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Chemiluminescent Ethanol Biosensor Development
James E. Atwater, James R. Akse, Jeffrey DeHart, and Richard R. Wheeler, Jr.
Umpqua Research Co.

Charles E. Verostko
NASA-Johnson Space Center

ABSTRACf The investigation and development of a chemiluminescence based ethanol detection concept into a biosensor system is descn1>ed. The biosensor uses alcohol oxidase to catalyze the reaction of short chain primary alcohols with elemental oxygen to produce hydrogen peroxide and the corresponding aldehyde. The reaction of hydrogen peroxide with an organic luminophore in the presence of a sufficient electric field results in emission of blue light with peak intensity at 42Snm. The chemiluminescent light intensity is directly proportional to the alcohol concentration of the sample. The aqueous phase chemistIy required for sensor operation is implemented using solid phase modules which adjust the pH of the influent stream, catalyze the oxidation of alcohol, provide the controlled addition of the luminophore to the flowing aqueous stream, and minimi7e the requirement for expendables. Precise control of the pH bas proven essential for the long-term sustained release of the luminophore. Electrocatalysis is achieved using a controlled potential across gold mesh and gold foil electrodes which undergo periodic polarity reversals. Ethanol concentrations (as C) in the range between 40 and 4.000 J1gILhave been determined. The performance features of this sensor are presented in this paper. INTRODUCTION For aqueous waste streams generated by a crew in regenerative life SUPPOrt. thanol is a major impurity. It is e poorly removed by physicochemical processes such as multifiltration (MF) and reverse osmosis (RO). and consequently forms a significant fraction of the efi1uent total organic carbon (fOC) from these processes. The inability of sorption and membrane based separations to remove ethanol and other low molecular weight water soluble organics bas led to the evaluation of alternative water reclamation methods such as electrochemical water recoveryl.2. and to the development of post-treatment technologies for RO and MF

systems such as low temperature aqueous phase catalytic oxidation3.4.and immobilized enzyme bioreactocs>7. The capability for quantitation of ethanol in waste water streams such as humidity condensate and in finished potable water can be used advantageously in the control of water processor operation. The initial chemiluminescent ethanol biosensor development effort was intended to demonstrate the feasi.oility of an accurate. precise. sensitive. and reliable means for the determination of ethanol and other low molecular weight straight chain alcohols within aqueous streams aboard spacecraft. A unique aspect of the ethanol biosensor operation is that all reagents are provided by solid state flowthrough modules. thus minimhing the storage and resupply logistic requirements for long term operation in earth orbit or on station within a Lunar or planetary outpost. Operation of the biosensor is based upon the oxidation of ethanol to acetaldehyde. and reduction of <>2to hydrogen peroxide ~Oi' which then reacts with a luminophore to produce light. The intensity of the light is directly proportional to the concentration of ethanol in the sample. Advantages inherent to the technique are the potential for detection of extremely low levels. the stability of the devices used to measure light intensity (photomultiplier tubes or charge coupled devices). and the use of solid state modules for the controlled release of reagents. This detection scheme may be conveniently embodied in either continuous on-line or flow injection analysis configurations. Ethanol is detected using the enzyme alcohol oxidase. Alcohol oxidase catalyzes the reaction of ethanol with molecular oxygen to form acetaldehyde and ~O2' H


I ~H


AIcchoI Oxida-.



~c ~H


(1) The ~O2 containing aqueous stream flows through a solid phase basification module where the :l1hlinity is adjusted to pH II:: 0. The basified efi1uentflows into a bed of 1

