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Development of the Indicator-PhotopolymerChemistries forMultianalyte Sensor ArraysBrian G. Healey, Suneet Chadha, and David R WaltMax Tishler Laboratory for Organic ChemistryTufts UniversityMedford, MA 02155,USAJamesB. Richards,Steve B. Brown,and Fred P. MilanovichMeasurement Sciences GroupEnvironmental Programs DirectorateLawrence Livermore National LaboratoryEvermore, CA 94550, USANovember, 1994This document has been submitted in fulfillment of Milestone 0 of theTechnical Task Plan SF 2310-04, "Multianalyte, Single Fiber Optical Sensor."Technical Program Manager/JesseL.#w

DISCLAIMERThis report was prepared as an account of work sponsoredby an agency of the United States Government. Neither theUnited States Government nor any agency thereof, nor anyof their employees, make any warranty, express or implied,or assumes any legal liability or responsibility for theaccuracy, completeness, or usefulness of any information,apparatus, product, or process disclosed, or represents thatits use would not infringe privately owned rights. Referenceherein to any specific commercial product, process, orservice by trade name, trademark, manufacturer, orotherwise does not necessarily constitute or imply itsendorsement, recommendation, or favoring by the UnitedStates Government or any agency thereof. The views andopinions of authors expressed herein do not necessarilystate or reflect those of the United States Government orany agency thereof.

DISCLAIMERPortions of this document may be illegiblein electronic image products. Images areproduced from the best available originaldocument.

Table of ContentsB A C K G R . D3INTRODUCTION.3INDICATORCHEM STRY. 5pH .5AI3 . 9Hydrocarbons .11REFERENCES. 152

BackgroundThe initiative towards remediation of ground and waste water facilities requires, as the foremoststep, identification and analysis of the numemus pollutant compounds present. In an effort to develop afield portable instrument for simultaneous monitoring of different contaminants, researchers at TuftsUniversity have been subcontracted by the Department of Energy to design and fabricate a multianalytefiber-optic chemical sensor. Fiber-optic chemical sensors, which allow remote detection of analytes haveproven to be an excellent alternative to traditional methods of analysis. A number of optical sensors havebeen described based on different chemical transduction mechanisms and optical properties'. 'Ihe small sizeof optical fibers, their insensitivity to electrical interference and the lack of the need for a reference sensormake these devices potentially suitable for remote applications*-'. Indicators that are sensitive to analyteconcentration and exhibit changes in absorbance or luminescence form the basis of such sensors.Traditionally, most multianalyte sensors have been merely several individual sensors fabricated into asensor bundle or array. This approach has limitations in the sense that the size of the array increasesproportionally with each added individual sensing component. Researchers at Tufts University havedemonstrated the capability of placing multiple indicator chemistries, which serve as discrete sensing sites,at the distal end of a single imaging fiber'. Typically an imaging fiber is 300-400 pm in diameter consistingof thousands of individual channels. The discrete sensing sites are the result of photopolymerizing differentindicators in a polymer matrix on multiple regions of the fiber. By coupling the imaging fibers to a chargecoupled device detector (CCD), one has the ability to spatially and spectrally discriminate the multiplesensing sites and hence monitor multiple analyte concentrations simultaneously. ?his report describes thedevelopment of the indicator chemistry and immobilization procedures developed for pH, AlS andhydrocarbons.3

lnfroducfionThere are several factors to be considered when immobilizing a ligand to serve as a sensor. Whilethe fluorescence intensity is proportional to the total number of immobilized ligands, in practice,concentration quenching or inner filter effects may occur when ligands are spaced too closely togethercausing a reduction in intensity. Consequently, the amount of ligand immobilized should be the smallestpossible amount that yields a sufficiently large fluorescence signal for the intended application.Immobilization introduces a new functional group on the ligand This may affect the fluomgenic propertiesof the ligand to the extent that an otherwise non-fluorescent Ligand may become fluorescent. 'Ihis isespecially observed for indicators which fluoresce upon binding to metal ions. In the present work, we havehad success immobilizing ligands via photopolymerization along with the polymer at the activated distalend of the fiber. Fluomphores are functionalized to bind selectively at the sensing site. For instance,labiledouble bonds are introduced on the fluorophore by reacting the amine or acid chloride derivative of theindicator with acryloyl chloride.The polymer matrix used is designed to facilitate mass transfer of the analyte to the immobilizedindicator. Consequently, immobilizing the pH and AIk sensitive indicators requires the polymer to behydrophilic so as to allow water and dissolved ions to easily penetrate into the matrix. Photopolymerizablehydrogels are most suitable for such applications. Polyacrylamide, poly-hydroxy ethyl methacrylate(HEMA) and poly vinyl pyrrolidone are some of the hydrogels that have been under consideration. E M Ahas proved the most favorable because of its considerable porosity, high tensile strength, pH stability andthe fact that it is easily photopolymerized at the tip of optical fibers. On the other hand, the differentpolymers used in case of the hydrocarbon sensor are primarily hydrophobic so as to allow only the volatilehydrocarbons to penetrate the matrix. Numerous siloxane based photopolymers have been identified for thisapplication.4

