ABSTRACf

The investigation and development of achemiluminescence based ethanol detection concept into abiosensor system is descn1>ed. The biosensor uses alcoholoxidase to catalyze the reaction of short chain primaryalcohols with elemental oxygen to produce hydrogen peroxideand the corresponding aldehyde. The reaction of hydrogenperoxide with an organic luminophore in the presence of asufficient electric field results in emission of blue light withpeak intensity at 42Snm. The chemiluminescent lightintensity is directly proportional to the alcohol concentrationof the sample. The aqueous phase chemistIy required forsensor operation is implemented using solid phase moduleswhich adjust the pH of the influent stream, catalyze theoxidation of alcohol, provide the controlled addition of theluminophore to the flowing aqueous stream, and minimi7ethe requirement for expendables. Precise control of the pHbas proven essential for the long-term sustained release of theluminophore. Electrocatalysis is achieved using a controlledpotential across gold mesh and gold foil electrodes whichundergo periodic polarity reversals. Ethanol concentrations(as C) in the range between 40 and 4.000J1gILhave beendetermined. The performance features of this sensor arepresented in this paper.

INTRODUCTION

For aqueous waste streams generated by a crew inregenerative life SUPPOrt.ethanol is a major impurity. It ispoorly removed by physicochemical processes such asmultifiltration (MF) and reverse osmosis (RO). andconsequently forms a significant fraction of the efi1uent totalorganic carbon (fOC) from these processes. The inability ofsorption and membrane based separations to remove ethanoland other low molecular weight water soluble organics basled to the evaluation of alternative water reclamation methodssuch as electrochemical water recoveryl.2. and to thedevelopment of post-treatment technologies for RO and MF

951686

Chemiluminescent EthanolBiosensor Development

James E. Atwater, James R. Akse,Jeffrey DeHart, and Richard R. Wheeler, Jr.

Umpqua Research Co.

Charles E. VerostkoNASA-Johnson Space Center

systems such as low temperature aqueous phase catalyticoxidation3.4.and immobilized enzyme bioreactocs>7. Thecapability for quantitation of ethanol in waste water streamssuch as humidity condensate and in finished potable watercan be used advantageously in the control of water processoroperation.

The initial chemiluminescent ethanol biosensordevelopment effort was intended to demonstrate the feasi.oilityof an accurate. precise. sensitive. and reliable means for thedetermination of ethanol and other low molecular weightstraight chain alcohols within aqueous streams aboardspacecraft. A unique aspect of the ethanol biosensoroperation is that all reagents are provided by solid state flow-through modules. thus minimhing the storage and resupplylogistic requirements for long term operation in earth orbit oron station within a Lunar or planetary outpost. Operation ofthe biosensor is based upon the oxidation of ethanol toacetaldehyde. and reduction of <>2to hydrogen peroxide

~Oi' which then reacts with a luminophore to producelight. The intensity of the light is directly proportional to theconcentration of ethanol in the sample. Advantages inherentto the technique are the potential for detection of extremelylow levels. the stability of the devices used to measure lightintensity (photomultiplier tubes or charge coupled devices).and the use of solid state modules for the controlled release ofreagents. This detection scheme may be convenientlyembodied in either continuous on-line or flow injectionanalysis configurations.

Ethanol is detected using the enzyme alcoholoxidase. Alcohol oxidase catalyzes the reaction of ethanolwith molecular oxygen to form acetaldehyde and ~O2'

H

I ~ '-./ ~~ ~c ~H~ ~ AIcchoIOxida-. II

H 0(1)

The ~O2 containing aqueous stream flows througha solid phase basification module where the :l1hlinity isadjusted to pH II::10. The basified efi1uentflows into a bed of

luminol (3-aminophthalhydrazide) crystals which slowlydissolve at a controlled rate. The pre-treated solutioncontaining all reagents necessary for the quantitative reaction,then flows into an Electrocatalyzed Chemiluminescence(ECL) cell. A controlled electric potential across the anodeand cathode catalyzes the oxidation ofluminol by Hz°2.