Both voltages were maintained relative to a AgIAgCI reference electrode by the potentiostat. The photomultiplier tube receives a positive bias of 1.luminol (3-aminophthalhydrazide) crystals which slowly dissolve at a controlled rate. a Baseline-Zeroing Circuit was designed and constructed as shown in Figure 3. By using an electrical potential across the ECL cell to catalyze the reaction of hydrogen peroxide with luminol. and luminol were obtained from Aldrich (Milwaukee). The differential amplifier also provides an overall gain of2. The differential amplifier output is a voltage which is proportional to the difference between the input and the offset voltages. High voltage was applied to the PMr using a Nucleus model 575 scalerratemeter power supply. EC 1.~~ Figure 1. is input to the Baseline Zeroing Circuit in the form of a voltage drop across a IOID 1% current sensing resistor. Polarity - Alcohol oxidase (alcohol oxygen SOLID PHASE BASIFICATION BEDS Two proprietaIy solid phase basification (SPB) materials were examined as candidates for the flow through pH conditioning bed. Flows were established using Masterflex model 7520-35 multichannel peristaltic pumps. The ECL apparatus consists of a three electrode electrolytic cell. and a Microelectrodes model MI 402 Ag/AgCI reference microelectrode. is used to compare the signal to an adjustable offset voltage.13) was purchased from Provesta (Bartlesville. The offset voltage is supplied by a voltage divider network through a potential follower. 1'. to less than ImV.Electrode potentials were controlled using a Pine Instrument Company model AFRDE potentiostat Light detection was achieved using a ten dynode fIamamatsu R878 5. dimethylsulfoxide. A potentiostat is used to maintain the desired potentials at the working (anode) and counter (cathode) electrodes versus a Ag/AgCI reference. A baseline corrected voltage is then output to the strip chart recorder. Application of this 'reagentIess' methodology to the determination of glucose and H2~ has been reported previously2. ~CH.A high input impedance DC Differential amplifier. Celite Bio-Catalyst Carrier R-648 (diatomaceous earth) was purchased from Manville (Denver). EnzymeImmobilization y TitaniumLinkage b to Diatomaceous Earth. HC-(CH.r°) ~ ~ '~ " / a H H. 1/ c-.. OK). The current through the PMr Base.ha-t . with charge proportional to light intensity. or by using fiber optics as a waveguide to carry the light to a remotely located PMT. ELECTRONIC CIRCUITS .An overview of the system electronics is shown in Figure 2. This feature gives the chemiluminescence biosensors the potential for extreme sensitivity. The intensity of the light emission is directly proportional to the ethanol concentration in the sample. Titanium oxychloride. The chemiluminescent reaction was electrocatalyzed in the ECL cell by a potential of approximately 0.NI"\ ~ '/ T~ /~~ ~0izNH. A fifteen turn 20ill potentiometer allows the experimenter to reduce the voltage output. ENZYME IMMOBJLlZATION Enzymes were immobilizedon titaniumactivateddiatomaceousearth using ethylene diamine and glutaraldehyde5-? s illustrated in a Figure 1. ethanol. ~O2 ~o c"""" II OH- ~ . corresponding to the PMr dark current. Light can be gathered efficiently by coupling the PMT directly to the ECL cell. - 2 . An end-on PMT is optically coupled to the cell. The pre-treated solution containing all reagents necessary for the quantitative reaction. A controlled electric potential across the anode and cathode catalyzes the oxidation ofluminol by Hz°2.08cm (2") diameter head-on type photomultiplier tube (PMT) with optimal spectral response at 420nm. In order to provide a signal that was proportional to small increases in current through the PMT base. ~ ~ " r ~.J.. glutaraldehyde. The light is detected and quantified by a photomultiplier tube (PMT).1.h-CH . since the excited metastable state of the reaction product has a short halflife9-ll. copper(ll) sulfate. APPARATUS . The PMT base was modified to provide a low voltage output across an external current sensing resistor. built using LM324N operational amplifiers and lOill 1% resistors. Overall PMr gains of 106 are typical. Conditioned PMr output was monitored using a Linear model 2030 chart recorder.6V applied across a gold foil working electrode (anode) and a gold mesh counter electrode (cathode). ethylene diamine. Electrocatalyzed chemiluminescence cells were constructed using a Bioanalytical model 94332 gold mesh electrode. - 0 Ni:! 0 (2) Light is emitted as a by-product of the reaction. EXPERIMENTAL CHEMICALS oxidoreductase. and a Nucleus TB-I photomultiplier tube base. /~~~~~ ~1'1'.OH c--OH ~ II ~ i -Oi.3.280V from the high voltage power supply. 0 ~ Q switching frequencies were synthesized using a Wavetek model 142 function generator. then flows into an Electrocatalyzed Chemiluminescence (ECL) cell. In-line pH was determined using Cole Parmer 05992-64 flow through pH electrodes. an Alfa model 14721 gold foil electrode.NCCH. i ~ 0 ~ / ~a-I:4r l~ 1-0. the reaction is confined spatially.

