10:

Halogenated hydrocarbons comprlsing chlorine, bromine, fluorine, or 15 iodine; and ;
Hydrocarbons of up to 25 carbon atoms containing one or more carbonyl groups (-CO) forming aldehydes and ketones.

wo 93/21513 Pcrluss3/o3448 37 2~1~838 111. Means for Immobilizing the Polarity-Sens~tive Dye and the Polymeric Materlal The manufacture of the fiber optic sensor as described herein requires that the polarity-sensltive dye and the polymeric material 5 each be deposited and Immobillzed at the Intended dlstal end of at least one optlcal flber strand. Not only must each of these components be lmmoblllzed at the tlp of the optlcal flber strand: but also It Is requlred that the immobillzed polymeric material enclose and encompass the entirety of the polarity-sensltlve dye to achieve the 10 Intended constructlon organlzatlon. Thus. a hlghly desirable approach and method for manufacture purposefully combines the polarlty-sensltive dye with monomers. or copolymers. or prepolymers to form the polymer; and then polymerizes or cross-l~nks the mixture in-situ directly at the intended dlstal end of the optlcal flber strand.
15 By thls method. the polarlty-sensitlve dye is Intimately intermixed and dlspersed within the substance and thickness of the polymerlc materlal and does not present any dlscrete format or layer as such.
The preferred method of depositlon and Immobillzatlon Is vla a coating polymerizatlon and employs an admL~cture of monomers 20 and/or prepolymers wlth one or more pre-chosen polarity-sensitive dye as a formulation. Such admixture preparations typically comprise solutions of several monomers and/or prepolymers in admixture and a concentration of at least one polarity-sensitive dye. A
representative listing of dlfferent monomer and prepolymer 25 compositions sultable for preparing an admixture are given by Table 5.
Such admixtures subsequently can be polymerized or solidifled by solvent evaporation to form the desired polymer matrix. An illustratlve listing of polarity-sensltive dyes ready for admixture and polymerization is glven previously by Table I and 2 above. It will be 30 appreciated that conventionally kllown technlques of polymerlzation including thermal. free radlcal. and photopolymerlzation are known and available to the user.

Wo 93~21513 ~ Pcr/uss3/o3~4x Table 5 A. Monomers acrylamide N.N-methylene bis(acrylamide) hydroxyethylmethacrylate styrene v~nyl acetate N-(3-aminopropyl) meth-acrylam~de hydrochlorlde IKodak, Inc.l 10 ~. Comonomer Wlth Dimethvlsiloxane (acryloxypropyl) methyl (15-20%) (aminopropyl) methyl (3-5%) (methacrylo~ypropyl) methyl (2-3%) C. T-Structure Polvdimethvlslloxanes methacryloxypropyl (2S-50%
Vinyl (50-75%) D . Waxes/Preformed Polvmers paraffln polyvinyl alcohol . .

WO93/21513 211 ~ 8 Pcr/usg3/o3448 It wlll be appreclated that the listings of Tables 1, 2, and 5 are merely exemplary of the many dlfferent composit~ons which can be usefully employed in admixture with one or mare solvachromic dves.
In addltion, the scientlflc and industrial literature provides ma~l~
5 alternative monomer preparations and admixtures which are also suitable for use in making the present ~nvention. Accordingly, all of these conventionally known monomer preparations are considered to be wlthin the scope of the present lnventlon.

