An optical multi-element fluorosensor for the simultaneous detection of

oxygen and pH in marine sediments

Jonas ArvidssonMaster of Science Project Report

June, 2000

SupervisorStefan Hulth

GÖTEBORG UNIVERSITYDEPARTMENT OF CHEMISTRY

Analytical and Marine Chemistry

ABSTRACTPlanar optical sensors are recently developed tools for detecting high resolution 2-dimensional distribution patterns of important solutes like oxygen and hydrogen ions inwater and sediments. The overall objective for this study was to develop a multi-elementfluorosensor for the simultaneous detection of both oxygen and pH in natural marinesediments. The optical pH and oxygen sensors were combined into one ”sandwichsensor”, instead of immobilising the different fluorophores in the same support.

The pH sensor is based on a well-studied ratiometric technique with the dualexcitation-single emission fluorophore HPTS (8-hydroxypyrene 1, 3, 6 trisulfonic acidtrisodium salt) covalently immobilised to a support foil. The oxygen sensor is also basedon a well studied fluorophore — Tris(1,10-phenanthroline)-ruthenium(II)chloride hydrate,with a single excitation-single emission characteristics. However, recent studies havefound indications of dual excitation maxima with this fluorophore, facilitating ratiometricdetection of fluorescence. Common for most studies with ruthenium complexes foroptical sensing of oxygen is the search for a suitable support into which the indicator canbe homogenously and evenly immobilised, but still maintain its spectral properties. Inthis study, I show the advantages of using a hydrogel with high water content as supportfor the ruthenium complex, in addition to a molecular sieve to reduce leakage of theoxygen indicator from the hydrogel.

Potential applications of this technique will be discussed and suggestions for furtherresearch will be given.

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CONTENTS

Abstract 2

Introduction 4•The oxygen discontinuity layer 4

•Bioturbation 4

•Detection methods for oxygen and pH 5

•Optical sensors 6

Materials and methods 8•Luminescence detection systems 8

•Different supports 10

•Multi-element fluorosensor 11

•Molecular seive 13

Results and discussion 14•Backgrund from the CCD camera system 14

•Leakage from different supports 15

•Optimal concentrations for HPTS and trishydrate 16

•Oxygen fluorophore - pH dependance 16

•Combined oxygen and pH fluorophores 17

•The combined oxygen and pH sensor 18

•2-D distribution of pH and oxygen in sediments 19

Conclusions 20

Acknowledgements 20

References 21

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INTRODUCTIONNumerous redox sensitive reactive pathways are found in the transitional degradation oforganic matter in sediments. Major oxidants (O2, NO3

-, Mn(IV), Fe(III), and SO42-) are

sequentially utilized as electron accptors by decomposing bacteria during earlydiagenesis. Growth efficiency and dominance for the microbial community follow thethermodynamic free energy yield for the various reactions. Reaction patterns and solutedistributions in sediment pore water are consistent with an overall stratification of redoxprocesses (Table 1). Oxic, suboxic and anoxic respiration therefore generally proceed ina succession, and the relative importance of the various reactions reflects quantity andquality of organic matter and oxygenation. Solute exchange across the sediment/waterinterface is enhanced by the bentic fauna through various combinations of excavation,burrowing, feeding and ventilation activities that transform surficial sediments fromhaving largely one-dimension to a complex web of micro-environments with oscillationsin time and space (Aller, 2000).

Bioturbation and bioirrigationMost benthic organisms can only dwell below theoxygenated surface layer of the sediment if they canoxygenate their immediate surroundings. The largerinfaunal animals construct burrow-systems whichare open at the surface, and by drawing a current ofwater into or through the burrow they can extendthe oxygenated surface layer several (10-100)centimeters down (FIG 1.) . The oxygen demand ofthe surrounding anoxic sediments continuallydepletes the burrow of oxygen and therefore themaintenance of an aerobic environment must bekept throughout the life of the burrow-dweller. Thewater current, however, may also be used to bring asupply of suspended food to the animal and maythus serve more than one function.

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TABLE 1. Standard state free energy for the most vital bacterialreactions in aquatic environments (Berner, 1980).

FIGURE 1. The redox structure of sedimentsis radically changed by the activities of theinfauna (redrawn from Aller, 2000).

Infaunal species and their bioturbation and bioirrigation activities are of interest notonly to biologists. For example; in order to interpret palaeo-ecological data it is necessaryto know how organisms become fossilized and under what conditions; burrowingactivities are of interest to sedimentological geologists because bedding layers aredisturbed as animals move through them; and transport of solutes and particles throughbioturbation and bioirrigation are of outmost importance for marine biogeochemists inorder to model and quantify rates and pathways for diagenesis in marine sediments.

Detection methods for oxygen and pHOxygen plays a key role in benthic ecology and early diagenesis of organic matter insediments due to the high energy yield in oxygen reduction. Common techniques toestimate sediment oxygen consumption include O2 exchange measurements in laboratory -incubated cores, or in-situ measurements using bentic chamber landers (Tengberg et al,1995). These techniques, however, do not probe into the sediment and hence give only alimited understanding of the processes taking place at the sediment-water interface orwithin the sediment. The introduction of O2 m i c ro - e l e c t rodes to marine ecology andb i o g e o c h e m i s t ry has allowed O2 distributions in sediments to be studied with a highspatial resolution which has subsequently increased our understanding of O2 dynamicsat the sediment interface (Jørgensen & Des Marais, 1990; Gundersen & Jørgensen, 1990).