A polarity switcher circuit. +sv KI K2 Potentiostat KI K2 ref reagent flow Function Generator n. driven by a function generator. INTEGRATED CHEMILUMINESCENCE BIOSENSOR PreliminaIy experiments were conducted using chemically catalyzedchemiluminescence detectionof hydrogenperoxide. A 25mm square by Imm thick gold foil electrode with gold contact wire was attached to the inside of the back plate. The PMI' was optically coupled to the surface of the front plate..015") thick PTFE gasket with a tortuous flow path was used as a spacer to separate the plates.08cm (2") in diameter. ~ -12v LM324N (Qu8d) Figure 3.73. i . oxidation step is followed by a corresponding reduction. Attached to the inside of the front plate was a 2. some of which are transmitted through the gold mesh to the PMr. was designed and constructed to provide this function.< ojfHtvolJ"Ie < 1..Polarity switching of the electrodes was incorporated into ECL cell operation to prevent deterioration of performance due to formation of an oxide coating on the gold electrode surfaces.. a flowing stream containing basified luminol and Cu+2catalyst is combined with a separate target analyte containing stream at the inlet to the cell.48cm (3/16") thick polycarbonate circular plates 5. each photoactive area was approximately 3cm2.. Baseline Zeroing Differential Amplification Circuit CHEMILUMINESCENCE CELLS - Two separate Figure 5. By maintaining a potential across the electrodes. A 0. and a gold contact wire.. To Gold Mesh Electrode Figure 4. photons are emitted from the flowing solution. The tortuous path configuration was found to improve overall performance by ensuring that air bubbles were not trapped within the cell.54cm (1") square gold mesh electrode with a mesh density of 39 lines per em. Polarity Switching Circuit The ECL cell is shown schematically in Figure 5. The chemically catalyzed luminescence detection cell was constructed using two polycarbonate circular plates 0..08cm (2") in diameter.. The Polarity Switcher circuit is shown schematically in Figure 4. These experiments were performed using the test stand .--= IOI:!U% Cumnt Sensing Resistor IIigb ~ I~ Baseline Zeroing CiIaJit To Recorder v Sv Zener To Gold Foil Electrode - .48cm (3/16") thick and 5. The ECL cell was also constructed from two 0. Photons emitted from the solution are transmitted through the front plate of the cell and detected by the PMr.n. separated by a silicone O-ring.n. Electrocatalyzed Chemiluminescence Detection.038cm (0. High Zin DC Differential amplifier OIrrDfl Sensntlll46e f=:~:=':---l i : 1__J +12v _NT 1. The PMr was optically coupled to the external surface of the front plate. In operation.PI8te - G88k8I Front late P Electrocatalyzed Chemiluminescence (ECL) Cell. +12 v 4"zero . The ECL cell functions by combining flowing streams of target analyte and basified luminol at the influent orifice and electrically catalyzing the luminol oxidation reaction within the cell. Under conditions of alternating polarity. chemiluminescentcells were designed and fabricated for chemically and electricallycatalyzed oxidation of luminol respectively. The internalvolumeof the cell was approximately500~ and the 3 . Figure 2.86. no DC DC BASE Tocal -.