IV. A Preferred Embodiment of the F~ber Opt~c Sensor To demonstrate a most desirable method of making the unique flber optic sensor comprlslng part of the present Invention: and as a demonstratlon of the utillty and effectiveness for making optical determinatlons using a fully constructed prepared embodiment of the i5 fiber opffc sensor, a detailed descriptlon of the components and manlpulatlve steps for maklng a sensor able to measure volatile organlc compounds tn ground water and soll samples Is presented. It wlll be expressly understood, however, that the deta~led descript~on whlch foUows hereinafter Is merely illustratlve and representative of 20 the many dlfferent klnds of sensors which can be made having one or more polarity-sensitive dyes and polymeric materlals lmmobllized o one optical strand end surface.
Flber-Optlc Materlals: The optical flber strand used to construct the sensor was 600 um in diameter, 5 m in length, having a 25 numerlcal aperture of 0.37 and coated wlth a protective plastic ~acket ~Ensign-Blckford Optlcs Co., Avon, CT). The proximal end was coupled to a fluorometer by an Optimate Connector (AMP Inc., Harrisburg. PA). The dlstal end was stripped, cleaved, pol~shed, and then' cleaned with concentrated snlfllric acld.
Surface Sllanization: The distal tip of the flber was soaked in - 10% (v/v) octadecyltriethoxvs~lal~e ~Petrarch Systems lnc., Brlstol, PA) in dry acetone overnlght to impro~ )ol~mer adheslon. The flber was rlnsed wlth acetone and dried ~ ell for I hour at 100-C.
Sensor Constructlon: The ~ or conflguration and materials WO 93/21513 3 8 pcr/us93Jo344x are shown ~n Flg. 4. The sellsing laver was appl~ed by solvent evaporatlon. The distal tip was dipped into a solutlon colltail1ing 0.314 mM Nile Red (Molecular Probes. Eugene. OR) and 10% (v/~) Dow Cornlng (Lansing, Ml) dispersion coating compound in toluel1e 5 and allowed to dry. The Dow compound is a dlmethylsilicone polvmer - that has an inflnlte molecular welght when cross-llnked. This procedure was repeated untll the flber~s dlstal face was coated wlth a llght-plnk layer, approxlmately 10-50 um thlck as observed with a microscope. The dellcate dlstal end of the flber was fltted with a llght 10 impermeable, porous, stainless steet sheath to protect the sensing layer.
Fleld-Portable Instrumentatlon: A block dlagram of an apparatus constitutlng the portable hydrocarbon fluorometer is shown in Flg. 5. The Illuminating llght &om a qua~z halogen lamp 15 was foc,used tnto a 600 um dlameter flber and conducted to an optical coupkr, where the approprlate excitatlon wavelength was selected b~, a 540 nm bandpass fllter. A beam splltter was used to dlscrlminate and dlrect returnlng auorescence llght through a 600 nm longpass fllter before It Imptnged on a photod~ode. The signal was condltioned 20 electronlcally and output to a hard-copy port. The instrument was packaged ~nto an aluminum case. having the dlmensions 46 cm long 36 cm wide x 18 cm h~gh. welghing 13 kg, re~u~rlng 100 V, 60 Hz power for operation, Measurements: The laboratory data were collected with both a 2S research grade flber-optic fluorometer lLuo, S. and D.R. Walt, Anal.
Chem. 61:174-177 (1989)1 and the field-portable fluorometer. All samples were measured, except those In the fleld. by a static headspace technlque IRoe et~l.. Allal. Chem. 61:2584-2585 (1989)1.
All mcasurements are of the headspace above the aqueous phase.
30 Single-component standard solut~olls were prepared by dlssolving the approprlate amount (micrograms) of aromatlc hydrocarbon In 1 L of dlsttllcd water. A 250 mL volume of stalldard solutlon was added to 400 mL glass ~ar. kaving a headspace ~olume of 150 mL. The ratlo t0.60) of headspace volume to salllple ~olume was kept constant in all ~O 93/21513 2 1 ~ I ~ 3 8 Pcr/US93,0~8 measurements. The jars were fitted with a cover containing an alr-tlght rubber septum. To measure each sample. the sensor was pusllec through the septum. exposing the sensor's tip to the organic vapor partltioned in the headspace volume. The baseline fluorescence was 5 recorded in a sampling vial contalning only dlstllled water. The --voltage of the photodlode was recorded over tlme from a dlgltal dlsplay. All laboratory sampks were tested at a constant 25C by ustng a thermostated bath to obtain constant vapor pressure. Field data were collected in cooperation wlth Moriock Envlronmental 10 (Lebanon. NH) at Pease Air Force Base. Dover. NH.