0 ~ Q" ~O2 ~o 1/

~ r ~.. ~. c-.OH ~c"""" OH- c--OHII II

~ 0 Ni:! 0 (2)Light is emitted as a by-product of the reaction. The

light is detected and quantified by a photomultiplier tube(PMT). The intensity of the light emission is directlyproportional to the ethanol concentration in the sample. Byusing an electrical potential across the ECL cell to catalyzethe reaction of hydrogen peroxide with luminol, the reactionis confined spatially, since the excited metastable state of thereaction product has a short half-life9-ll. Light can be gathered efficiently by coupling thePMT directly to the ECL cell, or by using fiber optics as awaveguide to carry the light to a remotely located PMT.

The ECL apparatus consists of a three electrodeelectrolytic cell. A potentiostat is used to maintain thedesired potentials at the working (anode) and counter(cathode) electrodes versus a Ag/AgCI reference. An end-onPMT is optically coupled to the cell. Overall PMr gains of106 are typical. This feature gives the chemiluminescencebiosensors the potential for extreme sensitivity. Applicationof this 'reagentIess' methodology to the determination ofglucose and H2~ has been reported previously2.

EXPERIMENTAL

CHEMICALS - Alcohol oxidase (alcohol oxygenoxidoreductase, EC 1.1.3.13) was purchased from Provesta(Bartlesville, OK). Titanium oxychloride, ethylene diamine,glutaraldehyde, ethanol, copper(ll) sulfate, dimethylsulfoxide,and luminol were obtained from Aldrich (Milwaukee). CeliteBio-Catalyst Carrier R-648 (diatomaceous earth) was pur-chased from Manville (Denver).APPARATUS - Electrode potentials were controlled using aPine Instrument Company model AFRDE potentiostat Lightdetection was achieved using a ten dynode fIamamatsu R8785.08cm (2") diameter head-on type photomultiplier tube(PMT) with optimal spectral response at 420nm, and aNucleus TB-I photomultiplier tube base. High voltage wasapplied to the PMr using a Nucleus model 575 scaler-ratemeter power supply. Conditioned PMr output wasmonitored using a Linear model 2030 chart recorder. In-linepH was determined using Cole Parmer 05992-64 flowthrough pH electrodes. Flows were established usingMasterflex model 7520-35 multichannel peristaltic pumps.Electrocatalyzed chemiluminescence cells were constructedusing a Bioanalytical model 94332 gold mesh electrode, anAlfa model 14721 gold foil electrode, and a Microelectrodesmodel MI 402 Ag/AgCI reference microelectrode. Polarity

switching frequencies were synthesized using a Wavetekmodel 142 function generator.ENZYME IMMOBJLlZATION - Enzymes wereimmobilizedon titaniumactivateddiatomaceousearth usingethylene diamine and glutaraldehyde5-?as illustrated inFigure 1.

~ ~~'~ H,NCCH,J.NI"\~

' /~~-Oi.r°) / " / T~

a ~0izNH.

i i

~H ~

HC-(CH,h-CH , ~CH,ha-t.. 1'.

/ ~a-I:4r0

l~1-0, /~~~~~~1'1'.~~Figure 1. EnzymeImmobilizationby TitaniumLinkage

to Diatomaceous Earth.

SOLID PHASE BASIFICATION BEDS - Two proprietaIysolid phase basification (SPB) materials were examined ascandidates for the flow through pH conditioning bed.ELECTRONIC CIRCUITS - An overview of the systemelectronics is shown in Figure 2. The photomultiplier tubereceives a positive bias of 1,280V from the high voltagepower supply. The current through the PMr Base, withcharge proportional to light intensity, is input to the BaselineZeroing Circuit in the form of a voltage drop across a IOID1% current sensing resistor. A baseline corrected voltage isthen output to the strip chart recorder.

The PMT base was modified to provide a low voltageoutput across an external current sensing resistor. In order toprovide a signal that was proportional to small increases incurrent through the PMT base, a Baseline-Zeroing Circuitwas designed and constructed as shown in Figure 3.A highinput impedance DC Differential amplifier, built usingLM324N operational amplifiers and lOill 1% resistors, isused to compare the signal to an adjustable offset voltage.The differential amplifier output is a voltage which isproportional to the differencebetween the input and the offsetvoltages. The offset voltage is supplied by a voltage dividernetwork through a potential follower. A fifteen turn 20illpotentiometer allows the experimenter to reduce the voltageoutput, corresponding to the PMr dark current, to less thanImV. The differential amplifier also provides an overall gainof2.

