Amperometric tetrathiafulvalene-mediated lactate electrode using lactate oxidase absorbed on carbon foil

Analytrca Chrmtca Acta, 234 (1990) 459-463 Elsevler Science Publishers B V , Amsterdam

459 - Prmted

m The Netherlands

Short Communication

Amperometric tetrathiafulvalene-mediated lactate electrode using lactate oxidase absorbed on carbon foil G PALLESCHI

*.a and A P F TURNER

Cran/reld Inslrtuie of Technology, Bmtechnology Centre, Cranjeld, Bedford MK 43 OAL, (Great Brttarn) (Received

1st August

1989)

ABSTRACT The features of a new sensor for determmmg L-lactate are reported The enzyme lactate oxldase and the mediator, tetrathlafulvalene (‘ITF), are absorbed on carbon foil disks previously bonded onto the ends of glass tubes Lmear cahbratlon graphs were obtamed m the range 10-4-10-3 M with p h yslologcal phosphate buffer (pH 7 35) and at 30 ’ C with a response time of a few seconds. Cahbratlon graphs m the range 10 -3-10m2 M were also obtained and the difference m response times between these two ranges were mvestlgated The results are pronusmg for assembling disposable lactate sensors for m vitro or for m or ex VIVOmeasurements Keywords Lactate,

Enzyme

electrode,

Tetrathafulvalene

mediator

Amperometric enzyme electrodes are becoming mcreasmgly popular, with emphasis on biosensors that can solve analytical and clinical problems such as the measurement of metabolites in biochemical analysts [l-3]. More recently, the need for simple, cheap and precise analyses in clinical chermstry has led to a search for new methods of sensing glucose in whole blood. One successful result has been the development of the “glucose pen”, using a carbon electrode and the enzyme glucose oxidase (GOD) electrochemically coupled with the mediator ferrocene [4]. Lactate m blood and m food analysis is another important metabohte for which a reliable sensor is required. Lactate sensors based on oxygen, hydrogen peroxtde and chemically modified electrodes have been developed [5-81 and successfully applied m clmical analysis. These methods are pre-

cise and accurate, but they do not solve the problem of oxygen fluctuation and deficiency, especially m whole blood or in fermentation analysis. Moreover, some of them involve lengthy procedures. A ferrocene-based lactate electrode has been developed [9] showing that lactate oxtdase could be coupled to ferrocene derivatives. Tetrathiafulvalene (TTF) has been shown to be a very good mediator facilitating electron transfer from glucose oxidase to graphite electrodes [lo]. In this paper the coupling of TTF with the enzyme lactate oxidase is described. This resulted in the development of a lactate probe based on a carbon foil electrode. Several calibration graphs were obtained and studies of electrode stability, reproducibility and sensitivity were done. The reactions involved are the following: lactate

* Present address Dlpartlmento Chlmlche, UmversltSi dl Roma Ralmondo, 00173 Rome, Italy 0003-2670/90/$03

dl Sclenze e Tecnoloue “Tor Vergata”, Via Orazlo

50 0 1990 - Elsevler

Science Publishers

BV

2TTF lactate

+ 2TTF+

+ pyruvate

+ 2TTF + 2H+

-+ 2TTF+ + 2e (200 mV vs. Ag/AgCl) + 0, + pyruvate

+ H,O,

(I) (2) (3)

460

Reactions 1 and 3 are catalysed by lactate oxidase. As is shown in reaction 3, oxygen competes with TTF for the substrate. However, this phenomenon, as explained later, occurs in a range of lactate concentrations not investigated m this work. A carbon foil sensor [ll] is held at an appropriate potential to monitor reaction 2 and the current produced by the reoxidation of TTF is correlated with the lactate concentration. Expenmental Materrals. Graphite foil sheets, 1 mm thick were obtained from Le Carbone, Portslade, Sussex. The epoxy resin to glue the discs was Araldite from Ciba-Geigy, Duxford, Cambs., and the silver epoxy adhesive used for the inner electrode electrical contact was from RS Components Corby, Northants. TTF was supplied by Aldrich Chemical, Gillingham, Dorset, and used as received. Lactate oxidase and lactic acid (lithium salt) were obtained from Sigma Chemical, Poole, Dorset. The buffer and all other chermcals were of analytical-reagent grade. Electrode construction. Graphite foil discs, 4 and 6 mm m diameter, were glued to glass tubes using the epoxy resin. This resin was hardened at 100” C for 20 mm. Insulated wires were fixed to the inner side of the graphite foil discs with silverloaded epoxy adhesive. The silver resin was allowed to set at 100°C for 20 mm. The wire was then fixed by filling the tube with 9 parts of Epon resin (grade 815) to 1 part of trtethylenetetramine and heating at 100 o C for 30 min. Preparation of lactate sensor. The mediator, TTF, was dissolved m acetone (l%, w/v). Carbon foil electrodes were placed in this solution for 2 h at room temperature, removed and allowed to dry m the air for 60 min. The electrodes were then immersed in a solution of lactate oxldase (25 mg ml-‘) for at least 24 h. After use the electrodes were stored refrigerated in the same enzyme solution. For blank measurements three electrodes were prepared with only TTF and three electrodes with only lactate oxidase. All the electrodes were stored refrigerated at 4°C. The three electrodes with only TTF were stored dried. The sensor was operated using a BBC 32K rmcrocomputer via a pro-