Selection of Fluorophorc-Polymer Combtnation: The optical properttes of Nile Red allow It to be used readlly tn the detection of organic compounds. It has been used commonly as a lipophilic dye in staintng cells and membranes and as a solvent polarity indicator. -15 Although Its solvachromlc beha~or has been descrtbed. it has not been investtgated extensively. Its fluorescence excltatlon and emisslon maxirna va~y wtth the hydrophoblctty of lts mlcroenvlronment. For example. the emlsslon maxlma of Nile Red i heptane and acetone arc 525 and 605 nm. respectively. These optical 20 properties may be explolted by creating a microenvironment that is sens1tlve and susceptlble to changes in hydrophoblclty.
Slllcone polymers are highly permeable to gases and organic solvents IKesttng. R.E.. Svnthetic Polvmer Membranes: A Structural Perspective. Chapter 4. Wiley & Sons. New York. 19851. Therefore. an organlc-vapor senslng layer can be constructed by incorporating Nile Red tnto a thin slloxane polymer layer on the distal face of an optical flber: As the polymer layer Is exposed to organlc vapors, absorption causes the mlcroenvlronment of Nile Red to become more nonpolar.
res~tflg tn fluorescence enhancelllellt of the fluorophore.
Spectral Characteristlcs: Flg. 6 displays the Increase ln tntenslttes of both the excttattotl alld et~l~sslon spectra of a sensor placed In the headspace above tllr~ lifferent concentratlons of bcnzcne acqulrcd wlth thc resear~ ;trllment; and show the excltatlon and emlsston spectra ol .~ msor exposed to 0. 100~ and WO 93~21513 PCr/US93/034~8 211183~ 42 200 ppm benzene. The excitation spectra were collected by sca~ g the excitatlon monochromator from 400 to S50 nm and mollitorillg the emisslon at 580 nm. The emission spectra were collected by measuring the emisston from 530 to 650 nm~ using an excitation 5 wavelength of S00 nm. These spectra were taken by the method of sampling described under Measurements. The em~ssion slgnal Increases from 238.000 cps in 0 ppm benzene to 625.000 cps in 200 ppm benzene. This dramatlc increase in Intensity is caused by absorption of benzene Into the polymer mlcroenvtronment of Nile Red.
10 resulting in enhanced fluorescence.
Morcover. the fluorescence emlsslon maxlmum shlfts from approximately 560 to 570 nm. corresponding to the solvachromic sensit1v~ty of Nile Red. Thls shlft could be attributed to the benzene-absorbed polymer mlcroenvironment stablllzing the exclted state of 15 the fluorophore. shifting the wavelength maximum to lower energv and. therefore. Ionger wavelengths. This effect is consistent wlth stabltization of the exclted state In n-~T or tr~ eloctronlc trans~tlons.
Gencral Response Characterlstics: Flg. 7 shows the responses of a sensor to the indlvldual components of the.conventional BTEX
20 series (benzene. toluene. ethylbenzene. xylene) and unleaded gasollne at 100 ppm with the fleld portable instrument. The concentration of gasoline. being a multicomponent species. is def~ned as the number of microliters of gasoline per liter of water. No attempt was made to calculate the vapor-phase concentration. Although the sensor is 25 most sensltive to xylene ln the BTEX ser~es, It responds equally well to gasollne. indicatlng that the sensor responds generally to a wide variety of volatile organ~c vapors. These unequal responses to the BTEX series cannot be explained by the dlfferences in vapor pressure of t~ç BTEX series components~ but are most likely due to differences 30 of the indlvldual compounds ln their permeablllty coefflcients and solublllties in the polymer.
A typical sensor response to illcreaslng concentratlons of p-~ylene as a function of time can be seell ~n Fig. 8. The baseline response was measured in the headspace of a flask containing on~

