Electrochemical Methods for the Determination of Glucose

Transcription

1 Electrochemicl Methods for the Determintion of Glucose Adrin W. Bott, Ph.D. Bionlyticl Systems, Inc. West Lfyette, I E-mil: w@ionlyticl.com Some of the more commonly used methods for the electrochemicl determintion of glucose re discussed, with prticulr emphsis on pulsed mperometric detection nd enzyme-sed sensors. Due to the pivotl role of glucose in physiologicl processes, much effort hs een devoted to the development of methods for detecting glucose in food nd iologicl mtrices. However, the development of direct methods hs een hindered y the lck of suitle chromophore or electrophore, nd hence more complicted methods hve een dopted for glucose determintion. Two of these methods, pulsed mperometric determintion nd enzyme-sed iosensors, re discussed in this rticle. Pulsed Amperometric Detection Electrochemicl detection using fixed potentil works est with molecules for which the products of the electron trnsfer rection re stilized y delocliztion through conjugted π system. Such stiliztion is not possile for glucose, nd hence the free rdicls formed y oxidtion or reduction re high energy; tht is, these processes hve high ctivtion energies. Therefore, lrge overpotentil is required, which is disdvntge in electrochemicl detection. Ctlytic oxidtion t moderte potentils y pltinum nd gold electrodes is possile through mechnism tht involves dsorption of glucose to clen electrode surfce (F1). Unfortuntely, the products of this ctlytic oxidtion cn remin dsored to the electrode surfce, which locks the ctlytic sites nd prevents further oxidtion. Consequently, the ehvior of glucose t pltinum nd gold electrodes t fixed potentil is chrcterized y decrese in the ctivity of the electrodes (F2). The deleterious effects of product dsorption cn e reversed nd the ctlytic ctivity of the electrode surfce cn e restored y two-step clening process following detection step (F3) (1). The first step is t potentil more positive thn the potentil used for detection, nd the second step lies t more negtive potentil. The effect of these two potentil steps cn e understood y exmining the rotting disk voltmmogrm of gold electrode in.1 M sodium hydroxide in the sence () nd in the presence of glucose () (F4). When no glucose in present, there re two mjor fetures in the rotting disk voltmmogrm. The nodic current on the forwrd scn t A is due to the formtion of surfce oxide lyer, nd the cthodic current on the reverse scn t C is due to dissolution of this lyer. The ddition of glucose leds to considerle increse in the nodic current on the forwrd scn t E nd F due to the oxidtion of the ldehyde nd lcohol groups t the re electrode surfce. The current for these ctlytic oxidtions is ttenuted t more positive potentils since the formtion of the oxide lyer locks the ctive sites on the electrode surfce. However, the current t G is still lrger in the presence of glucose due to oxidtive desorption of the dsored oxidtion products. The dissolution of the oxide lyer 25 Current Seprtions 17:1 (1998)

2 F1 Schemtic representtion of ctlytic oxidtion of glucose t gold or pltinum electrode (reprinted with permission from reference 1). F2 Comprison of A) pulsed mperometric detection nd B) constnt potentil detection of solution contining 3 ppm lysine, 1 ppm glucose, nd 4 ppm sucrose (reprinted with permission from reference 1). F3 Potentil wve form for pulsed mperometric detection. 2 nc 2 nc Potentil / V (A) (B) I I on the reverse scn llows the ctlytic oxidtion to recommence, nd this is shown y the oxidtive current t H. The optimum detection potentil for glucose is in the potentil region in which there is no oxide formtion nd no oxygen reduction (oxygen reduction occurs t potentils more negtive thn -.1 V). The dsored oxidtion products tht result from this ctlytic oxidtion cn e oxidtively desored y pplying more positive potentil. R c H 2e- [A] - 2 e RO + 2H + P fouling c c c c I I I I c I I c I c I c c Time (min) E det Time (s) E oxd E red However, the ppliction of this potentil lso leds to the formtion of n oxide lyer, which leds to pssivtion of the surfce. The ctlytic ctivity of the electrode surfce is restored y the ppliction of more negtive potentil, cusing the dissolution of the oxide lyer. Detection of glucose (nd other sugrs) using this triple-pulse potentil wve form is referred to s Pulse Amperometric Detection (PAD) or Pulsed Electrochemicl Detection (PED) (1) (the ltter term is lso used for other pulsed detection techniques). The effectiveness of PAD is illustrted in F2, which compres the reproduciility of the chromtogrms using PAD (F2A) nd constnt potentil detection (F2B). It cn e redily seen tht the pek currents for the constnt potentil detection decrese with time, wheres those for PAD remin constnt. Ctlytic oxidtion of glucose hs lso een reported using electrodes modified with copper slts or fricted from copper metl (2). The optimum potentil for the detection of glucose is independent of the method used to mke the copper electrode, which suggests common mechnism (3) (it hs een proposed tht this mechnism involves the copper(iii) oxidtion stte (4)). One mjor dvntge of the copper electrodes is tht oxidtion products re not dsored. Therefore, there is no need for pulsed potentil wve forms, nd constnt potentil cn e used for detection (F5). The ctlytic oxidtions discussed ove re specific only for the pproprite functionl group tht is, the lcohol or ldehyde groups. Therefore, there is no discrimintion etween glucose nd other sugrs or lcohols inherent in PAD, nd chromtogrphic seprtion is required. The other commonly used method for the determintion of glucose is sed on the rection of glucose with vrious enzymes. This pproch hs the dvntge of the specificity inherent in enzymes (5). Enzyme-Bsed Sensors The enzyme most widely used for glucose detection is glucose oxidse. The ctive site of this enzyme is flvin denine nucleotide (FAD), which exists in one of two forms oxidized (FAD) or reduced (FADH 2 ). FAD oxidizes glucose to gluconic cid, nd the FADH 2 generted y this rection cn e oxidized to FAD y oxygen (hydrogen Current Seprtions 17:1 (1998) 26