Typical O2 microelectrodes have the advantages of a small tip diameter of <10 µm, ashort response time of <1 s, and a stirring sensitivity of <1 % (Revsbech, 1989). Thesecharacteristics make O2 micro-electrodes ideal for many applications in benthicbiogeochemistry. A type of fiber optic O2 micro-sensor (micro-optode), which cancomplement the O2 micro-electrode, was introduced in 1995 into the field of aquaticresearch. The main advantages of this sensor compared to O2 micro-electrodes are amore simple and less time consuming construction and a superior long-term stability(Klimant & Wolfbeiss, 1995, Klimant et al, 1995). Moreover, the O2 optical sensor does notconsume the analyte.

Both micro-electrodes and micro-optodes measure the O2 saturation in onedimension, and vertical distibutions are obtained by stepwise manipulations of thesensor through the sediment. Measuring several profiles in order to describe temporaland spatial heterogenity is time-consuming and work-demanding. Simultaneousmeasurements of temporal changes in O2 concentrations in one or two dimensionsrequire a series of sensors with associated recording devices, which is expensive and inmost cases impractical. Describing 2-dimensional O2 dynamics in heterogeneouscommunities, as around inhabited animal burrows or in heavily bioturbated communitiesusing conventional microsensors, is thus a very difficult task.

The acidity of a solution has been an important parameter since the early days ofchemistry. Different dyes, from litmus to more sofisticated formulas, sensistive to pHhave over the years found extensive use as indicators. Today, pH electrodes are the mostwidely used and appreciated tool to determine pH since they are reliable over a wide pH

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range and fairly easy to use. However, the pH electrodes suffer from disadvantages in thatthey require a reference electrode, they are prone to disturbances from perturbations inthe electrolytes and surronding electric fields, they are fairly expensive and they sufferfrom the same limitations as the oxygen electrodes for simultaneous measurement oftemporal and spatial changes in heterogenous samples (Wolfbeiss, 1991).

Optical sensorsA sensor is defined as ”a device that is capable of continuously and reversibly recording aphysical parameter or the concentration of a solute” (Wolfbeiss, 1991). The mainadvantages using sensors is that measurements can de done directly in the sample. Nosampling, addition of reagents or dilution is normally required. Optical methods haveplayed a dominant role in analytical sciences for a long time, for example colorimetry,photometry and spot tests have been used to determine chemical species (Wolfbeiss,1991). The best known example of an optical detection method is probably the pHindicator strip, containing immobilised colour indicators sensitive to changes in pH. A pHstrip is, however, not per definition a sensor since the process is not reversible. Thefundamental laws of light have more or less remained the same over the years, buttechniques and instrumentation have improved dramaticallly the last decades. Opticalsensors are therefore nowadays frequently used to perform direct measuremenst ofsolutes. The reagent mediated sensors are preferably called optodes(greek for ”opticalway”) or sometimes optrode(the optical analog to eletrode).

A variety of fluorescent indicators is known, but only a few meet the requirements of

an excitation maximum beyond 400 nm to allow the use of inexpensive optics and light

guides, as well as inexpensive light sources such as halogen lamps. Large Stokes' shifts

are often required in order to separate scattered exciting light from fluorescence light.

Further requirements are the lack of toxicity, photostability, and the presence of

functional chemical groups suitable for covalent immobilization onto the support

(Wolfbeiss, 1991).The measuring principle of the O2 optode is based on the ability of oxygen to act as a

dynamic fluorescence quencher that decreases the fluorescence quantum yield of animmobilised fluorophore (Kautsky, 1939). The fluorophore is excited by light with a welldefined wavelength, and in the presence of O2 the intensity of the emitted light decreasesin a predictable way due to the quenching process. In contrast to O2 micro-electrodes thecalibration curve for fluorescent O2 sensors is most often nonlinear, but can in idealsystems be described by the Stern-Volmer equation:

I0/I = I + Ksv C

where I0 and I are the fluorescence intensities in the absence and presence of O2respectively, Ksv is the quenching constant expressing the quenching efficiency, and C isthe oxygen concentration (Stern & Volmer, 1919).

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Ruthenium(II) complexes are by far the most widely used O2 fluorophore since thesemetal complexes in general have efficient luminescences, relatively long-life metal-to-ligand charge-transfer excited states, fast response times, strong absorbtions, largeStokes’ shifts and high photochemical stability. Furthermore, their long excitation andemission wavelenghts are readily compatible with current solid state opto-electronicsand monitoring technology (Choi & Xiao, 2000). One of the most widely used rutheniumcomplexes for O2 detection is ruthenium(II)-tris-4,7-diphenyl-1,10-phenathrolineperchlorate (”trishydrate”), that has been found to have maximum excitation at 450 nm,and emission recorded at 610 nm (Glud et al, 1996, Klimant & Wolfbeiss, 1995).However,recent findings suggest that this ruthenium complex has two excitation peaks, at 340 nmand 500 nm, and emits light with an intensity maximum at 590 nm (Ohlsson, 2000). Thiswould allow for ratiometric measurements, which is generally believed to compensate fordrawbacks like photobleaching, uneven distribution of the dye, dye leaching anddisturbances in the excitation light (Wolfbeiss, 1991).