wo 93/21513 ~ 3 gcr/us93/o3448 distllled water. When the sellsor is placed in a flask col1taining 1~-xvlene. a sharp rise ~n voltage occurs, followed by a slower leve~ g off as equllibrium is established between the headspace alld sensing-la~er vapor concentrations. Flg. 8 indicates that the sensor response time 5 is established ~n less than 2.5 mlnutes as deflned by the signal reachlng 90% of Its flnal value. The recovery times, defined by the signal decreaslng to wtthin 10% of the starting baseline values. are longer: for example, for 10 and 160 ppm the recovery times are 2.5 and 10 minutes, respectlvely. The desorption process is retarded probably 10 by nonspeciflc hydrophobic ~nteractlons between the absorbed organic vapor and the hydrophoblc polymer/dye layer. The rate-de~terminlng p~rocess Is the diffuslon of the vapor Into and out of the polymer laver.
restrlcting the sensor's response and dictating the frequency of sampling.
Flg. 9 shows callbration curves for xylene and gasoline, indlcating very good linearity in the concentration range of 10-160 ppm. The variation In slopes ~s due to sensitivtty differences of the sensor to ~ylene and gasoline. Below 10 ppm, vapor detection is posslble, but nonlinear behavior Is observed. The sensor can detect 1 ppm gasollne but cannot be used to make quantltative measurements due to the nonllnearit!y of the calibratlon curve In this reglon.
However. it is stlll very useful in situations that require Informat~on as to the presence or absence of a contaminant, such as in leak detection from underground storage tanks.
Temperature Dependence: The sensor response is related dlrectly to the vapor pressure of the organ~c component. During data colleetion on the samples Investlgated above, a constant temperature was maintained throughout the measurement process.
~ To investlgate thc effect of temperature on the baseline slgnal, a sensor was placed tn a sampllng v~al contaln1ng only dlstilled water and was submerged lnto a temperature-controlled water bath. As expected, the fluorescence slgnal decreased due to acceleratlon of the thcrmal relaxatton processes as the ten~perature was ra~sed from 4 to 30C as shown In Fig. 10. In contrast. ill the presence of xvlene -WO 93/21513 PCI`/USg3/03448 3 3 ~
vapor, the sensor shows an increase in response as temperature increases. Thls Is illustrated by Fig. l 1. This result can be explained by four effects influencing sensor response s~multaneously. F~rst, the vapor pressure of the organlc component increases wlth temperature.
5 providlng a greater headspace concentration. Second. the polymer layer structure may become more amorphous, causlng a decrease in poroslty and a greater exclusion of water vapor. Water vapor could act as an interference by lncreasing the polarity of the membrane.
Thlrd, ln most cases. hlgher operatlng temperatures Increase the 10 permeablllty coefflclent and decrease the actlvatlon energy of the dlffuslon process. Fourth, Is the temperature dependence of the fluorophore.
Initlal F~eld Data: The purpose of the Inlt~al fleld work was to -show that the sensor responds qualitatively to in-situ fleld 15concentratlons and that the system was field-hardened. No attempt ~;
was made to critlcally evaluate the sensor's performance wlth that of established fleld methods (I.e., gas chromatography or photoionization probe tPlDI). The field studies were performed In cooperation wlth ~orlock J3nvlronmental and were conducted at Pease 20 Alr Force Base. NH, at a slte contamlnated wlth JP4 ~et fuel. Four Indivldual wells were measured In-sltu wlth the flber-optlc sensor and Its supportlng instrumentatlon and these measurements were compared to slmultaneous readings from a portable Photovac TIP PID, with a 10.2-eV lamp. The PID measurements were used as a relative 25 indlcator of contarnlnation between sltes, allowing us to test the response of the sensor to in-sltu samples of different concentrations.
The sensor was calibrated with bellzene by the method described under Mèasurements and the PID was spanned between air and 100 ppm~aqueous solution) benzene standard. Thus, the reported values 30 In Fig. 12 for the PID are "benzelle equivalents," whlch should approximate the extent of contalllillatiol~ In each well. Flg. 12 shows the response of a sensor in eacl~ ll at a depth of 3,5 m below ground levcl, compared to concentratiol~ .sllred concurrently with the PID. The PID measurements ill(ll~ e ttlat the four drill sltes have wo 93/21513 ~ 8 3~/US93/03448 4, vary~ng degrees of contaminatiol1. The fiber-optic sensor responded comparably to in-situ concentrations of JP4 in each monltoring well.
Moreover. the sensor responded semiquantitattvely to the differel~t degrees of contarnination as deflned by the PID.
Attempts to compare critically the measurements between the two instruments must be preceded by a thorough tnvestlgation takis1g into account the varlous problems of calibration and sampllng. For example, PIDs are compound-dependent. being more sensitlve to aromatics than aliphatlcs. On the other hand. the sensor is less compound dependent because It Is based on analyte polarlty.
Therefore. any comparison must account for thls callbratlon disparit~.
The callbratlon Issue ts especlally Important In monltorlng multicomponent contaminatlon sltes. Since JP4 is composed of alkanes. alkenes. and aromatlcs. the response would depend on the sens~tivity of the sensor both to the individual components and to the components collect~vely.
The approach of uslng a microenvironmentally sensltive fluorophore and orgànlc vapor~ permeable polymer as a sensing mechanlsm has proven successful in the laboratory and from inltial fleld studies. The sensor responds to environmentally slgnlflcant .
levels of llght mononuclear aromatics lBTEX sertes) and gasoline in the laboratory and responds to in-situ samples of VOCs. From the wlde range of compounds studled. the sensor should generally respond to vlrtually any organlc volat~le compound. The approach descrlbed 2~ has several advantages: the sensors are inexpensive to construct and provlde true real-time. in-sltu measuremenes; sensors respond almost instantaneously to the presence of VOCs. enabling a large number of samples to be measured; their small slze allows smaller dlameter sampllng wells to be drllled; and sensors can be used in sltuations 30 where electrlcal devices pose risks.

