9 LW c&AS1:FICArION OF MHIS -PAGE S8.2m 6Ap~ prIfo ved "" REPC " OMB No. 0704-0188 REPORT SECURITY CLASSIFICATION :4 KINGS UNCLASSIFIED nurit SECURITY CLASSIFICATION AUTHO l DISTRIBUTION /AVAILABILITY OF REPORT "APPROVED FOR PUBLIC RELEASE AND SALE: ). DECLASSIFICATION/DOWNGRADING LE its distribution is unlimited PERFORMING ORGANIZATION REPORT NUMBER(S) _. - S. MONITORING ORGANIZATION REPORTNUMBER(S) TECHNICAL REPORT NO. 0 ," . a. NAME OF PERFORMING ORGANIZATION | 6b. OFFICE SYMBOL 7a. NAME OF MONITORING ORGANIZATION OI f applicable) DEPARTMENT OF SPONSORED PROJECTS JNIVERSITY OF TEXAS AT AUSTIN THE UNIVERSITY OF TEXAS AT AUSTIN c ADDRESS (City, State, and ZIPCode) 7b. ADDRESS (City, State, and ZIP Code) Department of Chemical Engineering P.O. Box 7726 Austin, TX 78712-1062 Austin, TX 78713-7726 Ia. NAME OF FUNDING/ISPONSORING 8b. OFFICE SYMBOL 9. PROCUREMENT INSTRUMENT IDENTIFICATION NUMBER ORGANIZATION (If applicable) . OFFICE OF NAVAL RESEARCH P ADDRESS (City, State, and ZIP Code) 10. SOURCE OF FUNDING NUMBERS 800 N. Quincy Street PROGRAM IPROJECT I TASK IWORK UNIT Arlington, VA 22217 ELEMENT NO. NO. NO, ACCESSION NO. 11. TITLE (Include Security Classification) 0.3 -m Diameter Flexible Amperometric Lactate Probe UNCLASSIFIED 12. PERSONAL AUTHOR(S) Dan Li Wang and Adam Heller 13a. TYPE OF REPORT 13b. TIME COVERED 14. DATE OF REPORT (YearMonth,Day) I IsPAGE COUNT TECHNICAL FROM .JQ92 1/J.~ 1993 January 26 2 16. SUPPLEMENTARY NOTATION j 17. COSATI CODES 18. SUBJECT TERMS (Continue on reverse If necessary and identify by block number) FIELD GROUP SUB-GROUP Amperometric Lactate Probe; electrode; poly(vinylimidazole); ethylene glycol diglycidyl ether; immobilized horseradish peroxidase (HRP)-glucose oxidase (GOX); enzymes; (Cont.) 19. ABSTRACT (Continue on reverse if necessary and identify by block number) A flexible lactate electrode was made of 400±100 7 ptm diameter carbon fibers, epoxy embedded in a 0.3 mm diameter polyimide tubing. The electrode was modified by precipitating on it the relatively insoluble complex formed between 1100 kd partially N-ethylamine quaternized poly[(vinylpyridine)Os(bipyridine) Cl] Cl (POs-EA) and lactate oxidase. The steady-state lactate 2 electrooxidation current, at 2mM lactate concentration and at 220 C, was 400nA. The 50±10 tAcm-2 current density and the 20mAcm-2M-1 sensitivity decreased only by 5% upon increasing the partial pressure of oxygen from 0.0 to 0.2 atm. The electrode retains its sensitivity after dry storage at 40C for 4 months in air but loses at 370C half of its sensitivity in 7 hours through polymer desorption when operated at 0.4V (SCE). To eliminate interference by species that are electrooxidized at 0.4V (SCE), the lactate sensing probe was (a) electrically insulated with an epoxy made of poly(vinylimidazolc) cross- (Continued) 20. DISTRIBUTION /AVAILABILITY OF ABSTRACT 21. ABSTRACT SECURITY CLASSIFICATION FINUNCLASSIFIED/UNLIMITED 0 SAME AS RPT 0 DTIC USERS UNCLASSIFIED 22a. NAME OF RESPONSIBLE INDIVIDUAL 22b. TELEPHONE (Include Area Code) 22c OFFICE SYMBOL Adam Heller ((5 12) 471-8874 . .. DO Form 1473, JUN 86 I',ev,ouj editonj ire obtolete SECURITY CLASSIFiCATION OF THIS PAGE UNCLASSIFIED DISCLAIMER NOTICE THIS DOCUMENT IS BEST QUALITY AVAILABLE. THE COPY FURNISHED TO DTIC CONTAINED A SIGNIFICANT NUMBER OF PAGES WHICH DO NOT REPRODUCE LEGIBLY. S t.% (CONTINUED: BLOCK 18): oligosaccharides; aldehydes. (CONTINUED: BLOCK 19): linked with ethylene glycol diglycidyl ether, and (b) coated with an immobilized horseradish peroxidase (HRP)-glucose oxidase (GOX) film. The latter film was formed by co-immobilizing the two enzymes through periodate oxidation of their oligosaccharides to aldehydes and forming Schiffs-bases between the polyaldehydes and the enzymes' lysyl amines. In the presence of 1 mM glucose and in air, the interfering electrooxidation of 0. 