The last two decades there has been much interest in the development of optical pHsensors, mainly for biomedical applications. There are several advantages using optodescompared to conventional electrochemical sensors; the use of plastic optical fibresallows a high degree of mechanical flexibility combined with a small size and low cost,and most of the limitations for pH electrodes mentioned above are absent with optodes(Chun et al, 1998). Optical pH sensors are based on pH dependent changes of theabsorbance or fluorescence of certain indicator molecules (Wolfbeiss, 1991). Forabsorbance-based sensors, the color of the dye changes with pH. For fluorescence pHsensors, the difference in spectral properties of the conjugate acid-base pair of the dye isdetected. In this analytical application, the fluorescence method has the advantages ofhigher sensitivity and selectivity over theabsorbance method (Wolfbeiss, 1991). The majorlimitation of most pH optodes is the narrow pHrange covered: most dyes are only sensitive tochanges in pH values over 2 - 4 pH units (mostoften pKa ± 1 unit).

A number of fluorescent dyes have been usedas indicators for optical pH sensing. HPTS (8-hydroxypyrene 1, 3, 6 trisulfonic acid trisodiumsalt) is one representative from a class of dyescommonly used for pH optodes (Wolfbeiss, 1991).Its major advantages include high fluorescencequantum yield, visible excitation and emissionwavelengths, and a large Stokes' shift. It has a pKaof about 7 which is ideal for both physiological andseawater measurements. It may be excited at either405 nm or 450 nm to give an emitted fluorescence

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FIGURE 2. Excitation spectra of glass-immobilized HPTS at various pH values(phosphate buffer, 23°C, emission takenat 520 nm). (Wolfbeiss, 1991 andreferences therein)

at 520 nm (FIG. 2). The two excitation wavelenghts make HPTS an ideal fluorophore forratiometric measurements. It is generally believed that this approach compensate fordrawbacks like photobleaching, dye leaching, uneven distribution of the dye anddisturbances in the excitation light (Wolfbeiss, 1991).

The use of a 2D plate fluorosensor hasthe potential to greatly improve the detectionof temporal and spatial variations of solutes in the sediment since these sensors does notrequire the labour extensive manipulations of an electrode, and facilitate the research ofbioturbation and the distribution of oxygen in the water above the sediment surface, atthe water/sediment interface and in the sediment itself. An optical 2D sensor thatcombines the simultanueous measurements of two or more solutes has obviousadvantages in active service use, especially when the ambition is to describe complicatedprocessess in the sediment. The resulting images could be animated into a film showingthe actual course of events. The overall goal of this project was to find a way to combinethe optodes for oxygen and pH into one optical sensor that can be used to describesolute distributions i two dimensions.

MATERIALS AND METHODSLuminicence detection systemsFLUOROMAX2 — To reveal important characteristics, such as optimal concentrations forthe fluorophores and the response of the oxygen fluorophore to variations in pH, a highlysensitive spectrofluorometer (FluoroMax-2, SPEX Int´l Inc.) was used. See figure 3 for adespricption.

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FIGURE 3. Simplified block diagram over the FlouroMax2 optical setup. Light from the 150 W Xenon lamp (1)enters the excitation spectrometer, which delivers monochromatic light to the sample compartment (4).However, prior to reaching the sample compartment, 8% of the light is directed to the reference photodiode (5)via a quarts beam splitter (3). The beam splitter also acts as a transparent barrier to prevent dust from gettinginside the delicate optical components. Light emitted from the sample is dispersed by the emissionspectrometer (6) and directed to the signal photomultiplier detector (7). This signal is then amplified (8) anddisplayed on the computer monitor (9) (redrawn after Strömberg, 2000).

The FluoroMax-2 is designed for fluorescencemeasurements 90° to the emitted light, which isimpractical and sometimes impossible for non-liquid samples. The foil supports were tested byinserting a strip diagonally in the cuvette (FIG 4).The characteristics, such as backgrundfluorescence, of the hydrogels were also testedby stabilising a slice of the gel that fitteddiagonally in the cuvette with a thin frame madeof the same foil as the support for the HPTS . Thefoil had a rektangular hole cut to minimizeinterference from reflection of light from the foil.

2-D CCD CAMERA SYSTEM —The planar optode wasexcited by light from a 300 W Xenon (UV/VIS) arclamp equipped with a dual filter changer holdingappropriate narrow bandpass filters (Omega).The filtered light is led by a liquid guide through a lens assembly, collimating the lighttowards the optode (FIG.5) The angle between the excited and emitted light varied fromabout 20° to 30°, depending on distance to the aquarium.

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FIGURE 4. A typical set-upused for quantification offluorescence from afluorophore immobilisedin hydrogel, or tomeasure backgrundfluorescence from thehydrogel. Later researchhas shown that an angleof 30 - 40° instead of 45°to furthter reduce thebackground andreflexion. (Hulth, perscomm.)

FIGURE 5. Schematicdrawing of the experimentalsetup for the CCD-camerasystem. See text for details.

FIGURE 6. Since I have used normal SLR lenses for 35 mm cameraswith a chip of only 6.4 x 4.8 mm, the useable distances and magnifi-cations must be recalculated. My choice of lenses is the NIKON55mm f/2,8 macro lens and the NIKON 35 mm f/2,8 lens for optimalflexibility in resolusion, distance to the object and image area.

The light emitted by the planar optode was collected by a 8-bit COHU 4910 highperformance CCD camera (chip resolution 753 x 582 pixels) via different NIKON standardSLR (Single Lens Reflex) lenses (Fig. 9) to give optimal size of the image and spatialresolutions for the purpose of the test. The setup gives a theoretical pixel by pixelresolution of about 8.5 x 8.2 µm. The signal from the CCD camera was processed by aWindows-based PII-400 Mhz computer. The camera and light was placed in a custommade rack to allow for stable conditions during measurements and to give means forcontrolled movements in two dimensions.