lndicafor ChemistryMost pH indicators possess a limited dynamic range. As a result, multiple indicators must be employedto obtain seflsofs that cover a wider pH range'".Fluorescent pH indicators are typically weak acid dyes whosedissociated and undissociated forms have different absorption or fluorescent properties in the pH range of interest. nmy cases, fluorescence occurs only from the excited state of the base form, e.g. fluorescein and certainmar in ' - . Some indicators such as hydmxypyxme trisulfonic acid (HPTS) are fluorescent in both acid andParent FluorophoreTypical pKaTypical MeasurementEosin2.0-5.0excitation ratio 450/520 nmRhodols (NEBF)4.5-6.0excitation ratio 440/500nmFluoresceins5.0-7.0excitation ratio 450/490 nm8-Hydroxypyrene 1,3,6-trisulfonicacid (HPTS)I7.2-7.6Iexcitation ratio 450/400 nm7-Hydroxy methykoumarin7.0-85excitation ratio 390 nmSNm'7.0-7.8excitation ratio 490/540 tun oremission ratio 450/500 nmS N MI7.0-7.8I emission ratio580/630nmTable 1. Fluorescent pH indicators,classified by their parent fluorophores 16.base forms", allowing pH changes to be followed by measuring the relative emission of both forms rather thanonly one. Most investigations of optical pH sensors have concentrated on developing sensors for b i i c a lapplications because of the narrow pH range covered, with emphasis on achieving improved sensitivity andprecision As a result, fluorescent indicatorsdeveloped outside this range are few. Table 1 lists some of the parentfluorophores used as fluorescent pH indicators.5

The multiple pH indicators used should have a strong absorption within the wavelength range 400-700nm to allow the use of inexpensive optics. Furthermore, indicators should possess considerable pbto- andchemical stability, lack of toxicity, a functional group capable of chemical immobilization certain coumarirrshave proven to be usem pH indicators. However the spemd properties are disadvantageous because of the lowexcitation wavelength HIT'S has many advantages including high fluorescence quantum yield, visible excitationand emission, large Stokes shift and ability for dual excitation for precise calibration Howater, it is not suitablefor the present application. This is due to the fact that it cannot be spectficany functionalized for immobilizationwithout sacrificing its fluorescent properties. Eosi and fluorescein have been identifiedas fluorescent indicatmsensitivein the pH range 1-9pH units and which may be easily W o n a l i z d to facilitate immobilization.Both eosin and fluorescein are Commercially available as the amine derivative. The amine isfunctionalizedto a vinyl p u p via reaction with acryloyl chloride. The respective dyes, WfKn photo-polymerizedwith HEMA at the distal end of the fiber, are immobilized in the polymer matrix.Prior to immobilization of thedye, the distal end of the fiber is functionalizedto pennit covalent bonding. The various steps developed, namely9functionalization of the dye, silanization of the optical fibers, and immobilization of the dye along with they-polymer are shown below.Functionalizationof Indicators0q-qC'0NH2/Fiuoresceinamime6

qC'0Brp-".Br.O*Silanization of Fibers3-(trimethoxysilyl) propylmethacrylateImmobilizationr 7 4 Fp [email protected] CH2ICHflCOCH CH20I IndicatorCH3hydroxy ethyl methacrylateethylenegylcol dimethyacrylateInitiator, hvDye immobilized on FiberFigure l a shows the emission spectra obtained for eosin at different pH levels. The emission bandat 580 nm shows a steady increase with increasing pH over the 2-8 pH range. As is evident, maximumsensitivity would be obtained with the emission being monitored at 545 nm. Similarly, in the emissionspectra obtained for fluorescein (Figure lb), the intensity of the 520 band increases with increasing pH.The basic forms of both eosin and fluorescein have higher quantum efficiencies leading to increased7

.57s oo.500I600I520560540WawlengthFigure 1. Emission Spectra of eosin (a) and fluorescein (b) at respective pH valuesfluorescence at higher pH. In practice, to compensate for photobleaching, the fluorescence intensity ismeasured as a ratio for excitation at its excitation maxima vs excitation atitwavelength outside theexcitation spectra. Shown in figure 2 are the typical intensity vs pH response for eosin and fluoresceinimmobilized on a 0.1 -I0.55135PH79456PHFigure 2. Normalized emission intensity vs pH plots for eosin (a) and fluorescein(b) immobilized on individual fibers with excitation at 490 nm.878

Eosin shows maximum sensitivity in the 2-6 pH range, while fluorescein is most sensitive in the 58 pH range. Consequently, pH measurements over the range of 2-8 pH units can be achieved usingimmobilized eosin and fluorescein Further research will now focus on developing fluorescent indicatorchemistries to cover the higher end of the pH range (FY 1995 Milestone 4). In addition, poly-HEIvlA, thehydrogel currently being used to immobilize fluorescein and eosin, hydrolyses at pH 9. As a result, amore rugged matrix also needs to be identified for such a applicationAn aluminum ion sensor can be made by immobilizing a non-fluorescent ligand that forms afluorescent complex in the presence of the metal ion As mentioned earlier, concentration of the fluorophoreand immobilization procedures play a crucial role in the fluorescence intensity of the indicator.Furthermore, complex formation for most ligands involves displacement of one or more protons. Thismeans that the equilibrium for the indicator reaction is described by a pHdependent conditional formationconstant. The measurement, therefore, must be done at a constant pH or be corrected for variations in pH.Aluminum determination using fluorescence measurements has been widely studied and numerousmethods have been proposed". Aluminum forms fluorescent complexes with morin, salicylidene-2thiophenol, and a variety of hydrazones and Schiff's bases. To date, all sensors designed for A19 use theparent fluorogenic compound in solution or adsorbed on a solid support like cellulose or an ion exchangeresin'*. Immobilization of the indicator in such a non-specific manner often leads to a deactivation of theAik binding sites and an increase in background fluorescence. Furthermore, as the complex formation isnot reversible, the initial increase in fluorescence intensity observed in the presence of Alh, rapidly plateausonce the binding sites get saturatedWe have designed a sensor that not only has the indicator chemically immobilized on the distal endof the optical fiber but also exploits the diffusion of the Aik ions through the polymer matrix to extend the9

life of the sensor. Lumogallion, a non-fluorescent azo compound, forms stable fluorescent complexes withAlk in slightly acidic media showing an emission maxima at 580 nm for excitation at 490 mI