1 mM ascorbate was reduced by a factor of 20. This reduction results from formation of hydrogen peroxide in the glucose catalyzed reaction, and H 0 oxidation of the 2 2 ascorbate in a reaction catalyzed by. HRP. Acoesslon fPo NTIS GRA&I DTIC TAB 0 UnsanoLneed 0 JustLf 1catlon wV. By ITyrw QUALIT? r-SPEUED 3 DtutSPSo(cid:127)_ ,. 6.Availability Oodef &Tal Mp..l. %*S A - * tA.a - o. -. HMISTRY, 1993. , I 0.3 mm Diameter Flexible A..mperometric Lactate Probe Dan Li Wang and Adam Heller* Department of Chemical Engineering The University of Texas at Austin Austin, Texas 78712-1062 Abstract: A flexible lactate electrode w.as made of 400::100 7 l.m diameter carbon fibers. epoxy embedded in a 0.3 mm diameter polvimide tubing. The electrode was modified by precipitatini on it the relatively insoluble complex formed between 110(0 kd partially N-ethvlamine quaternized poly[( vinyipyridine)Os(bip~vridine ,'ClI CI (POs-EA, and lactate oxidase. The steady-state lactate electrooxidation current. at 2rmM lactate concentration and at 220 C. \%as 4Jt(nA. The 50=10 uAcm- 2 current densitx and the 20mAcm--N.l-ý ,ensti\it\ decreased only by 5% upon increasing the partial presure (it oxx,'en from 0.0 to 0.2 aim. The electrode retains its sensiti\ it\ after dr, _,toraze at 40C for 4 months in air but loses at 370C half or its ,ensitivit\ in 7 hours through polymer desorption when operated at ().'V (SCE). To eliminate interference by species that are electrooxidized at 0.4V (SCE). the lactate sensing probe was (a) electrically insulated with an epox\ made of poly(vinvlimidazole) crosslinked with ethylene glycol diglycidyl ether, and (b) coated with an immobilized horseradish peroxidase (HRP)-glucose oxidase (GOX) film. The latter film was formed by co-immobilizing the two enzymes through periodate oxidation of their oligosaccharides to aldehydes and forming 93-02078 01,1 ,- '""0 4 1111111l11\ L Schiffs-bases between the polyaldehydes and the enzymes' lysyl amines. In the presence of I mnM glucose and in air, the interfering electrooxidation of 0.1 mM ascorbate was reduced by a factor of 20. This reduction results from formation of hydrogen peroxide in the glucose catalyzed reaction, and H202 oxidation of the ascorbate in a reaction catalyzed by HRP. Introduction Simple glucose electrodes have been made by adsorbing a polyt( viny'lpyridineOs bipyridine )CII Cl derivative on graphite, rinsing. then complexing glucose oxidase to the adsorbed polymer.1 Upon complexing, the adsorbed redox polymer binds and penetrates the enzyme and establishes electrical contact between the redox centers of the enzyme and a graphite electrode.- Here we apply such a complexing process to formin2 lactate electrodes, on flexible bundles of epoxy-embedded. polyimide tubing-contained. carbon fibers. The electrodes are coated with an interferant eliminating laer. containin_ coimmobilized horseradish peroxidase (HRP) and gluco.,e oxidase (GOX). In an aerated glucose solution H,O', is ,enerated within the film through the GOX catal\zed r, action. The H 202 oxidizes the interterantN. but not lactate. in the HRP catalyzed reaction. The interference elimination process is built on that described for glucose electrodes. ', here lactate oxidcase has been used to generate H202 in a lactate containin,_ aerated solution..' Lactate oxidase from Pcdioco~''Us Np commonly used in amperometric lactate sensors.