G

PALLESCHI

AND

A P F TURNER

grammable biosensor interface (Artek, Olny, Bucks.). A three-electrode system was also used. In this case a potentiostat (Ministat, H.B. Thompson, Newcastle upon Tyne) was used. The output current was recorded on a Euroscribe recorder (Gallenkamp, London) via a resistance board (J.J. Instruments, Southampton). For the three-electrode configuration a saturated calomel electrode (SCE) and a platinum wire were used as the reference and auxiliary electrodes, respectively. The sensors were immersed m 15 ml of a phosphate physiological buffer contained m a 20-ml glass water-Jacketed cell (Joham Scientific, Ely, Cambs.) thermostated at 30 f 0.5 o C. The applied potential was 200 mV vs. Ag/AgCl and 160 mV vs. SCE m the three-electrode configuration system. Results and dlscusslon Preliminary measurements were carried out m order to see if lactate oxidase was able to couple with TTF giving a significant signal when n-qectmg lactate at and over the physiological range (ca. 1 mM). The pH and temperature were fixed at 7.35 and 30 ’ C, respectively. Calibration graphs were obtained by nqectmg lactate m order to give a final concentration of 1 mM for each iqection. Figure 1 shows a lactate calibration graph for two electrodes prepared as described under Ex20 7 ‘A

.

.

MA

. . .

ii ii

.

lo-

5 0

f

.* .

.

.

.

.

.

;;> . t )(:I: OO

t

:

: 10

:

LACTATE

:

:

l

B

D :c, 20

mmol/l

Fig 1 Lactate cahbratlon graph for two carbon fad electrodes m buffer (pH 7 35) at 30° C A and B, electrodes 1 and 2, respectwely, cahbratlon graph m presence of enzyme and mediator, C, electrode 1, cahbratlon graph m presence of enzyme and no mediator, D, electrode 2, cahbratlon graph m presence of mediator and no enzyme

TETRATHIAFULVALENE-MEDIATED

LACTATE

mmol/l

LACTATE

ELECTRODE

LACTATE

Fig 2 Cahbratlon graphs for lactate at low concentrations oxygen-saturated buffer solution; B, mtrogen-saturated buffer

penmental. The range investigated was from 1 to 20 mM and the signal obtained compared with the background noise (0.1 PA) was very promising. Calibration graphs obtained using electrodes with the enzyme only or with TTF only gave negligible current variations (curves C and D). A calibration graph for lactate was obtained in air-saturated buffer over the range 1 X 10P4-2 X lop3 M, representing the physiological range of lactate in whole blood. Figure 2A shows the nonlinearity of the first part of the graph (from 0 to 0.3 mM lactate), which may be due to the oxygen competing with TTF+ at low concentrations of lactate. At higher concentrations of lactate, oxygen was rapidly depleted and the mediator became the principal electron acceptor. On the latter part of the graph the slope decreases, presumably owing to the enzyme becoming saturated with substrate. In order to evaluate the oxygen interference, a calibration graph with nitrogen-saturated buffer was studied. Figure 2B shows linearity in the range O-2 x lop3 M lactate, supporting the explanation advanced above for the effect of oxygen on the previous calibration graph. Figure 2B also demonstrates that the lactate probe can be used in media wtth low oxygen pressure. The reproducibility of this lactate sensor was also studied. Since the enzyme and the mediator were not physically or chemically immobilized, loss of TTF and enzyme from the carbon electrode was expected, with consequent irreproducibility of the lactate electrodes. The procedures for covalently immobilizing enzymes on carbon foils are well described [ll]. This step was not made, however, because the intention was to develop

mmol/l

m the presence solution

and

absence

of oxygen

LACTATE

(pH

735)

at 30°C

A,

mmol/l

Fig 3 Reproduclbdlty of a carbon fad lactate sensor wth the enzyme lactate oxldase and TTF mediator absorbed A, first cahbratlon graph, B, second cahbratlon graph run after 15 mm, C, third cahbratlon graph run Immediately after the second. Buffer, pH 7 35, temperature, 30 o C

disposable sensors for lactate m which covalent immobilization is not used. Moreover, absorption of the enzyme on the carbon foil avoids the deacttvation winch often occurs during chemical immobilizations. Figure 3 shows successive calibration graphs obtained with the same electrode. After the first calibration graph the electrode was washed with buffer and kept for 15 mm in buffer. The second and third calibration graphs were separated by the washing step and a 5-min period m buffer. The non-reproducibility of the graph was presumably due to the loss of enzyme. The similanty of the second and third calibration graphs suggests that the rate of loss of enzyme decreased with time, presumably because the remammg enzyme was more strongly absorbed deep m the