The chemiluminescent reaction was electrocatalyzedin the ECL cell by a potential of approximately 0.6V appliedacross a gold foil working electrode (anode) and a gold meshcounter electrode (cathode). Both voltages were maintainedrelative to a AgIAgCI reference electrode by the potentiostat.

2

Polarity switching of the electrodes was incorporated intoECL cell operation to prevent deterioration of performancedue to formation of an oxide coating on the gold electrodesurfaces. Under conditions of alternating polarity, each

Potentiostat

KIK2

ref

reagentflow

;, iIOI:!U%

BASE CumntSensing

Tocal Resistor- -- -=IIigb~ I~

ToRecorder

BaselineZeroingCiIaJit

Figure 2. Electrocatalyzed Chemiluminescence Detection.

oxidation step is followed by a corresponding reduction. Apolarity switcher circuit, driven by a function generator, was

designed and constructed to provide this function. ThePolarity Switcher circuit is shown schematically in Figure 4.

High Zin DC Differential amplifier

f=:~:=':---lOIrrDfl Sensntlll46e i :

_NT 1__- J+12v

"zero ..

1.86..< ojfHtvolJ"Ie < 1.73.. +12 v

4-

~ -12v

LM324N (Qu8d)

Figure 3. Baseline Zeroing Differential Amplification Circuit

CHEMILUMINESCENCE CELLS - Two separatechemiluminescentcells were designed and fabricated forchemicallyand electricallycatalyzed oxidation of luminolrespectively. The chemically catalyzed luminescencedetection cell was constructed using two polycarbonatecircular plates 0.48cm (3/16") thick and 5.08cm (2") indiameter, separatedby a silicone O-ring. The PMI' wasoptically coupled to the surface of the front plate. Theinternalvolumeof the cell was approximately500~ and the

photoactive area was approximately 3cm2. In operation, aflowing stream containing basified luminol and Cu+2catalystis combined with a separate target analyte containing streamat the inlet to the cell. Photons emitted from the solution aretransmitted through the front plate of the cell and detected bythe PMr.

+svKI K2

FunctionGenerator

n.n..n...

DCDC

no

SvZener

v To - . ToGold GoldFoil MeshElectrode Electrode

Figure 4. Polarity Switching Circuit

The ECL cell is shown schematically in Figure 5.The ECL cell was also constructed from two 0.48cm (3/16")thick polycarbonate circular plates 5.08cm (2") in diameter.Attached to the inside of the front plate was a 2.54cm (1")square gold mesh electrode with a mesh density of 39 linesper em, and a gold contact wire. The PMr was opticallycoupled to the external surface of the front plate. A 25mmsquare by Imm thick gold foil electrode with gold contactwire was attached to the inside of the back plate. A 0.038cm(0.015") thick PTFE gasket with a tortuous flow path wasused as a spacer to separate the plates. The tortuous pathconfiguration was found to improve overall performance byensuring that air bubbles were not trapped within the cell.The ECL cell functions by combining flowing streams oftarget analyte and basified luminol at the influent orifice andelectrically catalyzing the luminol oxidation reaction withinthe cell. By maintaining a potential across the electrodes,photons are emitted from the flowing solution, some of whichare transmitted through the gold mesh to the PMr.

- PI8te G88k8I FrontPlate

Figure 5. Electrocatalyzed Chemiluminescence (ECL) Cell.