3 F4 Hydrodynmic cyclic voltmmogrms t gold electrode in deoxygented.1 M OH ) in sence of glucose (dshed line), nd ) in presence of.2 mm glucose (solid line) (reprinted with permission from reference 1). F5 Chromtogrm of grpe juice recorded using BAS LC-4C Amperometric Detector with BAS copper electrode (MF-11). F6 Rection scheme for the ctlytic oxidtion of glucose y glucose oxidse. F7 Schemtic digrm of n electrochemicl enzyme sensor: A) electrode, B) enzyme lyer, C) memrne, D) solution. peroxide is y-product of this rection). This mechnism is shown in F6. namps +.8V B µa G.4 A C F A schemtic digrm for n enzyme-sed sensor is shown in F7. The enzyme is immoilized on i c H i glucose -.4 E fructose sucrose -.8V Minutes Glucose FAD Gluconolctone FADH 2 O 2 Sustrte A B C D Rectnt Enzyme H O 2 2 Products the surfce of n electrode, nd this immoilized lyer is covered y memrne. The function of the memrne is to provide stility, nd it cn lso e used to prevent potentil interfernts from recting with the enzyme. The electrode ssemly is plced in the solution contining the nlyte, which cn redily diffuse through the memrne, nd into the immoilized enzyme lyer. In order to e used s glucose sensor, glucose oxidse ctivity must e converted into n nlyticl signl. The erliest glucose sensors were sed on mesurements of either the decrese in the oxygen (rectnt) concentrtion or the detection of hydrogen peroxide (product) (the concentrtions of oth oxygen nd hydrogen peroxide cn e determined electrochemiclly). However, the ccurcy of oxygen concentrtion mesurements is limited y the nturl vritions in oxygen concentrtion. The detection of hydrogen peroxide y its oxidtion t pltinum electrode requires potentil of V (vs. silver/silver chloride), nd hence is suject to interference y scoric nd uric cids. An lterntive pproch is to use oxidnts other thn oxygen to regenerte the oxidized form of FAD. The idel method would e direct electron trnsfer etween the ctive site nd the electrode surfce, ut this is not possile for glucose oxidse since the ctive site is emedded within the protein. Hence, electron trnsfer must e chieved through the use of meditors. Commonly used meditors include ferrocene, potssium ferricynide, nd ruthenium nd osmium complexes. The cyclic voltmmogrms of sensor sed on ferrocene is shown in F8 (6). In the sence of glucose (A), the reversile cyclic voltmmogrm of ferrocene is seen. The ddition of glucose is ccompnied y n increse in oxidtion current (B), which is chrcteristic of ctlytic oxidtion of glucose oxidise y ferrocene. However, there re 27 Current Seprtions 17:1 (1998)