relative to the AgIAgCI reference electrode. This was followed by fabrication and testing of a fully integrated chemiluminescent ethanol biosensor system. mixed with the carrier stream immediately prior to entry into the chemiluminescence detection cell. Solid phase basification beds were sized to consistently produce an alkaline efiluent. Flow ratios of 1:1 were used for the carrier and analytical streams. the components were integrated into a fully operational biosensor system. H202 Integrated ECL Chemiluminescent Sensor. Chemically Catalyzed Chemiluminescence.6 volt potential difference. Independently controlled peristaltic pumps were used to establish carrier stream and analyte stream delivery to the electrocatalyzed luminescent (ECL) cell. The analytical stream consisted of varying levels of hydrogen peroxide produced after passage through a 1. The integrated test stand for determination of ethanol by the ECL and with all solid state reagent addition modules is show in Figures 7. a solid phase luminol bed. controlled release of luminol into the aqueous stream. SOLID PHASE BASIFICATION MODULES Solid phase basification (SPB) beds13. using in-line beds of immobilized enzymes. Operational electrode potentials and means for polarity switching required for stable detection and quantification of ~O2 were studied. procedures and ~O2 production rates for alcohol oxidase were investigated sufficiently to demonstrate production of ~O2 proportional to influent ethanol. These two streams were combined to form the carrier stream. The resultant carrier stream contained 50mgIL luminol at pH 10. 2) controlled dissolution of a sufficiently high concentration of luminol in basified DI water using a flowthrough module. The carrier stream consisted of degassed deionized water which had passed sequentially through a 2. shown in Figure 8.illustrated in Figure 6.14containing a range of particle sizes of a proprietary crystalline material were tested. A peristaltic pump was used to feed the chemically catalyzed chemiluminescent cell with three flowing liquid streams. electrical catalysis of chemiluminescence. The carrier to analytical stream flow ratio was 1. 4) electrocatalyzed chemiluminescent oxidation of luminol by hydrogen peroxide and. 3) detection of luminescence. each unit operation required for sensor performance was refined to ensure successful operation.024mM copper sulfate in DI water.2cm3 bed of immobilized alcohol oxidase. The third stream. The analyte stream consisted variously of deionized water blanks and samples with differing levels of hydrogen peroxide dissolved in deionized water. Samples were analyzed by operating the carrier and analyte pumps for ten minutes prior to application of potential to the electrodes. the analyte stream. Several designs for electrocatalyzed luminescence cells were evaluated. a 0. Enzyme immobilization - - - 4 .5cm3 crystallized luminol bed and a 5. The results. A voltage peak from the PMT circuit proportional to the ~O2 concentration in the analytical stream was measured relative to baseline.5:1. The catalyst stream consisted of a 0. 5) enzyme catalyzed reaction of ethanol to produce hydrogen peroxide. EXPERIMENTAL APPROACH Each of the unit operations required for fiber optic chemiluminescent biosensor detection of ethanol were investigated sequentially. was applied between the gold foil anode and the gold mesh cathode for a period of one minute. and a second SPB bed. Solid phase modules for the controlled release of luminophore were investigated. PMr high voltage bias . The alkaline luminol stream was prepared by sequential flow ofDI water through a SolidPhase Base (SPB) bed. a 0. and detection circuit requirements were evaluated. These unit operations include: 1) solid phase basification ofDI water to provide an efiluent pH in the range of 10-11. Feasibility of the sensor system was ultimately demonstrated by the development of calibration curves for ~O2' and ethanol. The carrier stream and the analytical stream were mixed inside the ECL cell. and quantification of chemiluminescent light intensity. Effluent Cell PMT pH Alcohol Oxidase Column Peristaltic Pumps Deionized Water In Tedlar Gas Bag Ethanol In DIWater Figure 7. These unit operations include: enzyme catalyzed substrate oxidation to produce ~O2' solid phase basification of the flowing aqueous stream to pH~10. Following the investigation and refinement of individual unit operations.3.0cm3 SPB bed. After this equilibration period. RESULTS AND DISCUSSION Prior to assembly and testing of the integrated ethanol chemiluminescent biosensor. clearly indicate that efiluent pH is directly proportional to contact Hp Figure 6.5cm3 SPB bed. Preliminary investigations were conducted using chemically catalyzed chemiluminescence. Each bed was challenged with DI water at varied flow rates. corresponding to a range of empty bed contact times between 1 15 minutes. using crystalline media in a packed flowthrough bed.

