Biosensor Based on Cellobiose Dehydrogenase for Detection of

Anal. Chem. 2004, 76, 4690-4696

Biosensor Based on Cellobiose Dehydrogenase for Detection of Catecholamines Leonard Stoica, Annika Lindgren-Sjo 1 lander, Tautgirdas Ruzgas, and Lo Gorton*

Department of Analytical Chemistry, Lund University, P.O. Box 124, SE-22100 Lund, Sweden

A cellobiose dehydrogenase (CDH)-modified graphite electrode was designed for amperometric detection of catecholamines in the flow injection mode, by their recycling between the graphite electrode (+300 mV vs Ag|AgCl) and the reduced FAD cofactor of adsorbed CDH, resulting in an amplified response signal. The high efficiency of the enzyme-catecholamine reaction leads to a detection limit below 1 nM and a sensitivity of 15.8 A‚M-1‚cm-2 (∼1150 nA/µM) for noradrenaline, with a coverage of less than 2.5 µg of CDH adsorbed on the electrode surface (0.073 cm2). Working parameters such as pH, cellobiose concentration, carrier buffer, and applied potential were optimized, using hydroquinone as a model analyte. The sensitivity, linear range, and amplification factor can be modulated by the steady-state concentration of cellobiose in the flow buffer. The response of the sensor decreases only 2% when run continuously for 4 h in the flow injection mode. The response peak maximum is obtained within 6 s at a flow rate of 0.5 mL/ min, representing the time of the entire sample segment to pass the electrode. CDH enzymes from Phanerochaete chrysosporium and Sclerotium rolfsii were investigated, providing different characteristics of the sensor, with sensors made with CDH from P. chrysosporium being the better ones. The importance of monitoring catecholamines (CAs) originates from the wide range of neural pathways employing biogenic amines as neurotransmitters or as hormones. For this reason, a large proportion of clinically effective drugs for treating neurological and psychiatric disorders affects one aspect or another of the catecholamine transmitter system. Due to their physiological importance, the determination of biogenic amines is a challenging task for clinical analysis. Their concentrations (subnanomolar range) in the blood or close to the neuron cell (or nerve tissue) need a very sensitive method of analysis with low detection limits. As a nonenzymatic method, amperometric detection of CAs at glassy carbon electrodes1,2 for HPLC is used for classical clinical analysis. On the other hand, microfiber electrodes for capillary electrophoresis (CE) reached astonishing limits of detection * Corresponding author. Phone: +(46)-46-222 75 82. Fax: +(46)-46-222 45 44. E-mail: [email protected]. (1) Kissinger, P. T. Curr. Sep. 1986, 7, 73-75. (2) Cheng, F.-C.; Kuo, J.-S. J. Chromatogr., B 1995, 665, 1-13.