462

G

6

LACTATE

mmol/l

Fig 4. Reproduclblhty of three different lactate sensors measured at the same time m the same bulk solution Buffer, pH 7.35, temperature, 30 o C n = Electrode 1, 0 = electrode 2, * = electrode 3

porous carbon electrode. Loss of TTF over this period from similar TTF-mediated glucose electrodes did not affect the reproducibility of the calibration graph, TTF bemg present in large excess. Moreover, the electrochemistry of the process was unaffected [12]. For a disposable single-use sensor, it is the reproducibility of different sensors tested with the same concentration of lactate that is important. Although the electrode area for each sensor could

LACTATE

mmol/l

Fig. 5 Behavlours of two different lactate electrodes dunng lactate cahbratlon at low and high lactate concentrations All the points before the first pseudo-steady state were obtamed with a response time of a few seconds The pomts obtained to reach the second steady state were obtamed \nth a response time of 1-4 mm.

PALLESCHI

AND

A P F TURNER

not be perfectly defined, three electrodes which had the same nommal area were treated with the same solutions of TTF and enzyme and tested simultaneously. The calibration graphs obtained (Fig. 4) showed satisfactory reproducibility. Variations in the response time with these electrodes were indicative of the underlymg process of mediation. It was observed that during the mitral lactate mlections over the range 10-4-10-3 M, a steady-state current was reached m less than 5 s. When the lactate concentration was increased to 1.5 mM, however, a two-step response was observed (Fig. 5). Mediated enzyme electrodes reported m the literature generally have a response time of l-2 min [1,2,4,9-121; a behaviour m winch two steps can be discerned has not been previously observed. In this case two different pseudosteady states were observed (Fig. 5), the first at low lactate concentration with a response time of a few seconds and the second at high lactate concentration with a response time of several mmutes. This electrode behaviour could be due to direct interaction between TTF and the enzyme absorbed m the carbon foil. It may be hypothesized that there is some enzyme m intimate contact with the mediator and the initial lactate injected reacts with this form, which is rapidly saturated. Following this, the system continues to react in the normal way accordmg to the classical enzyme and mediator interaction (second saturation). This phenomenon, observed many times during cahbration, requires a more detailed kmetic analysis. In conclusion, it has been illustrated that the combination of carbon foil, enzyme, TTF and electrochenncal detection can be apphed to lactate determination and may find widespread applications with other enzyme substrates, providing a range of useful and mexpensive electrochemical biosensors.

G.P. gratefully thanks the CNR Consiglio Nazionale delle Ricerche Progetto Finahzzato Materiah e Disposittvi per 1’Elettronica dello Stato Sohdo, Sottoprogetto Sensort, for a grant to work in Canfield. The hospitality of Prof. Higgins and his staff at Cranfteld is also gratefully acknowledged.

TETRATHIAFULVALENE-MEDIATED

LACTATE

ELECTRODE

REFERENCES 1 A P F Turner, I Karube and G S. Wrlson (Eds.), BIOsensors, Fundamentals and Apphcatrons, Oxford SCI Publ., Oxford, 1987 2 K Mosbach (Ed ), Methods in Enzymology, Vol 137, Immobrhzed Enzymes and Cells, Part D, Acadenuc Press, New York, 1988 3 M. Mascrm and G Palleschr, Sel. Electrode Rev, 11 (1989) 191-264 4 D G Francis, H.A.O. Hill, W J Aston, I J Hrggns, E V Plotkm, L D.L. Scott and A.P F Turner, Anal Chem , 56 (1984) 667 5 M Mascuu, S Fortunate, D. Moscone, G Palleschr, M Mass.1 Benedettr and P Fabtettl, Chn Chem, 31 (1985) 451

463 6 G. Palleschr, M Mascuu, L Bemardr, G Bombardlen, A M De Luca, Anal Lett 22 (1989) 1209-1220 7 L Gorton and A Hedlund, Anal Chum Acta, 213 (1988) 91 8 C M Battersby and P. Vadgama, Drab Nutr Metab., 1 (1988) 43 9 A.Z Preneta. Ph.D Thests, Cranfreld Instrtute of Technology, 1987 10 M F Cardosl and A.P F Turner, m A.P F Turner, 1 Karube and G S Wilson (Eds ), Brosensors, Fundamental and Apphcatrons, Oxford Scl Publ, Oxford, 1987, pp 257-275 11 A.P F Turner, Methods Enzymol , 137 (1988) 90 12 S.P Hendry, Ph D Theses, Cranfleld Instrtute of Technology, 1989