:

WO 93/21513 PCr/USs3~03448 V. Alternative Embodiments of the Fiber Optlc Sensor The various polarity-sensitive dye/polymeric materlal combinations that were used are listed in Table 6. Each successive dye/polymer pair reflects and demonstrates the basic result.

_ WO 93/21513 Pcr/us93/03448 3 ~

Table 6 Palr Polarltv-Sensltive Dve Polvmeric Materlal Fluorescein dlssolved parafllm 2 Acrylodan dlssolved parafllm 3 Dansyl Iyslne dlssolved parafllm 4 Anthracene-9-carbox- dimethyl methylvinyl aldehyde carbohydrazone slloxance Octadecyl rhodamlne dlmethyl methylvinyl slllcone 6 Nlle Red dlmethyl sillcone , WO 93/21513 PCl~/US93/03448 A. The Acylodan-Pa_afilm Combination Uslng a vlscous solutlon of polymer and dye. a bare optlcal flber was dlp-coated to produce a thin layer of acrylodan/parafllm.
Acrylodan was selected because It is envlronmentally sensitive and 5 has been used to test llgand blnding. Parafllm was used because it is a wax-ltke substance and allows the absorptton of hydrocarbons.
Furthermore. It was readlly avallable. Flg. 13 shows the response of the fully constructed acrylodan/parafllm sensor to gasollne exposure.
The emlsslon spectrum decreases in Intenslty on exposure to toluene.
10 Thls sensor was tested~ on a xenon arc lamp research grade flber optlc --fluorometer. The values given by Table 7 show about a 16% decrease to Intenstty on exposure to 200 ul of toluene tn a 1.0 Iiter flask of alr.

~0 93/21513PCr/U~93/03448 49 ~1118~
Table 7 VALUES OF THE ACRYLODAN/PARAFILM SENSORS
EXPOSED TO TOLUENE

Concentra,tlon Time Kcps Concentratipn Time Kcps 3 207 15 l86 7 170 ~ 19 232 12 195 24 216 : :~

_ .

WO 93/21513 2 l l l 8 3 8 Pcr/us93/o3448 B. The Dansvl Lvsine-Parafilm Combination ~ig. 14 empirically shows that the fluorescence of the solvachromic dye decreases Ol1 e.Yposure eo pure toluene. This combination suf~ered from the same troubles as the previous acrvlodan/parafllm signal sensor - such as poor solubility itl the polymer; weak fluorescence: and a decreased signal on exposu~re to organic vapor. The data of Fig. 14 shows the emission spectrum of a dansyl Iysine/parafllm sensor exposed to tohtene.

C. The Anthracene-9-Carbox~taldehYde CarbohvdrazonelDimeth~l and Methvlvlnvl Slllçone Comb~natlon A third solvachromlc fluorophore. anthracene-9-carboxaldchvde carbohydrazone or "ACC" was used flrst with parafilm and then with a gas chromatography copoîymer. dimethyls~loxane and methylvinylslloxane. The emplrlcal results shown by ~ig. 15 shows the response of a sensor made out of the copolymer when exposed to 333 ul of toluene In 1.0 llter of air. It was evident that the large Increase in slgnal, averaglng 14%. was a vast improvement that could be attributed to the change in polymer.