4 F8 Cyclic voltmmogrms of glucose oxidse/ ferrocene glucose iosensor A) in sence of glucose, nd B) in presence of 1 mm glucose (dpted from reference 6). F9 Structure of polyvinylimidzole polymer ound to n osmium complex. (A) 1 µa (B) 1 µa.5.5 ( ) m Cl some prolems ssocited with this method. Since meditors re smll molecules, they cn diffuse out of film immoilized on the electrode surfce, resulting in loss of ctlytic ctivity. In ddition, the oxidized meditor nd oxygen cn compete for oxidtion of the ctive site. One pproch tht hs een well studied t BAS is tht of wired enzyme electrodes (7-12). PVIn- m = 1 nd n = 3-1 ( ) n.. The meditors for these systems re osmium ipyridine complexes, which re ctionic nd ind electrostticlly to the nionic glucose oxidse. This llows fcile exchnge of electrons etween the osmium centers of the complexes nd the ctive site of the enzyme. One of the coordintion sites of the osmium complex is occupied y the -tom of n imidzole or pyridine moiety of polyvinylimidzole or polyvinylpyridine polymeric unit (F9). These polymeric units rect with diepoxide to form crosslinked, three-dimensionl redox polymer which remins immoilized on the surfce of the electrode (F1). Electron trnsfer etween the ctive site nd the electrode surfce is chieved y electron-hopping etween the osmium centers ttched to polyvinyl units; hence, the ctive sites re considered to e wired to the electrode surfce. Some typicl results for the chrcteriztion of wired glucose oxidse re shown in F11-13 (9). The redox polymer for these studies ws sed on polyvinylimidzole polymer ound to n osmium center coordinted to methyl-sustituted ipyridine lignds. The hydrodynmic voltmmogrms of this electrode in the sence nd in the presence of glucose re shown in F11. When there is no glucose present, the hydrodynmic voltmmogrm is chrcteristic of surfce-ound redox species (the osmium complex); however, the ddition of glucose cuses n increse in the oxidtion current tht is chrcteristic of ctlytic process. The effect of oxygen on the vrition of the limiting current with glucose concentrtion is shown in F12. At low glucose concentrtion, the competition with oxygen cuses significnt decrese in the ctlytic current, ut there is essentilly no effect t higher glucose concentrtions. The effects of interferents (5 µm scorte,.4 mm urte nd 1 mm cetminophen) is shown in F13. Lck of interference from these molecules cn e ttriuted to the low potentil required for oxidizing the osmium meditors (i.e., it is not positive enough to cuse oxidtion of ny interfernts). Detection of Hydrogen Peroxide As mentioned ove, one method for the determintion of glucose concentrtion is the mes- Current Seprtions 17:1 (1998) 28

5 F1 Schemtic representtion of cross-linked polymer network in wired enzyme electrodes. ote the mechnism for the trnsfer of electrons etween the electrode surfce nd the ctive site of the enzyme. F11 Hydrodynmic cyclic voltmmogrms of wired glucose oxidse electrode sed on polyvinylimidzole ) in sence of glucose, nd ) in presence of 48 mm glucose (dpted from reference 9). F12 Stedy-stte response of wired glucose oxidse electrode under nitrogen ( ) nd under ir ( ) t different glucose concentrtions (dpted from reference 9). urement of the concentrtion of hydrogen peroxide generted y the Current Density / µa cm-2 Current Density / µa cm HO O H 2 GOX O reoxidtion of the ctive site of the enzyme. Oxidtion of hydrogen OH H OH OH Potentil / mv vs SCE Glucose Concentrtion / mm peroxide t pltinum electrode is prone to interference from scoric cid nd uric cid, nd hence other methods for hydrogen peroxide determintion hve een investigted (1,11,13-18). One pproch hs een the use of n enzyme electrode sed on the reduction of hydrogen peroxide y horserdish peroxidse. This enzyme is different from glucose oxidse in tht direct electron trnsfer etween the ctive site nd the electrode surfce is possile ( meditorless electron trnsfer) (13-15). However, there re dvntges to using meditorsed enzyme electrodes, some of which hve een illustrted y studying system sed on wired peroxidse electrode consisting of cross-linked polyvinylpyridine (1). The hydrodynmic voltmmogrms for the wired peroxidse electrode with no coordinted osmium complexes in the sence () nd in the presence () of hydrogen peroxide re shown in F14. The ddition of hydrogen peroxide does cuse n increse in the limiting current, which is consistent with direct electron trnsfer etween the electrode surfce nd the ctive site. However, the ddition of hydrogen peroxide to solution in contct with wired peroxidse electrode contining coordinted osmium complexes cuses much lrger increse in the oxidtion current (F15) (out two orders of mgnitude lrger). In the first cse, electron trnsfer cn only occur etween the electrode surfce nd the ctive sites of enzymes tht re in direct contct with the electrode surfce, wheres in the second cse, ctive sites throughout the whole immoilized lyer re wired to the electrode surfce. Mny more ctive sites re therefore ville for the ctlytic process, nd the current is correspondingly greter. The wired peroxidse electrode hs een used for detection of glucose following seprtion y liquid chromtogrphy, nd its performnce hs een compred with 29 Current Seprtions 17:1 (1998)