INTEGRATED CHEMILUMINESCENCE BIOSENSOR-PreliminaIy experiments were conducted using chemicallycatalyzedchemiluminescencedetectionof hydrogenperoxide.These experiments were performed using the test stand

3

illustrated in Figure 6. A peristaltic pump was used to feedthe chemically catalyzed chemiluminescent cell with threeflowing liquid streams. The alkaline luminol stream wasprepared by sequential flow ofDI water through a SolidPhaseBase (SPB) bed. a solid phase luminol bed. and a second SPBbed. The catalyst stream consisted of a 0.024mM coppersulfate in DI water. These two streams were combined toform the carrier stream. The third stream, the analyte stream.mixed with the carrier stream immediately prior to entry intothe chemiluminescence detection cell. The analyte streamconsisted variously of deionized water blanks and sampleswith differing levels of hydrogen peroxide dissolved indeionized water. Flow ratios of 1:1 were used for the carrierand analytical streams.

The integrated test stand for determination ofethanol by the ECL and with all solid state reagent additionmodules is show in Figures 7. Independently controlledperistaltic pumps were used to establish carrier stream andanalyte stream delivery to the electrocatalyzed luminescent(ECL) cell. The carrier stream consisted of degasseddeionized water which had passed sequentially through a2.5cm3 SPB bed. a 0.5cm3 crystallized luminol bed and a5.0cm3 SPB bed. The resultant carrier stream contained50mgIL luminol at pH 10.3. The analytical stream consistedof varying levels of hydrogen peroxide produced after passagethrough a 1.2cm3 bed of immobilized alcohol oxidase. Thecarrier to analytical stream flow ratio was 1.5:1. The carrierstream and the analytical stream were mixed inside the ECLcell.

H202

Hp

Figure 6. Chemically Catalyzed Chemiluminescence.

Samples were analyzed by operating the carrier andanalyte pumps for ten minutes prior to application of potentialto the electrodes. After this equilibration period, a 0.6 voltpotential difference, relative to the AgIAgCI referenceelectrode, was applied between the gold foil anode and thegold mesh cathode for a period of one minute. A voltage peakfrom the PMT circuit proportional to the ~O2 concentrationin the analytical stream was measured relative to baseline.EXPERIMENTAL APPROACH - Each of the unitoperations required for fiber optic chemiluminescentbiosensor detection of ethanol were investigatedsequentially. These unit operations include: enzymecatalyzed substrate oxidation to produce ~O2' solid phasebasification of the flowing aqueous stream to pH~10,controlled release of luminol into the aqueous stream.electrical catalysis of chemiluminescence, and quantificationof chemiluminescent light intensity. Enzyme immobilization

procedures and ~O2 production rates for alcohol oxidasewere investigated sufficiently to demonstrate production of~O2 proportional to influent ethanol. Solid phasebasification beds were sized to consistently produce analkaline efiluent. Solid phase modules for the controlledrelease of luminophore were investigated. Preliminaryinvestigations were conducted using chemically catalyzedchemiluminescence. Several designs for electrocatalyzedluminescence cells were evaluated. Operational electrodepotentials and means for polarity switching required for stabledetection and quantification of ~O2 were studied. PMr highvoltage bias .and detection circuit requirements wereevaluated. This was followed by fabrication and testing of afully integrated chemiluminescent ethanol biosensor system.Feasibility of the sensor system was ultimately demonstratedby the development of calibration curves for ~O2' andethanol.

CellpH Effluent

PMT

EthanolInDIWater

Alcohol OxidaseColumn

PeristalticPumps

Deionized Water

In Tedlar Gas Bag

Figure 7. Integrated ECL Chemiluminescent Sensor.

RESULTS AND DISCUSSION

Prior to assembly and testing of the integratedethanol chemiluminescent biosensor, each unit operationrequired for sensor performance was refined to ensuresuccessful operation. These unit operations include: 1) solidphase basification ofDI water to provide an efiluent pH in therange of 10-11, using crystalline media in a packed flow-through bed; 2) controlled dissolution of a sufficiently highconcentration of luminol in basified DI water using a flow-through module; 3) detection of luminescence; 4)electrocatalyzed chemiluminescent oxidation of luminol byhydrogen peroxide and; 5) enzyme catalyzed reaction ofethanol to produce hydrogen peroxide, using in-line beds ofimmobilized enzymes. Following the investigation andrefinement of individual unit operations, the componentswere integrated into a fully operational biosensor system.SOLID PHASE BASIFICATION MODULES - Solid

phase basification (SPB) beds13,14containing a range ofparticle sizes of a proprietary crystalline material were tested.Each bed was challenged with DI water at varied flow rates,corresponding to a range of empty bed contact times between1 - 15 minutes. The results, shown in Figure 8, clearlyindicate that efiluent pH is directly proportional to contact