The general operation of the optical system was controlled by the InCytIm2™software, along with the quantification of the detected fluorescence. The pictures fromthe sediment setups were analysed in the NIH Image software (version 1.58), which is afull-featured image analysis program written by Wayne Rasband, NIH, Washington, DC,USA. The MacIntosh version of the program is freely available from the FTP sitersbweb.nih.govon the internet. A Windows version with the main features of the NIHsoftware can be found on www.scioncorp.com.

Different supportsCommon for most studies with ruthenium complexes for optical sensing is the search fora suitable support that maintains the properties of the fluorophore. Oxygen sensingproperties, such as sensitivity, selectivity, dynamic range, response time, photostability,shelf lifetime, durability, and manufacturing reproducibility, have complex dependencieson the support structure into which the optical indicator is embedded. The solutespecific indicator may be chemically bound, physically adsorbed, or simply dissolved inthe support matrix. Many oxygen fluorosensors have been described that make use ofsilicone films, plasticized PVC, cellulose or polystyrene foils, sol-gel materials, fumedsilica gel disks, or porous glass powders as supports. Desirable properties of the sensingmatrix include high permeability to oxygen, adequate mechanical, thermal, and chemicalstrength, good transmittance in the visible region of the spectrum, and proper functionalgroups for electrostatic or covalent immobilization of the dye. Moreover, inexpensive andreproducible manufacturing of the indicator layers is preferrable for practicalapplications under active-service conditions (Garcia-Fresnillado et al, 1999). In this study Ihave used a hydrogel with very high water content as support, along with a molecularseive to reduce leakage.

HYDROGELS — Standard structural hydrogels on the market are usually covalentlycrosslinked. Such crosslinking renders them both water and solvent insoluble and givesthem high chemical, thermal and UV stability. Covalent crosslinking also makes themthermosets, which means that their processing methods are limited, and makes themweak in the swollen state, especially at high water contents.

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HYPAN™ polymers (www.hymedix.com) were designed specifically to have all theadvantages of covalently crosslinked hydrogels, but without their disadvantages. Theyare hydrophilic acrylate derivatives with a unique multiblock structure. Each polymerchain of HYPAN™ is composed of several sequences of units with pendant hydrophilicgroups (soft blocks) such as -OH, -COOH, -CONH2, -CONH, SO3H, etc., and severalsequences of units with pendant nitrile groups (hard blocks)(FIG.7). By changingproduction conditions, it is possible to obtain polymers not only with varying hydro-

p h i l i c i t y, but also to change the lengths of the blocks and/or the nature of the side gro u p s .HYPAN™ polymers are made from a polymeric base, using water based reactants.

They do not contain free monomers, initiators, plasticizers or stabilizers, and aretherefore extremely pure. The reaction proceeds on the side groups and thus does notchange the chain length and distribution of the starting polymer, and preserves itscarbon-carbon backbone, resistant to degradation and enzymatic cleavage.

The oxygen flourophore was immobilised in HYPAN™ hydogels of three differenttypes — HN-80, HN-90 and AA-90. The structural diffrences between these hydrogels areprotected by U.S patent, but the HN-80 and HN-90 only differs in water content (watercontent in percent). The AA-90 contains more soft blocks than the HN series hydrogels.

Multi-element fluorosensorThe aim of this project was to find a way to combine theoptodes for oxygen and pH into one multi-elementfluorosensor. Initial tests were done in the FluoroMax2 byimmobilising both fluorophores in the same hydrogel. Isoon realised that this is not possible (see Results andDiscussion). The fluorophores were therefore kept indifferent supports in a combined sandwich optode (FIG. 8).The optical sensor was clamped with a grey PVC frame tothe inside of different small (to suit the aim of theexperiment) aquariums constructed of transparent glass.

PH OPTODE — Because HPTS has three sulfonate groups, itcan be immobilized conveniently and essentiallyirreversibly on certain supports, e.g. an ordinarytransparency film for InkJet printers (Hulth et al, 2000).

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FIGURE 7. The chemical structure of the HYPAN™ hydrogels (A). Structure of polyarylonitrile shown forcomparison (B). (From www.hymedix.com)

A B

FIGURE 8. The different dyedsupports were clamped with aPVC frame to the inside of the testaquarium.

Because of the small thickness of the sensing layer (100-200 µm) equilibration with the sediment pore water isextremely rapid (less than 1s). Further development of theindividual pH sensor was not an objective of this project.

HPTS (FIG. 9) dissolved in Milli-Q has been found toabsorb light at 405 nm and 450 nm, and emit light with anintensity maximum at 520 nm (Wolfbeiss, 1991). Rescentfindings confirm the spectral properties also afterimmobilisation to the transparency film (Hulth et al, 2000).The fluorescence signal from immobilized HPTS decreaseswith increasing pH when excited at 405 nm and the signali n c reases with increasing pH when excited at 450 nm (FIG. 2).

During pH sensor preparation, the foil was immersedin a 5 µM solution (the found optimal concentration) of HPTS dissolved in filtered seawater (0.3 µm) of appropriate salinity (28-32), and left to soak overnight. The sensing foilwas prepared by carefully dipping it a few times in sea water to remove any excessfluorophore. The foil was then clamped with a PVC frame to an appropriate aquarium forthe tests to be done (FIG. 8). During all stages of the optode preparation, care was takento avoid unnecessary illumination to reduce the risk of photobleaching.