Th.30mgIL obtained using this method is given in Figure 10. The solubility of luminol is known to be strongly pH dependent. A 0. ISO.. ECL DETECTION . This pH value was judged much better for promoting efficient luminol chemiluminescence. Based on these data. The results of these experiments also prompted th( development of baseline compensation and amplificatiOl circuitry for the PMI' output so that much lower levels of the target analytes could be detected and quantified accurately. The same bed of crystalline luminol was subsequently challenged with the effiuent from an SPBbed. This ensured that the chemiluminescence was confined within a small volume in close proximity to the PMI'. Luminol is sparingly soluble in aqueous solutions. A cah1>rationcurve for ~O2 in the concentration range between 0 .3.4 9. This was followed by similar experiments using SPB and crystalline luminol beds. Upon disassembly of the ECL cell a discoloration of the foil electrode surface was noted.8 9. of the solid phase system effiuents. formation of an oxide coating of the gold surface wa suspected.3 to 7.Ocm3/min.2 0 1 2 3 4 5 6 7 8 9 v 0 0 10 7S. Evidently.2 c: . with aqueous eu+2 introduced into the basified luminol containing stream. The results obtained by UV spectrophotometry are shown in Figure 9.Ocm3 SPB bed was placed downstream from the luminol bed. pH versus Contact Time for SPB Size Fractions. and for this reason is compatI1>le with use in a solid phase module.8 10.6 .0 10. Luminol dissolved in an ethanol-dimethyl sulfoxide mixture.5cm3 bed of crystalline luminol was prepared and used to determine the washout characteristics of this material with a DI water influent at flow rates between 1. cell reassembled. When a second 5. Crystalline luminol with a more favorable particle size distribution was obtained by recrystallization using a proprietary process. the 75-106~ particle size fraction was selected for testing in series with a luminol bed at a flow rate of 2mUmin. The resultant pH was -7. Hydrogen peroxide was also mixed with the luminol-catalyst solution just prior to injection into the chemiluminescent chamber adjacent to the PMI'. the aqueous luminol concentration can be controlled by the pH adjustment of the SPB to produce a nearly constant level for prolonged periods of sustained flow. potentiostat and ECL cell electrodes to determine preciseI: the sign and magnitude of electrode potentials which resulte in oxidation and reduction of the gold electrodes. The chemically catalyze< luminescence experiments demonstrated the quantitativI utility of the chemiluminescent oxidation reaction betweer ~O2 and luminol. ECL system response decreased by a factor 0 approximately one third. The surface contamination was removed and th. Cyclic voltammetry (CV) was performed using th. the quantity of luminol that dissolves depends more on the influent pH than on the bed size (contact time). efi1uentpH increased to 10.4 :I: 2" 10. since the formation of the insoluble hydroxide if favored at these high pHs. ::J IE 10. This behavior is ideal for application in reagentless chemiluminescent biosensors. Under continuous flow conditions.0 w 9. and also confirmed the adequacy of the PMI' and the optical coupling between the cell and the detector. 300 JIIII Empty Bed Contact Time (minutes) V/Q - 11 12 13 14 15 Figure 8. CHEMICALLY CATALYZED LUMINESCENCE - - 11. small variations in the pH of the water produced by the SPB bed produced only minor variations in aqueous luminol concentration even though the bed size was 5 Chemically catalyzed chemiluminescence was first investigated using prepared aqueQJlSreagents. In the present application.time and inversely proportional to particle size. The C1 experiments were also expected to indicate if other oxidatior reduction reactions were 0 ccurring dwing ECL determin- - . The efi1uentluminol concentration ranged between 42-59mgIL. The two fold increase in th( effective range for the solid phase reagent system over th( liquid reagent system was most probably due the increased pF. Differing amounts OJ Cu+2 between the two systems might also have beer responsible.5 for the 2.5cm3 luminol configuration. continually decreasing throughout the experiments.2. After three hours of operation however.6 9.6 10.I06J1111 300-600 JIIII 6OO-IOOOJIIII I!J. and aqueous eu+2 catalyst was added to the basified solution.5 after passage through the luminol bed indicates pH control of the dissolution process. greater than 30 fold improvement in the minimum detectio! limit of hydrogen peroxide over the previously use< chemically catalyzed system. The module must release enough luminol so that the quantitative chemiluminescent reactions can proceed over the desired concentration range for the target analyte.The first ECL experiment provided . SOLID PHASE MODULES FOR CONTROLLED RELEASE OF LUMINOL The controlled release of luminol into the sample stream using an in-line solid phase module greatly simplifies the analytical process!5-!7. The drop in pH from 10. DI water was basified by passage through a SPB bed. The module must also exhibit favorable stability and life characteristics so that near constant aqueous luminol levels can be sustained over prolonged periods of continuous flow. Luminol is commercially available as a fine powder which is not usable for an in-line module due to the inordinately high inlet pressures required to sustain flow.5cm3 SPB0. The cah1>ratioD curve for hydrogen peroxide over the concentration spaD between 0 6OOmgIL generated using the solid phase modules is shown in Figure II.