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between attomoles to zeptomoles.3,4 Limited by the volume of the sample (e.g., brain dialysis) or by the concentration of the sample (e.g., from blood), the determination of catecholamines requires high sensitivity. For amperometric detection, the highest sensitivity is achieved in two modes: (i) by an increase in the coulometric efficiency (hydrodynamic perspective); (ii) by an increase in the number of recycles of the same analyte molecule at the electrode surface. The maximum extrapolated sensitivity in amperometric methods represents the situation when all the analyte molecules participate in the reaction at the electrode in a limited time scale (instantaneous electrolysis). The coulometric oxidation efficiency for a classical flow-through setup is very low in the majority of cases (estimated less than 20%5). To illustrate the importance of the coulometric oxidation efficiency, besides the more common expression of the sensitivity related to the molar concentration of injected analyte, molar sensitivity, SM as A‚M-1‚cm-2, a second mode of expressing the sensitivity in relation to moles of the injected analyte, mole sensitivity, Sm as A‚mol-1‚cm-2 is used. A higher Sm will mean an increased oxidation efficiency of the amperometric system. In the particular case for a microfiber electrode in capillary electrophoresis, the sensitivity was increased to (SM/Sm) 10-2/1.25 × 109 6 or to 1.58/8 × 104,7 by improving the coulometric oxidation efficiency to more than 50% in the first case.6 This is achieved by decreasing the distance between the capillary wall and the microfiber electrode. Recycling of CA using an interdigitated electrode array (IDA)8,9 (longitudinal recycling) represents a second way to enhance the sensitivities SM/Sm (2.4/108) of the detection in CE (∼4 redox cycles).9 A recent example of an IDA system led to a signal of ∼8 nA at a steady-state concentration of 100 nM dopamine at a flow rate of 2 µL‚min-1, resulting in an SM of 20.8 In the present paper, modification of the electrode with cellobiose dehydrogenase (CDH) is proposed. The recycling of the analyte between the enzyme and the electrode produces a signal of 18 nA for a injection (loop volume of 50 µL) of 10 nM (3) Hochstetler, S. E.; Puopolo, M.; Gustincich, S.; Raviola, E.; Wightman, R. M. Anal. Chem. 2000, 72, 489-496. (4) Chen, T. K.; Luo, G.; Ewing, A. G. Anal. Chem. 1994, 66, 3031-3035. (5) Stulik, K.; Pacakova, V. Electroanalytical measurements in flowing liquids; Ellis Horwood Limited: Chichester, England, 1987. (6) Woods, L. A.; Roddy, T. P.; Paxon, T. L.; Ewing, A. G. Anal. Chem. 2001, 73, 3687-3690. (7) Marazuela, M.; Agui, L.; Gonzalez-Cortes, A.; Yanez-Sedeno, P.; Pingarron, J. M. Electroanalysis 1999, 11, 1333-1339. (8) Hayashi, K.; Iwasaki, Y.; Kurita, R.; Sunagawa, K.; Niwa, O. Electrochem. Commun. 2003, 5, 1037-1042. (9) Liu, Z.; Niwa, O.; Kurita, R.; Horiuchi, T. Anal. Chem. 2000, 72, 13151321. 10.1021/ac049582j CCC: $27.50