4

time and inversely proportional to particle size. Based onthese data, the 75-106~ particle size fraction was selectedfor testing in series with a luminol bed at a flow rate of2mUmin. The resultant pH was -7.5 for the 2.5cm3 SPB-0.5cm3 luminol configuration. When a second 5.Ocm3SPBbed was placed downstream from the luminol bed, efi1uentpHincreased to 10.3. This pH value was judged much better forpromoting efficient luminol chemiluminescence.SOLID PHASE MODULES FOR CONTROLLEDRELEASE OF LUMINOL - The controlled release ofluminol into the sample stream using an in-line solid phasemodule greatly simplifies the analytical process!5-!7. Themodule must release enough luminol so that the quantitativechemiluminescent reactions can proceed over the desiredconcentration range for the target analyte. The module mustalso exhibit favorable stability and life characteristics so thatnear constant aqueous luminol levels can be sustained overprolonged periods of continuous flow.

11.0

10.8

10.6

10.4:I:2" 10.2c:..::JIE 10.0w

9.8

9.6 v 7S.I06J1111

I!J. ISO. 300 JIIII

0 300-600 JIIII

0 6OO-IOOOJIIII

9.4

9.20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Empty Bed Contact Time (minutes) -V/Q

Figure 8. pH versus Contact Time for SPB Size Fractions.

Luminol is sparingly soluble in aqueous solutions,and for this reason is compatI1>lewith use in a solid phasemodule. Luminol is commercially available as a fine powderwhich is not usable for an in-line module due to theinordinately high inlet pressures required to sustain flow.Crystalline luminol with a more favorable particle sizedistribution was obtained by recrystallization using aproprietary process. A 0.5cm3 bed of crystalline luminol wasprepared and used to determine the washout characteristics ofthis material with a DI water influent at flow rates between1.6 - 2.Ocm3/min. The results obtained by UVspectrophotometry are shown in Figure 9. The same bed ofcrystalline luminol was subsequently challenged with theeffiuent from an SPBbed. The efi1uentluminol concentrationranged between 42-59mgIL. The solubility of luminol isknown to be strongly pH dependent. The drop in pH from10.3 to 7.5 after passage through the luminol bed indicatespH control of the dissolution process. Under continuous flowconditions, small variations in the pH of the water producedby the SPB bed produced only minor variations in aqueousluminol concentration even though the bed size was

continually decreasing throughout the experiments.Evidently, the quantity of luminol that dissolves dependsmore on the influent pH than on the bed size (contact time).In the present application, the aqueous luminol concentrationcan be controlled by the pH adjustment of the SPB to producea nearly constant level for prolonged periods of sustainedflow. This behavior is ideal for application in reagentlesschemiluminescent biosensors.CHEMICALLY CATALYZED LUMINESCENCE -Chemically catalyzed chemiluminescence was firstinvestigated using prepared aqueQJlSreagents. DI water wasbasified by passage through a SPB bed. Luminol dissolved inan ethanol-dimethyl sulfoxide mixture, and aqueous eu+2catalyst was added to the basified solution. Hydrogenperoxide was also mixed with the luminol-catalyst solutionjust prior to injection into the chemiluminescent chamberadjacent to the PMI'. This ensured that thechemiluminescence was confined within a small volume inclose proximity to the PMI'. A cah1>rationcurve for ~O2 inthe concentration range between 0 - 30mgIL obtained usingthis method is given in Figure 10.