the anode voltage was reduced to +O. Washout Curve - CIYSfalline Luminol Module. Is. 210 ~ ~19) ::> 0 11) ~ 50 Q8 1.'8 .0 '8:6. 7 a. The voltage sweep rate was set at 20m. This was accomplished. Potential of both the working and counter electrodes was controlled relative to the reference. 9 '(. As a result.5 '.2 to 30 seconds.. with peaks at higher and lower voltages representing the oxidation and reduction of water. 8 ::> . the optimal voltage could only be determined empirically using the ECL cell and reference electrode. 'E i Periodic switching of electrode parity was initially considered a more elegant solution to the gold oxidation problem. and in subsequent runs. Subsequently.0 051015202530354045505511085 HydIOgen l51.!5 0.5 0.45V and +O.2 ! 'E 15 c 0 0 . Continuous oxidation of the working electrode surface was judged responsible for degradation of the capacity to catalyze luminescence between luminol and hydrogen peroxide. Cu(ll) Catalyzed~O2 CL (AqueousLuminol) 6 During DC operation of the ECL cell.0 eoo Peroxide (mgL) 850 Figure 9. The combined effects of the new timing format and the reduced anode potential resulted in an increase in the . ECL Cell -Cyclic Voltammogram. the gold mesh as the counter electrode. The lack of success for this approach most probably lies in the asymmetry between the electrodes.20 c .. 0 0 5 10 15 20 25 30 35 40 45 Throughput (UIersIcm') . with the V gold foil as the working electrode.25 ~ CI 715 7.0 !6.0 -2.0 f! 2.0 \tI.a Figure 12.a 5. A significant problem in the determination of suitable non-oxidizing electrode potentials originates from the low conductivity of the supporting electrolyte in the chemiluminescent biosensor configuration. The CV trace illustrated in Figure 12 shows symmetrical oxidation-reduction reactions at +O.11) . This was attempted when ECL cell performance degradation was first observed and abandoned because of instability in the output signal.0 ~3.0 -E 415 10 ::> 4.. The switching interval used varied between 0. a 1-4 ~ ~ ~3 'E 122 ~s c 1 Ou 0 5 10 15 20 25 30 35 Hydrogen Peroxide (mgIl) Figure 10.5.. These results indicated that the anode potential of +O. <i6 . Figure 11.8V applied to the foil was too high..5 E .6V. 50 ~u 55 ~ 3.. Based upon these results it was concluded that continuous powering of the electrodes was not only unnecessary but also detrimental to long term sensor stability. promoting oxidation of the gold electrode.175V. the PMr voltage reached its peak value within one minute of sample introduction.19) .S . Cu(ll) Catalyzed ~O2 CL (Luminol-Bed) anons. the ECL electrodes were powered for one minute intervals at the end of which peak voltage was measured. This results in a significant (but unknown) potential drop between the reference and working electrode(s). The primary delays in the detector response were due to the residence times within the solid phase beds and interconnecting tubing. and the AgI AgCI reference electrode.J 'E g E ~ e w 8 5 .