During pH response experiments, the pH of the seawater in the aquarium wasregulated by adding a few drops of 0,1 M solutions of either HCl or NaOH. The resultingpH was controlled before and after the individual tests with a conventional pH electrodestandardized in Hansson buffer solutions. Measurements were normally performed within10 minutes after the initial setup.

OXYGEN OPTODE — The oxygen-quenchable fluorophore ruthenium(II)-tris-4,7-diphenyl-1,10-p h e n a t h roline perchlorate (”trishydrate”) (FIG. 10)was dissolved in hydrogels from Hymedix. Thisfluorophore has been found to absorb light at 340nm and 500 nm, and emit light with an intensitymaximum at 590 nm (Ohlsson, 2000). Initial testswere done in the FluorMax2 to investigate howtrishydrate reacts under varying pH.

The hydrogel supports for the oxygen-quenchable fluorophore were supplied byHymedix in pre-fabricated sheets of 0,1 mmthickness. The hydrogel was immersed in a 0,5mM solution of the fluorophore dissolved infiltered sea water (0.3 µm), and left to soak

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FIGURE 9. Chemical structure of8-hydroxypyrene 1, 3, 6 trisulfonicacid trisodium salt (HPTS). Mw =524.37 daltons. Excitation maximaat 405 nm and 450 nm. Emission at520 nm (Wolfbeiss, 1991).

FIGURE 10. Chemical structure ofruthenium(II)-tris-4,7-diphenyl-1,10-phenathroline perchlorate (”trishydrate”).Mw = 712.61 daltons. Excitation maxima at340 nm and 500 nm. Emission at 590 nm(Ohlsson, 2000).

overnight. The optode was thereafter prepared by placing the hydrogel in sea water for 1-2 hours to remove any excess fluorophore. The optode was clamped with a PVC frame toan appropriate glass aquarium for the test to be done (FIG. 8). When using a molecularsieve, the dyed hydrogels were just dipped a few times in seawater to rinse off excessfluorophore and then clamped to the aquarium with the sieve between the seawater andthe hydrogel (FIG. 8). During all stages of the optode preparation care was taken to avoidunnecessary illumination to reduce the risk of photobleaching.

During oxygen response experiments, the oxygen concentration of the seawater inthe aquarium was regulated by flushing the aquarium with air or 100% N2 for 10 - 15minutes through a bubbling stone. Further analysis of the resulting oxygen saturationwith Winkler titration or Clark electrode was not made. Mentioned treatment wasassumed to give 100% or 0% O2 saturation respectively. Measurements were normallyperformed within 10 minutes after the initial setup.

Molecular sieveSince the oxygen fluorophore is not covalently bond to the hydrogel support, amoluecular sieve (Molecular/Por®) was used in some experiments to reduce previouslyobserved leakage and to give mechanical stability. The Molecular/Por® cellulose estermembranes from Spectrum® (www.spectrum.com) are composed of solubilized cellulosematerial (cellulose acetate) cast on a thin polypropylene backing material. They areavailable in a wide range of well defined MWCO (Molecular Weight Cut Off) between 100and 50 000 daltons. In this study we used a filter with MWCO of 500 daltons (the Mw oftrishydrate is 712.61 daltons). The membranes are hydrophilic which provides lowadsorption and they may be used in solutions ranging in pH from 2 to 9 and temperaturesup to 37°C.

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RESULTS AND DISCUSSIONBackground from the CCD camera systemFor most of the initial tests in the optical system, it has been assumed that the internalbackground from the CCD camera system to be negligible compared to the fluorescencefrom the optical sensors. However, inconsistensies in some results, especially att lowintensities, nourished a suspicion of an internal background. A sheet of support foil wasprepared with HPTS in the normal manner and clamped with a PVC frame on the theinside of the test aquarium (FIG. 8) . The pH was monitored during the test series andfound the be constant (pH 7.51 ± 0.06). The optodes were excited at 405 nm and 450 nmat different exposure times (FIG. 11). In theory, increasing the exposure time with a factor2 would result in twice the amount of recieved light. Hence, the increase of intensitywould be 2. This was not so for the uncorrected values, but close to 2 for the correctedintensities (Table in FIG 11). There was an internal blank of about 25, which is aconsiderable amount at low intensities/short exposure times. Therefore more informationis needed about the system background and how to best compensate for it.

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0

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0 0,5 1 1,5 2 2,5 3 3,5 4

FIGURE 11. Background from the CCD camera system. When plotting differentexposure times against resulting intensities for exactly the same conditions, theresulting linear function does not pass origo. This is due to a system dependentbackground.The corrected intensity increase deviates significantly from 2, butthe corrected values are quite close to 2, especially at exposure times above 2 sfor 405 nm and 0.8 s for 450 nm.