This was followed by similar experiments using SPBand crystalline luminol beds, with aqueous eu+2 introducedinto the basified luminol containing stream. The cah1>ratioDcurve for hydrogen peroxide over the concentration spaDbetween 0 - 6OOmgIL generated using the solid phasemodules is shown in Figure II. 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.of the solid phase system effiuents. Differing amounts OJCu+2 between the two systems might also have beerresponsible, since the formation of the insoluble hydroxide iffavored at these high pHs. The chemically catalyze<luminescence experiments demonstrated the quantitativIutility of the chemiluminescent oxidation reaction betweer~O2 and luminol, and also confirmed the adequacy of thePMI' and the optical coupling between the cell and thedetector. The results of these experiments also prompted th(development of baseline compensation and amplificatiOlcircuitry for the PMI' output so that much lower levels of thetarget analytes could be detected and quantified accurately.ECL DETECTION - The first ECL experiment provided ;greater than 30 fold improvement in the minimum detectio!limit of hydrogen peroxide over the previously use<chemically catalyzed system. After three hours of operationhowever, ECL system response decreased by a factor 0approximately one third. Upon disassembly of the ECL cella discoloration of the foil electrode surface was noted. Th,formation of an oxide coating of the gold surface wasuspected. The surface contamination was removed and th,cell reassembled.

Cyclic voltammetry (CV) was performed using th,potentiostat and ECL cell electrodes to determine preciseI:the sign and magnitude of electrode potentials which resultein oxidation and reduction of the gold electrodes. The C1experiments were also expected to indicate if other oxidatiorreduction reactions were 0 ccurring dwing ECL determin-

5

25

.

~CI

..5.20c.2!'E 158c00

g 10E::>

..J'E8 5ew

.0

0 5 10 15 20 25 30 35 40

Throughput (UIersIcm')

45 50

Figure 9. Washout Curve -CIYSfalline Luminol Module.

anons. The voltage sweep rate was set at 20m.VIs, with thegold foil as the working electrode, the gold mesh as thecounter electrode, and the AgI AgCI reference electrode. TheCV trace illustrated in Figure 12 shows symmetricaloxidation-reduction reactions at +O.45V and +O.175V, withpeaks at higher and lower voltages representing the oxidationand reduction of water. These results indicated that the anode

potential of +O.8V applied to the foil was too high, promotingoxidation of the gold electrode. Continuous oxidation of theworking electrode surface was judged responsible fordegradation of the capacity to catalyze luminescence betweenluminol and hydrogen peroxide. A significant problem in thedetermination of suitable non-oxidizing electrode potentialsoriginates from the low conductivity of the supportingelectrolyte in the chemiluminescent biosensor configuration.This results in a significant (but unknown) potential dropbetween the reference and working electrode(s). As a result,the optimal voltage could only be determined empiricallyusing the ECL cell and reference electrode. This wasaccomplished, and in subsequentruns, the anode voltage wasreduced to +O.6V.

9

'(;;'8...

i 7a.<i6.

'E

~sa1-4~~3'E122~c 1

Ou0 5 10 15 20 25

Hydrogen Peroxide (mgIl)

30 35

Figure 10. Cu(ll) Catalyzed~O2 CL (AqueousLuminol)

55

7157.0

'8:6.5E 6.0!6.5';a 5.0

-E 415~::> 4.0

~3.S

~3.0-2.5f 2.0!~ul51.0

0.!5

0.0051015202530354045505511085

HydIOgen Peroxide (mgL)

850

..

eoo

Figure 11. Cu(ll) Catalyzed ~O2 CL (Luminol-Bed)

210

~~19)

~::>0 11)

50

Q8 1.0

\tI;ge

.11)

.19)

.a

Figure 12. ECL Cell -Cyclic Voltammogram.

Periodic switching of electrode parity was initiallyconsidered a more elegant solution to the gold oxidationproblem. This was attempted when ECL cell performancedegradation was first observed and abandoned because ofinstability in the output signal. The switching interval usedvaried between 0.2 to 30 seconds. Potential of both theworking and counter electrodes was controlled relative to thereference. The lack of success for this approach mostprobably lies in the asymmetry between the electrodes.

During DC operation of the ECL cell, the PMrvoltage reached its peak value within one minute of sampleintroduction. The primary delays in the detector responsewere due to the residence times within the solid phase bedsand interconnecting tubing. Based upon these results it wasconcluded that continuous powering of the electrodes was notonly unnecessary but also detrimental to long term sensorstability. Subsequently, the ECL electrodes were powered forone minute intervals at the end of which peak voltage wasmeasured.