0. For this reason development of suitable electrode regeneration methods were undertaken. Among these are short active lives. This resulted in the adoption of a new polarity switching strategy in which the system was operated normally for six to eight hours. IMMOBILIZED ALCOHOL OXIDASE BEDS Alcohol oxidase (AO) was assayed for enzyme activity by the disappearance of ethanol and the appearance of acetaldehyde as determined by gas chromatography.8150 . and the consequent need for frequent recalibration. Feasi. It was found that H202 recovery could be improved dramatically when flow rates and bed sizes were adjusted to give a better match between challenge ethanol concentrations and enzymatic activity. Expanded Scale Ethanol Calibration Curve - CONCLUSIONS Amperometric biosensors suffer from several problems which render their use impractical for deployment aboard spacecraft.4. Calibration curves have been generated using fully integrated reagentless test systems. high rates of signal drift. Although the overall stability was much improved using the new operating procedures. INTEGRATED BIOSENSOR TEST The components required to perform each unit operation were assembled into an integrated systems for quantitation of ethanol.viable operation time periods of over eight hours with only a 10% decrease in sensor response.1g/L(Ppb) is presented in Figure 14. corresponding to a carrier stream to analytical stream flow ratio of 1. In this configuration a challenge stream containing 395J. 410.1g/Lof hydrogen peroxide.5:1. an expanded scale version of this curve between 0-250J. For application in the integrated ethanol chemiluminescent biosensor. the ECL for the luminolhydrogen peroxide reaction proved stable and reproducible. a 1. Aqueous ethanol solutions ranging in concentration between 40 and 4114J. The system was operated in this manner for approximately 100 hours over a six week period with no discernible degradation in performance. incorporating solid phase media in replacement of aqueous phase reagents has shown potential as a means to overcome these difficulties.1g/L(ppb). The ethanol sensor was challenged with three replicates each of standards containing 40. and 98. resulting in standard deviations (lcr) of 3. AO on DES was found to produce stoichiometric conversion of 15. 200. The height of the signal above the baseline for this concentration suggests that even lower levels could be detected using the current relatively crude embodiment of the innovative technology. A novel chemiluminescence based approach to biosensor development. These data clearly demonstrate the sensitivity of this technique and the capacity to extend the lower limits of detection to even lower values with the current sensor configuration.1g/L (ppb) respectively. Ethanol concentrations as low as 40 J. Further 7 .1g/Lof ethanol produced 33 J. ~O2 is readily decomposed by transition metal impurities on the diatomaceous earth support. The resulting data were used to construct the smooth quadratic calibration curve over this range shown in Figure 13. Because the ability to detect very low levels is an important feature of the ethanol biosensor system.g c g 8100 "0 c ffi 50 0 2 3 4 5 6 7 8 9 10 11 12 PMT Response (mV) Figure 14.1g/L have been detected. Improvements in sensitivity are also achievable using this technique.6. this level of degradation was still considered unacceptable.100J. 250 6 :::200 ~ c . 17. 4500 4000 ~3500 as ~300) c: 0 2500 ~ 82000 8 1500 "0 ~ 1000 W 500 0 0 10 20 30 40 PMT Response 50 (mY) 60 70 80 90 - Figure 13.8mg/L ethanol to acetaldehyde in a IOcm3 bed with a flow rate of 2cm3/min.3%. powering the electrodes intermittently for one minute intervalsto perform analyses. and 4. This resulted in poor recovery of the hydrogen peroxide from the immobilized enzyme bed. .1>ility of the Ethanol Chemiluminescent Biosensor concept has been rigorously demonstrated. Using this slow switching operational protocol.4J.2cm3 AO bed was used at a flow rate of 3cm3/min. The operational period was followed by one hour of continuous reversed polarity which was found to fully return the ECL electrodes to their original performance level.1g/L(ppb) as C were determined using the integrated chemiluminescent ethanol biosensor. 25. Unfortunately. This corresponds to a H202 production efficiency of 11. Ethanol Calibration Curve.