Exposure time (s)

Intensity (arbitrary units)

Exposue time Intensity Increase Corrected Increase405 nm 0,5 143 55

1,0 216 1,51 128 2,332,0 348 1,61 260 2,034,0 587 1,69 499 1,92

450 nm 0,2 158 700,4 238 1,51 150 2,140,8 395 1,66 307 2,051,6 693 1,75 605 1,97

Leakage from different supportsAs discussed, there were indications that the hydrogel was not able to hold the oxygendye over time (FIG. 12). To further evaluate and confirm these indications, a series ofexperiments in the optical system was prepared to quantitatively and qualitativelyevaluate how the different hydrogels leaked over time. Three samples, one each of HN-80,HN-92 and AA-90 were prepared in a 0,5 mM solution of trishydrate and left to soakovernight. After a quick rinse in seawater they were clamped on the inside wall of the testaquarium with a PVC frame with three slots (FIG. 8). The fluorescence intensity atexcitation wavelenghts 340, 405, 450 and 500 nm was measured at emission wavelength610 nm over a period of nearly 4 days. These time series revealed AA-90 as the gel thatcould hold most dye (FIG. 12) . It was also the gel that released most dye initaially and thegel that retained most dye over time. From these results it was concluded that AA-90 wasthe preferred gel for further experiments and the preparred gel should be left to soak inseawater for 5 to 10 hours before use. A gel prepared in such a manner was used in asediment setup and gave a rather stable signal for at least a week.

Furthermore, a molcular sieve was used between the gel and the sea water in theaquarium, in order to retain the dye from leaking out of the hydrogel. A molecular sievewith MWCO of 500 daltons was selected. This will retain most of the the trishydratcomplexes with a Mw of 712 daltons, but will let smaller ions and molecules, like O2 andH+, pass freely. However, it was not selected to retain HPTS (Mw 424 daltons) because ofthe good covalent immobilisation of this dye to the support foil (Hulth el al, 2000).Initially there was a dramatic change in fluorescence intensities over time, with a rapidloss of about 35% over the first hours. After this initial loss of intensity, the signal becamestable over time at a 3 times higher intensity than for AA-90 without this molecular sieve(FIG. 12).

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FIGURE 12. To test the ability toimmobilise trishydrate and to retain itover time, the three different hydrogelsat hand were dyed and clamped to theglass in the same aquarium. Theresults show that the amount of immo-bilised oxygen fluorophore initiallyretained in the gel differs and also theability to keep the fluorophoreimmobilised in the hydrogel. Themolecular sieve makes a dramaticdifference in retaining the fluorophorein the hydrogel.

Optimal concentrations for HPTS and trishydrateTo evaluate and verify optimal concentrations of HPTS to be used in the multi-elementsensor, solutions with different concentrations of HPTS were prepared by diluting a stocksolution with filtered seawater (0.3 µm). Flourescence from these solutions were detectedin the FluoroMax2 at excitation wavelenghts 405 nm and 450 nm (FIG. 13). It was foundthat HPTS concentrations below about 4 µM was not affected by the ”inner-filter” effect.HPTS immobilised on the supporting transparency foil behaves in a similar way (data notshown). The selected optimal concentration for the HPTS solution to be used forimmobilisation on the support foil was therefore selected to 4 µM, which correspondswell to earlier results (Hulth et al, 2000). Optimal concentrations for trishydrate has beenevaluated in a similar way and found to be 0,5 mM (Ohlsson, 2000).

Oxygen flourophore – pH dependenceSix different solutions of 0,5 mM trishydrate wereprepared. The pH of the different solutions waschanged in the interval of 6 to 9 with additions of0,1 M HCl or NaOH, and the fluorescence detectedusing the FluorMax2 at excitation 340 nm and 500nm, emission 590 nm at both oxygenation anddeoxygenation (FIG. 14).

The oxygen flourophore seemed to beinsensitive towards shifts in pH within theselected pH range, which also corresponds to arange that is normally found in a naturalsediments and waters.

Response of the oxygen fluorophore has beentested for variations in ionic strenght (Ohlsson,2000). Her observations were similar as for thosefound for pH, i.e. a stable signal for varyingsalinities.

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HPTS at 405 nm

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FIGURE 13. To find out optimal concentration for HPTS, intensity was measured for different concentrations.The selected concentration should be in the linear range to avoid inner filter effect. The selected concentrationis 5 µM.

HPTS at 450 nm

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0% O2 saturation

FIGURE 14. One prerequiste for a suitable O2fluorophore to be included in the multi-element sensor is constant fluorescence withvariations in pH. No significant change influorescence (±2%) in the expected pH rangecould be detected.

Interactions between the flourophores foroxygen and pHThe selected optimal solutions for the two fluor-phores were prepared by diluting stock solutionswith filtered seawater (0.3 µm). Initially they weretested independantly to reveal their characteristics(FIG 15 A and B).

Trishydrate showed a strong emittance signalat 590 nm when excited at 500 nm and a weaker,but detectable emittance signal when excited at340 nm (FIG. 15A). This corresponds well to earlierfindings for this fluorphore, which has been foundto have dual excitation wavelenghts (Ohlsson, 2000).

HPTS gave strong signals at both excitationwavelenghts (405 nm & 450 nm) in conjunctionwith earlier findings (Hulth et al, 2000) (FIG. 15B).

The two fluorophores combined in the samesolution (at the same concentrations as above)gave a dramatic effect on the behavior of HPTS(FIG. 15C) . This is not yet well understood. Theemittance signal at 520 nm (emission maxima forHPTS) for the HPTS and trishydrate solutiondecreased significantly compared to the HPTSsolution, but at 590 nm (emission maxima fortrishydrate) the signal was fairly constantcompared to the solution with only trishydrate.

It was therefore concluded that: 1) HPTSaffects trishydrate in solution; 2) trishydrateaffects HPTS to a limited degree only. The excitedlight from HPTS at 520 nm will, to some degree, beabsorbed by trishydrate (FIG 15 C) but most likelynot enought for total exctinction (FIG. 15D).