The combined effects of the new timing format andthe reduced anode potential resulted in an increase in the

6

viable operation time periods of over eight hours with only a10% decrease in sensor response. Although the overallstability was much improved using the new operatingprocedures, this level of degradation was still consideredunacceptable. For this reason development of suitableelectrode regeneration methods were undertaken. Thisresulted in the adoption of a new polarity switching strategyin which the system was operated normally for six to eighthours, powering the electrodes intermittently for one minuteintervalsto perform analyses. .The operational period wasfollowed by one hour of continuous reversed polarity whichwas found to fully return the ECL electrodes to their originalperformance level. The system was operated in this mannerfor approximately 100 hours over a six week period with nodiscernible degradation in performance. Using this slowswitching operational protocol, the ECL for the luminol-hydrogen peroxide reaction proved stable and reproducible.IMMOBILIZED ALCOHOL OXIDASE BEDS -Alcohol

oxidase (AO) was assayed for enzyme activity by thedisappearance of ethanol and the appearance of acetaldehydeas determined by gas chromatography. AO on DES wasfound to produce stoichiometric conversion of 15.8mg/Lethanol to acetaldehyde in a IOcm3 bed with a flow rate of2cm3/min. Unfortunately, ~O2 is readily decomposed bytransition metal impurities on the diatomaceous earthsupport. This resulted in poor recovery of the hydrogenperoxide from the immobilized enzyme bed. It was foundthat H202 recovery could be improved dramatically whenflow rates and bed sizes were adjusted to give a better matchbetween challenge ethanol concentrations and enzymaticactivity. For application in the integrated ethanolchemiluminescent biosensor, a 1.2cm3 AO bed was used at aflow rate of 3cm3/min, corresponding to a carrier stream toanalytical stream flow ratio of 1.5:1. In this configuration achallenge stream containing 395J.1g/Lof ethanol produced 33J.1g/Lof hydrogen peroxide. This corresponds to a H202production efficiency of 11.3%.INTEGRATED BIOSENSOR TEST - The componentsrequired to perform each unit operation were assembled intoan integrated systems for quantitation of ethanol. Aqueousethanol solutions ranging in concentration between 40 and4114J.1g/L(ppb) as C were determined using the integratedchemiluminescent ethanol biosensor. The resulting data wereused to construct the smooth quadratic calibration curve overthis range shown in Figure 13. Because the ability to detectvery low levels is an important feature of the ethanolbiosensor system, an expanded scale version of this curvebetween 0-250J.1g/L(Ppb) is presented in Figure 14. Thesedata clearly demonstrate the sensitivity of this technique andthe capacity to extend the lower limits of detection to evenlower values with the current sensor configuration. Theethanol sensor was challenged with three replicates each ofstandards containing 40, 200, 410, and 4,100J.1g/L(ppb),resulting in standard deviations (lcr) of 3.6, 17.0, 25.4, and98.4J.1g/L(ppb) respectively.

4500

4000

~3500as

~300)c:0 2500~82000

8 1500"0

~ 1000W

500

6:::200

~c,8150,gc

g8100"0c

ffi 50

00 80 9010 30 40 50 60

PMT Response (mY)

7020

Figure 13. Ethanol Calibration Curve.

250

02 6 7 8 9 10

PMT Response (mV)

11 124 53

Figure 14. Expanded Scale Ethanol Calibration Curve

CONCLUSIONS

Amperometric biosensors suffer from severalproblems which render their use impractical for deploymentaboard spacecraft. Among these are short active lives, highrates of signal drift, and the consequent need for frequentrecalibration. A novel chemiluminescence based approach tobiosensor development, incorporating solid phase media inreplacement of aqueous phase reagents has shown potential asa means to overcome these difficulties. Improvements insensitivity are also achievable using this technique.