D. The Octadecvl Rhodamine/l)imethvl and Methvlvinvl Silicone Comblnat~on It was deemed that a lipophilic dye would be a better choice of solvachromic fluorophore due to its improved solubility in organic solvents and deslrable solvachromic behavior. The first lipophilic dve evaluated was octadecyl rhodamille B chloride salt or `'ODR" . This llpophllic dye has several advantages over dyes tested previously: it has a greater solubll~ty In organic solvents; It does not partltioll O~lt of th,e polymer due to 1ts long octadecyl tail; It has excltation and emission peaks at longer wavelengths ~ex S40. em 580); and It has good photostabilit!,r.
Spectral Characteristtcs: The e.Yc~tation and emission spectra of ODR in the polymer are shown ill ~igs. 16 and 17. ~Ig. 16 shows the exc~tatton spcctrum of octadec~lrllodam~ne In the copolymer WO g3/21513 Pcr/uss3/o3448 51 ~i 11838 dimethyl and methy1 vil1yl siloxane ol1 exposure to gasoltne ( 100 ul of gasollne In I llter of air) taken at dlfferent tlme illtervals: ( I ) after minute: (2) after 2 minutes; al~d ~3) after 3 minutes. Ill comparisot1.
Flg. 17 shows the emlsslon spectrum of octadecylrhodamlne in the copolymer dlmethyl and methylvinyl siloxane on exposure to gasoline taken as dlfferent tlme Intervals (100 ul of gasoline In I llter of alr):
- ( I ) after 1 mlnute: (2J after 2 minutes: (3) after 5 minutes: and ~4) after 7 minutes.
On exposure to gasoline. the excitatlon and emlssion spectra both lncrease in lntenslty. After seven minutes the photon intenslty increased rom 15.000 to 45.000 counts per second. Thls Increase ls ~ . , ~. . .
belleved attrlbutable to the lncreased sensltlvlty of ODR to changes In lts microenvlronment. The maxlmum emlsslon wavelength was shlfted from 630 nm to 590 nm. indicating that ODR's solvachromic behavior Is p~eserved In the polymer.
- Response Characterlstlcs:
The response profile of an ODR/DM MV slloxane sensor to three concentrations of toluene ~s shown in Flg. 18. An Immediate increase In intenslty occurred oll exposure to vapor; and after one mlnute the sensor was withdrawn from the sample and the intensity returned to inltial basellne values. Thls proves that the polymer absorptlon process essentlally is reversible.
Figs. l9A- l9C show the response of this sensor to three different concentratlons of gasoline. Fig. l9A shows exposure to 1 ul gasoline ln 6 llters of air: Fig. I9B shows exposure to 10 ul gasoline in 6 liters of air; and ~Ig. l9C shows e.Yposure to 20 ul gasoline ~n 6 liter& of air. As the sensor is exposed to organlc vapor, the lntenslty rises. reaching a plateau at 49 ~ utes for 0.16 ppm. 1 10 mlnutes for 1.6 ppm. and over 180 mlnutes for 3.2 ppm. Indlcating equlllbrium condltlons have been achleved across the membrane. The equillbrlum intensi~y values can be used to ~el~erate a callbratlon plot as shown by ~tg. 20. The data are the meall ~t.mcl~rd deviation of three measurements. The curve was lill~ (1 lo tlle equatlon: Y = 3.06 x 104 I X 3.75 X 104. Because the till~ ) r~ ch equlllbrlum is relatively :
.

WO 93~21513 PCI/US93/03448 ~11183~ 52 long. a kinetic callbration plot can quantitate concentration.
In comparison. Flg. 21 illustrates slope calibratlon measured after 90 mlnutes of four different ODR/DM MV slloxane sensors. The data are the mean standard devlation of three measurements. The data were fltted to the equations - Curve A: Y = -1.78 x 10-1 + X 5.77 x 10-1: curve B: Y = -1.78 x 10 1 + X 5.80 x 10-l; curve C: Y = -5.83 x 10 2 + X 5.85 x 10-1: curve D: Y = -2.98 x 10 1 + X 3.78 x 10-1. Note that Flg. 21 shows the changes in slope that occur over the first 60 mlnutes of exposure are responslve to the dlfferent concentration of gasoline used.
It was deemed Important to work with dye concentratlons that produced strong fluorescence slgnals, and to Identiiy those whlch were most sensitlve to small changes In the concentration of absorbed vapor. To test for maximum response, a hlghly concentrated solution of solvachromlc dye was serially diluted by add~ng aliquots of gasoline. ~Ig. 22 shows dye concentratlon as a function of fluorescence slgnal and demonstrates a maximum Intenstty at a dllution correspondlng to 1.15 x 1O 3 M/l,. The dye concentratlon that produces the largest slgnal change with the smallest change In exposure corresponds to the concentr~tlon in Flg. 22 at the left edge of the peak: 1.7 x 10-3 M/L. Table 8 shows the average ratio change on exposure to acetone of flve different groups of sensors made wlth an Increasing concentration of dye. The ~ncrease in ratio indlcate the dye concentration in the polymer is important and should be optimlzed. The data acqulred on ODR-DM MV siloxane combination demonstrated concluslvely that the sensor was both operational and capable of continuous monitoring.