To test how the fluorophores for oxygen andpH are immobilsed in the hydrogel, a strip of HN-90was left overnight to soak in a solution of thecombined fluorophores (0.4 µM for the pH fluoro-phore and 0.5 mM for the oxygen fluorophore).After rinsing the strip, it was secured in a cuvette(FIG. 4) with filtered seawater (0.3 µm) and testedfor emittance at excitation wavelengths 340, 405 450and 500 nm (FIG 15D). This shows that HPTS can not be immobilised in the HN-90 support, since thesignal at 520 nm is more or less absent.

Insted of mixing the two dyes in one support,the descision was to use separate supports for thetwo types of indicators for further experiments.

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FIGURE 15. Emission scans for excitationswavelenghts 340 nm, 405 nm, 450 nm and500 nm. A, trishydrate in solution; B, HPTSin solution; C, both trishydrate and HPTS insolution; D, both trishydrate and HPTSimmobilised in hydrogel HN-90. The concen-trations for trishydrate and HPTS are selectedoptimal values. Trishydrate clearly interferswith HPTS (C) and it is not practical to immo-b i l i s e HPTS in hydrogel due to leaching.

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The combined oxygen and pH sensorTo test the dual-element sensor the individual supports were prepared as previouslydescribed and clamped to the inside of the test aquarium (FIG. 8). The test aquarium wasfilled with seawater and to vary oxygen concentrations the aquarium was flusched withair or 100% N2, and to vary pH additions of 0,1 M HCl or NaOH were made. Test serieswas run for both oxygen and pH with the results shown in fig 16 and fig 17. The oxygensensistive part of the optode responded with a 34% change in signal intensity at 340 nmand 23% at 500 nm between deoxygenation and oxygenation (FIG 16) . The signal wasstable over at least 14 days (data not shown). Due to the reduced leaking, the signal fromthe combined multi-element fluorophore with a molecular sieve is about 3 times higherthan for the single optode. This is important, especially at excitation wavelength 340 nmwhere the output signal is quite weak.

For the pH sensitive part of the sensor to function as expected, the molecular sieveand the hydrogel must be permeable to small ions affecting pH. Preliminary findingsindicates that the multi-element fluorophore responds in a similar way as compared tothe tests in the FluoroMax2 (FIGS. 15C and D), which indicates that the hydrogel ormolecular sieve does not disturb the pH sensor.

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FIGURE 16. Comparison between output signal from a single oxygen optode and from the oxygen optodecombined in a multil-element fluorophore with molecular sieve. Data is corrected for background from thecamera system and supports. Solid lines respresents data for the combined multil-element fluorophore anddotted lines data for trishydrate in hydrogel AA-90 after being soaked in seawater for 26 hours.

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2-D distribution of pH and oxygen in sedimentsAn especially designed aquarium was filled with homogenous surface sediment togetherwith overlying water. Before adding the sediment and water, a dual-element pH andoxygen optical plate sensor was clamped to the inside of the aquarium (FIG. 8) . Thesensor was left to settle and to develop steady state conditions with the sediment porewater system. Measurements of the 2-D pore water distribution were then made for bothoxygen and pH after bubbling the overlaying water with N2 or air. The resluts are shownin Figs. 18A to 18F. The intensities are not quantified, but the images show how pH andoxygen distributions in the sediment are affected by the treatment in the overlayingwater.

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FIGURE 18. Images A to F were taken with the combined sandwich optode in a homogenised sedimentat equilibrium with the water above. No bubbling were made for 3 days. The images shows cleary thesediment surface and the transition to the oxygen discontinuity layer. The images were processed inthe NIH software.A) Natural light.B) Excitation 405 nm and emission 520 nm (pH). C) Excitation 450 nm and emission 520 nm (pH).D) Excitation 340 nm and emission 610 nm (oxygen).E) Excitation 450 nm and emission 610 nm (oxygen).F) Excitation 500 nm and emission 610 nm (oxygen).

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the oxygen discontinuity layer

CONCLUSIONSThe attempt to immobilise the fluorophores for oxygen and pH in the same supportfailed, but the strategy to use different supports was successful. The high water contentfor the hydrogel used as support for the oxygen fluorophore facilitates ion mobility andthereby does not interfere with the function of the pH sensor.

The molecular sieve proved very useful in preventing leakage of the oxygenfluorophore from the hydrogel. The relativeley large pore size of the sieve did notinterfere with the free mobility of the smaller solute ions to be measured.

The oxygen sensor responded well to changes in oxygen saturation of seawater. Thedifference in signal between deoxygenation and oxygenation was 23% at 500 nm and 34%at 340 nm, which corresponds well to earlier findings for this fluorophore and support.However, the suggested ratiometric possibilities with the used ruthenium complex gaveno clear indications since the signal obtained at 340 nm was too weak. Furtherinvestigations into this matter must be made, possibly with a more sensitive detectionsystem, e.g. a cooled CCD camera.

The pH sensor responded according to previous research, with no apparentinterferences from the oxygen sensor or molecular sieve.

The combined sensor used in a sediment setup gave indications that the multi-element pH and oxygen plate fluorosensor could be useful for the determination ofspatial and temporal solute distribution patterns in natural sediments. This would greatlyfacilitate investigations in bentic biogeochemistry, possibly with the addition of one ormore sensors in the combined setup. However, additional tests must be done to fullyunderstand the mechanisms and characteristics of the fluorophores like photobleaching,and possible ratiometric detection of the oxygen fluorophore. Investigations should alsofocus on a quick and reliable way to accurately quantify the results.