Feasi.1>ility of the Ethanol ChemiluminescentBiosensor concept has been rigorously demonstrated.Calibration curves have been generated using fully integratedreagentless test systems. Ethanol concentrations as low as 40J.1g/Lhave been detected. The height of the signal above thebaseline for this concentration suggests that even lower levelscould be detected using the current relatively crudeembodiment of the innovative technology. Further

7

development is expected to lower the ethanol detection limitby an order of magnitude. Detection limits are primarilydetermined by enzyme conversion efficiencies, dark currentlimitations, light leakage, detection circuitry, and backgroundchemiluminescence. Improvements in enzyme immobil-ization procedures and hardware design can certainly providemeans to significantly overcome these limitations.

Unit operations required for sensor operation havebeen developed sufficiently for demonstration of the conceptThese include pH adjustment using solid phase flow-throughmodules, immobilized enzyme catalyzed oxidation of ethanolto hydrogen peroxide, controlled release of luminol using asolid phase flow-through module, electrocatalyzedluminescence using a potentiostat and gold electrodes, andquantification of light intensity using a photomultiplier tube.The solid phase luminol beds were shown capable ofsustained controlled release of the luminophore at a sufficientconcentration for the biosensor systems to function over abroad span of target analyte concentrations. The ECL cellfabricated from transparent polycarbonate, and using a goldfoil working electrode, and a gold mesh counter electrode,was found to effectively initiate the reaction between luminoland hydrogen peroxide at an applied potential of 0.6 volts.The ECL cell design was also found to adequately confine thereaction spatially for efficient collection of the light eitherdirectly by an optically coupled photomultiplier tube.

Alcohol oxidase effectively catalyzes the oxidation ofseveral low molecular weight primary alcohols. Work to datehas examined the response of the chemiluminescence basedbiosensor to ethanol, although the sensor can be expected torespond to other alcohols. When applied to the examinationof reclaimed waters and wastewater streams associated withregenerative life support systems, the sensor response will beproportional to the sum of methanol and ethanol present Ifquantitation of individual alcohols is required, this techniqueis applicable as an alcohol selective detector which can beused in conjunction with liquid chromatographic separations.

NASA's current requirements for reclaimed potablewater specifies a maximum of SOOJ.1g/LTOC, of which amaximum of lOOJ,Lg/Lmay be uncharacterized, i.e., ofunknown composition. Current technologies such asaqueous phase catalytic oxidation systems (APCOS) arecertainly capable of meeting the 500J,Lg/Llimit, but may havedifficulty with the lOOJ,Lg/Llimit depending upon themagnitude and composition of the influent organics. Theproblem of uncharacterized TOC can be diminished by usingchemiluminescent alcohol biosensors to determine theconcentrations of low molecular weight straight chainalcohols in the product water.

ACKNOWLEDGMENT

This work was supported by the NationalAeronautics and Space Administration's Lyndon B. JohnsonSpace Center, Houston, Texas, under contract NAS9-19021.

~

REFERENCES

1. Akse, J.R, Atwater, J.E., Thompson, 1.0., and Wheeler,RR, Jr., A Breadboard Electrochemical Water RecoverySystem for Producing Potable Water from CompositeWastewater Generated in Enclosed Habitats, in WaterPurification by Photocatalytic, Photochemical, andElectrochemical Processes, Rose, T.L., Conway,B.E.,Mwphy, O.J., and Rudd, E.J, Eds., ElectrochemicalSociety, Hooksett, NIl, 1994.

2. Akse, 1.R, Atwater, JE., Schussel, L.J., and Verostko,CE., Development and Fabrication of a BreadboardElectrochemical Water Recovery System,SAE TransJ.Aerospace, 102, (1), 513-529, 1993.

3. Akse, J.R, and Atwater, J.E., Advanced CatalyticMethods for Destruction of Environmental CoDt~min:mts,AIAA-95-LS-70, presented at AIAA/NASALife Sciencesand Space Medicine Conference, Houston, April 3-5,1995.

4. Akse, J.R, Carter, D.L., Jolly., C, Thompson, J., andScott, B., Catalytic Methods Using Molecular Oxygen forTreatment of PMMS & ECLSS Waste Streams, presentedat the 22nd International Conference on EnvironmentalSystems, Seattle, Washington, July 1992.

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