- has been used lhrou,.hout this work. The 3 enzyme catalyzes reaction I and also the oxidation of the FADH by 02, 2 whereby H202 is formed. LOX-FAD + Lactate -, Pvruvate + LOX-FADH2 (1) Amperometric lactate sensors have been based on this reaction combined with reactions 2 and 3: LOX-FADH2 + 2-Mox LOX-FAD - 2Mre (2) M re + 2 e- (3) "where Mo\ and Mre are the oxidized and tile reduced forms of a diffusional mediator, such :is 0- H-,O, or Fc-/Fc (where Fc is a ferrocene derivativei.5-10 FAD/FADH- centers of LOX were also non-diffusionally electrically connected to electrodes through a 3-dimensional redox epoxy- based electron relaving network.,* Lactate mono-oxygenase troi .Ivcohacteriunl sine-ciaitis. having an FMN co-factor. has also been ipplied in lactate sensors. This enzyme catalxzes the oxidation or lactate bv oxv.Len to acetic acid and carbon dioxide.,I However. the enzxine is inhibited by phosphate,1 2 an ion present in blood and other tissues, whereas lactate oxidase from Pediococcus sp. is not inhibited b\ phosphate. Because the sensor is to be used in phosphate-containin, phl\, Iological solutions, the Pediococcus enzyme was used in this \\ork. 4 In vivo measurement of lactate is of clinical interest in monitoring shock, respiratory insufficiency and heart failure. 13 Miniaturization and flexibility of sensors is relevant to lactate monitoring at a specific site, or in an organ where tissue damage upon electrode insertion must be avoided. Miniature glucose sensors14-18 based on glucose oxidase. an enzyme of higher specific activity than that of LOX, have been reported.1 --18 One limit imposed on miniaturization is defined by (a) the achievable current density, which in turn can be limited by the specific activity of the enz\me in catal\zzinc reaction 1: or (b) b\ the electron transfer rate to a diffusional mediator or polymer bound relay (reaction 2); or (c) by the rate of mediator diffusion in solution or electron diffusion through the redox conducting polymer, that may iimit the rate of reaction The redox polynmer PO>-EA forms relati\el\ insoluble adducts with glucose oxidase and uttter lenz',me,,, that can be crosslinked with diepoxides.lz. 19. 20) The electronic conduction in the resulting redox epoxy network in glucose microelectrodes i], sufficient to allow a current density in excess of 0.5 mAcm-- ,:nd a ,,ensitivit\ in exce,. of 50mAcm-M -1 The glucose microelectrodes ..re relati\ el\ insensiti\ e i-_3 ') to aeration and deaeration. 1. In order to provide strength. flexibility and adequate currents. so that Faraday cac,,es will not be needed, carbon fiber bundles consisting of several hundred carbon libers \\ere used. .\ flexible polyimide tubing 21 provided insulation. 5 Experimental: Chemicals. Lactate oxidase (LOX. 35 units/mg) from Pediococcus sp. (EC number is not available) was purchased from Genencor Inc. and was used as received. Glucose oxidase (GOX, EC 1.1.3.4, type X, 128 units/mg), horseradish peroxidase (HRP. EC 1.11.1.7, type VI. 260 units/mo), L-lactic acid and ethvlene lvcol di+l.cidvl ether (EG) were from Si,,ma and were used a.,, receiled. The redox polymer. POs-EA was synthesized as previousl\ reported. 19. 20) Polkivinylimidazole) (PVI) was synthesized as described." Apparatus. An EG&G Princeton Applied Research 273 potentiostat/galvanostat and an En',man Instrumentation potentiostat with a con\entlonal electrochenmical :,e!l ,xere used respectively for the cyclic voltamnmetrv and for the ,ontant-potential experiments. All potentials are referenced to the cell',, ,aturated calomel electrode (SCE). A home-built flowý ce;1 built \%ith a rotarx injection valve (Beckman) was used for the flow injection analxses. The microelectrode was placed approximately 0.5cm from