in Chemiluminescence and Photochemical Reaction Detection in Chromatography. J. University oflllinois.R. AF. 8. Eds. J. Atwater.R. Chem.E. T. Van Dyke. RR.L..Lg/L limit. The problem of uncharacterized TOC can be diminished by using chemiluminescent alcohol biosensors to determine the concentrations of low molecular weight straight chain alcohols in the product water. Akse. Schussel. 1995. J.. Eds. RR.E.. JE. Akse.1g/L OC.SAE Trans J.. presented at the 22nd International Conference on Environmental Systems. 1. Advanced Catalytic Methods for Destruction of Environmental CoDt~min:mts. NIl. J. 1995.. Birks.E. FA.J.. 1995. B. Texas. Work to date has examined the response of the chemiluminescence based biosensor to ethanol. 1977. Warrendale.. and Wheeler. 30 (5).0.Lg/Llimit depending upon the magnitude and composition of the influent organics. and Dehart. 1993.. 10. 8 . and Atwater. 1991. and Carter. NASA's current requirements for reclaimed potable water specifies a maximum of SOOJ. L. controlled release of luminol using a solid phase flow-through module.. A Urease Bioreactor for Water Reclamation Aboard Spacecraft. 1989.L.J.. The ECL cell fabricated from transparent polycarbonate. 1994.6 volts. RP. Jolly. Akse. Hooksett. J. (1). C. detection circuitry. of unknown composition.E... CE.E.. electrocatalyzed luminescence using a potentiostat and gold electrodes. 13. B.. Conway. J. Unit operations required for sensor operation have been developed sufficiently for demonstration of the concept These include pH adjustment using solid phase flow-through modules.A.. Anal. in Electroanalytical Chemistry. J. Houston. A Breadboard Electrochemical Water Recovery System for Producing Potable Water from Composite Wastewater Generated in Enclosed Habitats. The solid phase luminol beds were shown capable of sustained controlled release of the luminophore at a sufficient concentration for the biosensor systems to function over a broad span of target analyte concentrations. 985-994. Bard. New York. Johnson Space Center. When applied to the examination of reclaimed waters and wastewater streams associated with regenerative life support systems. Akse.. 102. D. Urbana.. Cheng. E. ACKNOWLEDGMENT This work was supported by the National Aeronautics and Space Administration's Lyndon B. J.. SPIE Proceedings Series. July 1993. Schussel. J. VCR. Carter.. 1. Development and Fabrication of a Breadboard Electrochemical Water Recovery System.. 12. Photochemical.J. and Atwater. 1. Schussel. July 1992. Alcohol oxidase effectively catalyzes the oxidation of several low molecular weight primary alcohols. and Scott. and Rudd.. 2574. SP-873" Behrend. RD.J. this technique is applicable as an alcohol selective detector which can be used in conjunction with liquid chromatographic separations... 10. REFERENCES 1. Chemosphere..R. L. 7.. L. D. 2. and background chemiluminescence. E. 4.-Y. in Regenerative Lift Support: Systems & Processes. of which a T maximum of lOOJ.J. immobilized enzyme catalyzed oxidation of ethanol to hydrogen peroxide. The ECL cell design was also found to adequately confine the reaction spatially for efficient collection of the light either directly by an optically coupled photomultiplier tube.. 513-529.e. Nieman. Seattle. C. Ed. 932181. Akse. Techniques of Electrocatalyzed Chemiluminescence. Detection Based on Solution-Phase Chemiluminescence Systems. Jolly.R. 633. D. submitted to Enzyme Microb.. DeHart. 1995. 1989. L.D. and Verostko.A and H. was found to effectively initiate the reaction between luminol and hydrogen peroxide at an applied potential of 0. Atwater. 78-84. presented 23rd International Conference on Environmental Systems.J. Technol. A Series of Advances. Electrochemical Manipulation of Fluorescence and Chemiluminescence Signals at Fiber-Optic Probes. J. and Reysa. Catalytic Methods Using Molecular Oxygen for Treatment of PMMS & ECLSS Waste Streams. Colorado Springs. 9. Detection limits are primarily determined by enzyme conversion efficiencies. and Atwater.. 11. and Wheeler. although the sensor can be expected to respond to other alcohols. Atwater.. Applications ofElectrocatalyzed Chemiluminescence ofLuminol. Van Dyke. Rose. J. Electrochemical Society. dark current limitations. and a gold mesh counter electrode. Washington. A Continuous Alcohol Oxidase Bioreactor for Regenerative Life Support. Houston. under contract NAS9-19021.E. Reagentless Chemiluminescence Based Fiber Optic Sensors for Regenerative Life Support in Space.Ieffers.Lg/L may be uncharacterized... L. MacElroy. 3. Thompson. Jr. Faulkner. Improvements in enzyme immobilization procedures and hardware design can certainly provide means to significantly overcome these limitations.R. Mwphy. light leakage. in Water Purification by Photocatalytic. and quantification of light intensity using a photomultiplier tube. and Electrochemical Processes. 5... Schussel. 1986. 1. Current technologies such as aqueous phase catalytic oxidation systems (APCOS) are certainly capable of meeting the 500J. SAE Technical Paper Series No. Advanced Development of Immobilized Enzyme Reactors.. Jr.W.E. Atwater. Society of Automotive Engineers. AIAA-95-LS-70. 61. Thompson. New Technologies for On-Line Water Quality Monitoring. 6. and using a gold foil working electrode. April 3-5.A. Doctoral Dissertation. the sensor response will be proportional to the sum of methanol and ethanol present If quantitation of individual alcohols is required.~ development is expected to lower the ethanol detection limit by an order of magnitude. J. presented at AIAA/NASA Life Sciences and Space Medicine Conference. I.R and A1. i.. L. but may have difficulty with the lOOJ. T.L. Aerospace. O.

239. Houston..R. and T.. Immobilized and Solid-State Reagent Systems for Luminol Chemiluminescence in Flow Systems.A Nieman. Hool. 869. Chern. Jeffers. and T. T. 1988. Chemiluminescence Analysis in Flowing Streams with Luminol Immobilized on Silica and Controlled-Pore Glass. 59. 15. Hool. K. Anal. March 1993. Paxton. J. D.A. Anal. An On-Line Water Quality Monitor for Space Station Freedorn.L. Dougherty. 1988.A Nieman. 834. T. presented at AIChE Spring National Meeting. July 1993. Chern. E. 1987. paper 79c.E. 17. 16. and Atwater. Acta {Wien} lII. 60. Immobilized Luminol Chemiluminescence Reagent System for Hydrogen Peroxide Determinations in Flowing Streams.A.. . 14.Mikrochirn. K.. Nieman.-Colorado Springs.