6 F13 Stedy-stte response of wired glucose oxidse electrode in sence of ny dded interfernts (+), nd in presence of 5 µm scorte,.4 mm urte nd 1 mm cetminophen (O) t different glucose concentrtions (dpted from reference 9). F14 Hydrodynmic cyclic voltmmogrms of wired peroxidse electrode sed on polyvinylpyridine with no coordinted osmium complexes ) in sence of hydrogen peroxide, nd ) in presence of.1 mm hydrogen peroxide (dpted from reference 1). F15 Hydrodynmic cyclic voltmmogrms of wired peroxidse electrode sed on polyvinylpyridine contining coordinted osmium complexes ) in sence of hydrogen peroxide, nd ) in presence of.1 mm hydrogen peroxide (dpted from reference 1). Current Density / µa cm-2 Current / µa oxidtion Current / µa reduction oxidtion reduction Glucose Concentrtion / mm Potentil vs SCE / mv Potentil vs SCE / mv tht of pltinum electrode (11). The experimentl setup is shown schemticlly in F16. ote tht the elunt from the column first psses through n IMmoilized Enzyme Rector (IMER) contining glucose oxidse, which genertes the hydrogen peroxide to e detected t the electrode. A solution contining scoric cid nd uric cid in ddition to glucose ws pssed through the chromtogrphy column. The chromtogrms recorded using the pltinum electrode nd the peroxidse electrode re shown in F17A nd F17B, respectively. The sensitivity nd detection limit for glucose is etter for the peroxidse electrode. This electrode is lso more stle nd less susceptile to interfernts due to the lower potentil required for its opertion. In ddition, the equilirtion time required for the peroxidse electrode is less thn tht required for the pltinum electrode. Conclusion Although the glucose sensors discussed ove give good results in the reserch lortory, there is no gurntee tht the technology involved cn e dpted for commercil use. The fctors tht must e tken into considertion when developing commericlly vile sensor hve een previously discussed in this journl (19). References 1. W.R. LCourse, Pulsed Electrochemicl Detection in High-Performnce Liquid Chromtogrphy, Wiley, J. Ye nd R.P. Bldwin, J. Chromtogr. A. 687 (1994) 141 nd references therein. 3. P. Luo, S.V. Prhu nd R.P. Bldwin, Anl. Chem. 62 (199) J.M. Zdeii, J. Mrioli nd T. Kuwn, Anl. Chem. 63 (1991) J.M. Kuffmn nd G.G. Guilult in Bionlyticl Applictions of Enzymes Vol. 36 (C.H. Suelter, ed.), p M.A. Lnge nd J.Q. Chmers, Anl. Chim. Act 175 (1985) B.A. Gregg nd A. Heller, Anl. Chem. 62 (199) 258. Current Seprtions 17:1 (1998) 3

7 F16 Experimentl setup for the detection of glucose using n IMER followed y detection of hydrogen peroxide fter chromtogrphic seprtion. IMER Column Electrode Injector Liquid Chromtogrphy CC-5 Dt Acquisition Interfce DA-5 Interfce Amperometric Detector LC-4C Detector Pump PM-8 Pump Wste Moile Phse F17 Chromtogrms of 1 nmol glucose (G),.23 nmol scoric cid (AA), nd.24 nmol of uric cid (UA) t A) pltinum electrode, nd B) wired peroxidse electrode. Peroxidse Electrode (B) G 8. T.J. Ohr, R. Rjgopln nd A. Heller, Anl. Chem. 65 (1993) T.J. Ohr, R. Rjgopln nd A. Heller, Anl. Chem. 66 (1994) M. Vreeke, R. Midn nd A. Heller, Anl. Chem. 64 (1992) 384. (A) Pt Electrode G AA 5 na 2 na 11. L. Yng, E. Jnle, T. Hung, J. Gitzen, P.T. Kissinger, M. Vreeke nd A. Heller, Anl. Chem. 34 (1995) I. Willner, E. Ktz nd B. Willner, Electronl. 9 (1997) J. Wng, A. Ciszewski nd. ser, Electronl. 4 (1992) V. Kcniklic, K. Johnsson, G. Mrko-Vrg, L. Gorton, G. Jonsson-Pettersson nd E. Csorefi, Electronl. 6 (1994) E. Csoregi, L. Gorton, G. Mrko- Vrg, A.J. Tudos nd W.T. Kok, Anl. Chem. 66 (1994) 364. µa AA t/min t/min 16. H. Skslund, J. Wng, F. Lu nd O. Hmmerich, J. Electronl. Chem. 397 (1995) J. Wng, J. Liu, L. Chen nd F. Lu, Anl. Chem. 66 (1994) J. Wng, L. Chen nd J. Liu, Electronlysis 9 (1997) P.T. Kissinger, Curr. Seps.16:3 (1997) Current Seprtions 17:1 (1998)