ihe inlection \alve and the reference electrode about lcm from the workin,.2 electrode. Lactate microelectrode fabrication. The fiber bundle was made as follows: 300 to 500 carbon fiber, w\ere inserted into a 0.3m1m diameter 7cm long polvimide tubing. A t. I mm polviniide insulated platinum wire, auxiliary electrode, was alo in,,erted into the lubine. The tubing was 6 completely filled by capillary action wit, an epoxy "Super-Low Viscosity r Embedding Media" (Polysciences. Inc., Warrington. PA). The fiber-epoxy composite w z cured in the tubing at 800C for 2 days, then glued to a stainless steel wire with an electrically conducting silver epoxy. An approximately 450 beveled electrode tip was formed by polishing sequentially with three grades of diamond paste (15, 6 and I micron), sonicating and washing with alcohol and distilled water. A drop of 1% Triton X-100 was then placed on the cleaned bevel. After air drying (-lh. 22°C). the surface was washed ,aith deionized water. The purpose of the Triton X-100 treatment was to con\ert the hydrophobic epoxy surface to a hydrophilic for improved adorption of tile redox polymer and its adduct with LOX. The surface area of the electrode was determined as follows. DA thick zold laver wa,, sputter-deposited on the be\eled electrode tip. i.e. on the tops of the fibers and on the epoxy betveen the fibers. 3 Next the currents of the gold coated electrode and of an exposed 2mam diameter gold disk electrode were determined in 0.5mM ferrocenemethanol and measured by differential polaro,_,raphic \ olhammetrv iDPV . The electrode surface area %as then calculated from the ratio of the currents. 24 The actual surface area of the epo\.-embedded fiber bundle was 0.009cm2 -- 0.002cm ' nearly an order of magnitude higLher than the geometric area. showing that the surface \\a,, rou_'h. Electron microcopy showed that the fiber tips protruded. as hemipheri,.al domes. from the embedding epoxy. The lactate sensing, electrode \a\, formed b\ adsorbine the adduct of the redo\ po.lmner and lactate o\mdase. Phe adsorption process was the 7 following: One droplet (0.2 to 0.3 pl.) of a 10 p.g/,l solution in 0.02M HEPES buffer pH 7.0 was transferred from the tip of a capillary tube onto the bevelled surface, first of the polymer. then of the enzyme. The mixed solution was allowed to dry under air for > 3 days. Coulometry showed an osmium loadin- of 45 -mC cm-2 based on the electrochemically estimated 0.009 cm 2 surface area. No diepoxide or other crosslinker. 1 9 was used in making these electrodes. i.e. functionalization depended solely on adsorption of the precipitated POs-NH2-LOX complex. Before testing. the electrodes were soaked in (.02M pPH 7.01 sodium phosphate buffer containing 0.1.\1 NaCI for at least three hours to remove leachable components. The interferant eliminating laier was applied on top of a PVI-EG electrically insulating "barrier' Liver, formed o'er the lactate sensing laver '-,, an earlier described me'hod.3- The PVi-EG laver was then coated by app'~lng a 0_2 or droplk- ol a dissolved mi\ture of polvaldehvdic HRP ,:nd GOX.3 This mixture \(cid:127)t, prepared as folloý,,: ling HRP and 1mg GOX .,-ere dissolved in 50.UL of UI.M sodium bicarbonate, to which 5.iL of sodium periodate (1 2mgimL) \\a, added.> The solution was incubated in the dark. at room temperature. for -N' hours. After incubation, the solution was dialyzed against .u2.1. pH 7.01 sodium phosphate buffer containing O.1 M NaCG in order to eliminate the excess periodate. The polyaldehydic HRP and GOD were allo\\ed to self-,.rosslink by Schiff-base formation between the HRP aldehlde. and GOX lvs\ amines for two days. at 220C.