ACKNOWLEDGEMENTSThe outcome of this study would not have been possible without the achievements fromthe innumerable and anonymous scientists that over the years have given priority to ourunderstanding of nature instead of personal greed. We all owe a dept of gratitude to thesescholars and authors, whose laboriouos toils have led to the knowledge we have todayabout the sea and its inhabitants. No one mentioned, but no one forgotten. However, itwould not be fair to miss the oppotunity to thank my supervisor, Stefan Hulth, for hisnever ending optimism despite the numerous disappointments (for some reason theynormally occured on Fridays) during the project. My belief in a successful outcome of theachievements was unsettled several times, but I was always set back on track by thetransmission of his unwavering faith in our predestined path to the ultimate goal.

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REFERENCESALLER RC. Solute transport and biogeochemical reactions in the bioturbated zone: structure andbiogeochemicla function. In The bentic boundary layer. Transport, processes and biogeochemistry. Eds B.BJörgensen and B. Boudreau. Oxford University Press. 2000.

BERNER. Early Diagenesis - A Theoretical Approach. Princeton University press, 280 pp. 1980

CHOI MMF; XIAO D. Single standard calibration for an optical oxygen sensor based on luminescence -quenching of a ruthenium complex. ANALYTICA CHIMICA ACTA, Vol 403, Iss 1-2, pp 57-65. 2000.

CHUN MN; CHUN-SING F; KWOK-YIN W; WAIHUNG L. Evaluation of a luminiscent ruthenium complex immobilisedinside Nafion as a optical pH sensor. ANALYST, Vol 123, pp 1843-1847. 1998.

GARCIA-FRESNILLADO D; MARAZUELA MD; MORENO-BONDI MC; ORELLANA G. Luminiscent Nafion membranes dyedwith ruthenium (II) complexes as sensing materials for dissolved oxygen. LANGMIUR, vol 15, 6451-6459.1999.

GLUD RN; RAMSING NB; GUNDERSEN JK; KLIMANT I. Planar optrodes: A new tool for fine scale measurements oftwo-dimensional 02 distribution in benthic communities. MARINE ECOLOGY-PROGRESS SERIES, Vol 140, Iss1-3, pp 217-226. 1996.

GLUD RN; SANTEGOEDS CM; DE BEER D; KOHLS O; RAMSING NB. Oxygen dynamics at the base of a biofilm studiedwith planar optodes. AQUATIC MICROBIAL ECOLOGY, Vol 14, Iss 3, pp 223-233. 1998.

GLUD RN, KUHL M; KOHLS O; RAMSING NB. Heterogeneity of oxygen production and consumption in aphotosynthetic microbial mat as studied by planar optodes. JOURNAL OF PHYCOLOGY, Vol 35, Iss 2, pp270-279. 1999.

GUNDERSEN JK; JÖRGENSEN BB. Microstructure of the diffusive boundary layers and the oxygen uptake of thesea floor. NATURE, Vol 345, Iss 6276, pp 604-607. 1990.

HULTH S; ALLER RC; ENGSTRÖM ; SELANDER E. 2-D pH plate fluorosensor (optode) for early diagenetic studies ofmarine sediments. In preparation, 2000.

JØRGENSEN BB; DESMARAIS DJD. The diffusive boundary layer of sediments: oxygen microgradients over amicrobial mat. LIMNOLOGY AND OCEANOGRAPHY, Vol 35, Iss 6, pp 1343-1355. 1990.

KAUTSKY, H; WOLFBEISS OS Quenching of luminence by oxygen. TRANS FARADAY SOC. 35:216-219. 1939.

KLIMANT, I; MEYER V; KÜHL M. Fiber optic oxygen micro sensors, a new tool in aquatic biology. LIMNOL.OCEANOGR. 40:1159-1165. 1995.

KLIMANT I; WOLFBEISS OS. Oxygen sensitive luminiscnet materials based on silicone soluble ruthenium diiminecomplexes. ANALYT CHEM. 34:3160-3166. 1995.

OHLSSON Å. Distributions of solutes and particle associated compunds in marine sediments: a 2D approach.MSc Thesis, Göteborg University. 2000.

REVSBECH NP. An oxygen microsensor with guard cathode. LIMNOLOGY AND OCEANOGRAPHY, Vol 34, Iss 2,pp 474-478. 1989.

STERN O, VOLMER M. Über die Abklingziet der Fluorezenz. PHYS Z., 20: 183-188. 1919.

STRÖMBERG N. Optical fluorosensors for the detection of O2 and NH4+ in natural waters. MSc Thesis,

Göteborg University. 2000.

Tengberg, A., F. de Bovée, P. Hall, W. Berelson, B. Chadwick, G. Ciceri, P. Crassous, A. Devol, S. Emerson, J.Gage, R. Glud, F. Graziottin, J. Gundersen, D. Hammond, W. Helder, K. Hinga, O. Holby, R. Jahnke, A.Khripounoff, H. Lieberman, V. Nuppenau, O. Pfannkuche, C. Reimers, G. Rowe, A. Sahami, F. Sayles, M.Schurter, D. Smallman, B. Wehrli and P. de Wilde. Benthic chamber and profiling landers in oceanography -A review of design, technical solutions and functioning. Progress in Oceanography 35:253-294. 1995.

WOLFBEISS, O.S. Fiber Optic Chemical Sensors and Biosensors, volume 1. ISBN 0-8493-5508-7. 1991.

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