_ -WO 93/21513 PCr/US93/03448 21~183~

Table 8 SENSOR RATIO DATA OF OPTIMUM DYE CONCENTRATION

Dye Concentration Average Ratio . Group Molar (10-31 IY/In A 0.5 1.26 B 1.0 1.17 C 1.7 1.34 D 2.5 1.2 :
E 4.0 1.1 Sensors were exposed to pure acetone. The ratio ls lX/Io (lx is the intenslty on exposure to acetone and IO is the intensity before exposure.

_ WO 93/21513 Pcr/us93/o3448 The present inventton is not to be restricted in form nor limited in scope except by the claims appended hereto.

Claims

What we claim is:
1. A fiber optic sensor for detecting an organic analyte of interest in a fluid sample. said sensor comprising:
an optical fiber strand able to convey light energy of a predetermined wavelength. said optical fiber strand having a proximal end. a distal end. and a strand length:
at least one polarity-sensitive dye immobilized at the distal end of said optical fiber strand. said polarity-sensitive dye being able to absorb light energy of a predetermined wavelength; and at least one polymeric material immobilized at the distal end of said optical fiber strand such that said immobilized polarity-sensitive dye is contained within said polymeric material. through which at least a portion of such organic analyte as is presented by the fluid sample becomes absorbed by said immobilized polymeric material and a measurable change in the spectral properties of said contained polarity-sensitive dye is produced;
means for introducing light energy of a predetermined wavelength to the proximal end of said fiber optic sensor; and means for detecting light energy released by said contained polarity-sensitive dye.

3. The fiber optic sensor as recited in claim 1 or 2 wherein said optical fiber strand conveys exciting light energy of a first wavelength and emitted light energy of a second wavelength.

4. The fiber optic sensor as recited in claim 1 or 2 wherein said polarity-sensitive dye is a chromophore.

5. The fiber optic sensor as recited in claim 1 or 2 wherein said polarity-sensitive dye is a fluorophore.

6. The fiber optic sensor as recited in claim 1 or 2 wherein said polymeric material is a silicone based polymer.

7. The fiber optic sensor as recited in claim 6 wherein said contained polarity-sensitive dye is Nile Red.

8. The fiber optic sensor as recited in claim 1 or 2 wherein said polymeric material is selected from the group consisting of plyethylene, polypropylene, polymethylmethacrylate, polystyrene, polyhydroxyethylmethacrylate, polyurethanes, polyvinyl chlorides, polyvinylidene chloride, fluorinated polyolefins. parafilm. and chlorofluoro polyolefins.

9. The fiber optic sensor as recited in claim 2 wherein said means for detecting light energy is by detection of emitted light energy of another wavelength.

10. A method for detecting an organic analyte of interest in a fluid sample, said method comprising the steps of:
admixing the fluid sample comprising the organic analyte of interest with a fiber optic sensor comprised of:
an optical fiber strand able to convey light energy of a predetermined wavelength, said optical fiber strand having a proximal end, a distal end, and a strand length, at least one polarity-sensitive dye immobilized at the distal end of said optical fiber strand, said polarity-sensitive dye being able to absorb light energy of a predetermined wavelength.
and at least one polymeric material immobilized at the distal end of said optical fiber strand such that said immobilized polarity-sensitive dye is contained within said polymeric material, through which at least a portion of such organic analyte as is presented by the fluid sample becomes absorbed by said immobilized polymeric material and a measurable change in the spectral properties of said contained polarity-sensitive dye is produced;
introducing light energy of a predetermined wavelength to the proximal end of said fiber optic sensor whereby said light energy is conveyed to said distal end of said strand and said contained polarity-sensitive dye absorbs at least a portion of said light energy: and detecting light energy emitted by said contained polarity-sensitive dye at said distal end of said fiber optic sensor. said detected light energy being a measure of the organic analyte of interest in the fluid sample.

11. A method for making a fiber optic sensor able to detect a organic analyte of interest in a fluid sample. said method comprising the steps of:
obtaining an optical fiber strand able to convey light energy of a predetermined wavelength, said optical fiber strand having a proximal end, a distal end, and a strand length;
admixing at least one polarity-sensitive dye able to absorb exciting light energy of a predetermined wavelength with at least one polymerizable material to form a reaction mixture; and polymerizing said reaction mixture at the distal end of said optical fiber strand such that said polarity-sensitive dye is contained within an immobilized polymeric material, through which at least a portion of such organic analyte as is presented by the fluid sample becomes absorbed by said immobilized polymeric material and a measurable change in the spectral properties of said contained polarity-sensitive dye is produced.