© 2004 American Chemical Society Published on Web 06/29/2004

noradrenaline at a flow rate of 0.5 mL‚min-1. This represents a more efficient recycling of catecholamines (face-to-face recycling) in the narrow region between the electrode and the enzyme film (∼50 redox cycles), resulting in sensitivities SM/Sm of 24/4.8 × 105. In this respect, for a nonefficient oxidation system (from a hydrodynamic perspective), reflected by a smaller Sm, the CDH biosensor is able to provide the most efficient alternative of recycling among enzymatic/nonenzymatic sensors for detection of CA in a flow-through system and resulting in the highest SM of detection compared to any system previously reported. CDH is not a unique enzyme in the sense that it has been used to amplify the response signals for amperometric detection of CA. Other examples of redox enzymes that have been used to amplify the signal by recycling of CA are glucose PQQ-dehydrogenase (GDH),10-15 laccase,11,15-17 and tyrosinase (polyphenol oxidase).18-20 Another approach is the use of bienzyme systems, in which two kinds of redox enzymes, one reducing, for example, GDH15,21,22 or oligosaccharide dehydrogenase23 in combination with an oxidizing enzyme (i.e., tyrosinase, laccase), are coimmobilized on the membrane of an oxygen electrode. The recycling of CA between these two enzymes leads to a net consumption of dissolved oxygen. By monitoring the O2 consumption, the concentration of CA involved in the recycling can be estimated. Also, a trienzyme-based sensor has been suggested for detection of CA, consisting of diaphorase, tyrosinase, and glucose NAD-dehydrogenase.24 CDH is an extracellular hemoflavooxidoreductase catalyzing the oxidation primarily of cellobiose, cellodextrins, and some other low molecular saccharides, whereby the FAD cofactor is reduced.25,26 All CDH enzymes known25,26 have a two-domain structure, one larger domain containing FAD and another second smaller domain containing heme (cytochrome b type), both being in their oxidized form in the resting state of the enzyme. The FADcontaining domain supports all known catalytic and cellulose (10) Eremenko, A.; Makower, A.; Jin, W.; Ruger, P.; Scheller, F. W. Biosens. Bioelectron. 1995, 10, 717-722. (11) Lisdat, F.; Ho, W. O.; Wollenberger, U.; Scheller, F. W.; Richter, T.; Bilitewski, U. Electroanalysis 1998, 10, 803-807. (12) Wollenberger, U.; Neumann, B. Electroanalysis 1997, 9, 366-371. (13) Lisdat, F.; Wollenberger, U.; Paeschke, M.; Scheller, F. W. Anal. Chim. Acta 1998, 368, 233-241. (14) Rose, A.; Scheller, F. W.; Wollenberger, U.; Pfeiffer, D. Fresenius J. Anal. Chem. 2001, 369, 145-152. (15) Lisdat, F.; Wollenberger, U.; Makower, A.; Ho ¨rtnagl, H.; Pfeiffer, D.; Scheller, F. W. Biosens. Bioelectron. 1997, 12, 1199-1211. (16) Ghindilis, A. L.; Gavrilova, V. P.; Yaropolov, A. I. Biosens. Bioelectron. 1992, 7, 127-131. (17) Haghighi, B.; Gorton, L.; Ruzgas, T.; Jo ¨nsson, L. Anal. Chim. Acta 2003, 487, 3-14. (18) O ¨ nnerfjord, P.; Emne´us, J.; Marko-Varga, G.; Gorton, L.; Ortega, F.; Domı´nguez, E. Biosens. Bioelectron. 1995, 10, 607-619. (19) Deshpande, M. V.; Hall, E. A. H. Biosens. Bioelectron. 1990, 5, 431-448. (20) Cummings, E. A.; Mailley, P.; Linquetter-Mailley, S.; Eggins, B. R.; McAdams, E. T.; McFadden, S. Analyst 1998, 123, 1975-1980. (21) Ghindilis, A. L.; Michael, N.; Makower, A. Pharmazie 1995, 50, 599-600. (22) Szeponik, J.; Moller, B.; Pfeiffer, D.; Lisdat, F.; Wollenberger, U.; Makower, A.; Scheller, F. W. Biosens. Bioelectron. 1997, 12, 947-952. (23) Bier, F. F.; Ehrentreich-Foerster, E.; Scheller, F. W.; Makower, A.; Eremenko, A.; Wollenberger, U.; Bauer, C. G.; Pfeiffer, D.; Michael, N. Sens. Actuators, B 1996, 33, 5-12. (24) Lisdat, F.; Wollenberger, U. Anal. Lett. 1998, 31, 1275-1285. (25) Henriksson, G.; Johansson, G.; Pettersson, G. J. Biotechnol. 2000, 78, 93113. (26) Cameron, M. D.; Aust, S. D. Enzyme Microb. Technol. 2001, 28, 129-138.

binding properties.25 In the case of CDH from Phanerochaete chrysosporium, the two domains are connected through a linker polypeptide sequence of 26 amino acids, rich in threonine and serine residues. The heme cofactor of the smaller domain is known from its crystal structure to be surface exposed,27 and is also believed, but still in debate,28 to be the site, where one electron-no proton acceptors can pick up the charge from the reduced enzyme. In this work, CDH from two different fungi were studied, viz. from P. chrysosporium (P.c.) (white rot fungus, MW 89 000) and S. rolfsii (S.r.) CBS 191.62 (soft rot fungus, MW 110 000). Both forms of CDH present very good thermal stability with an optimum catalytic activity at ∼50 °C and between pH 4 and 5.25,29 Within this pH range, the intramolecular electron transfer between the two domains is also very efficient. However, the intramolecular electron-transfer rate between the FAD and heme domains is largely pH dependent,28 is decreased as the pH is increased, and is virtually switched off as the pH is increased above 6. Thus, at pHs above 6 and in the presence of cellobiose, the FAD domain remains in its fully reduced state, whereas below pH 6, the charge can be further transferred in single electron steps to the heme domain. We have investigated previously CDHs from both P.c. and S.r. for their ability for direct electron-transfer communication with electrodes30,31 and found that CDH from the white rot (P.c.) is much more efficient than that from soft rot (S.r.). For applications such as described in this paper, it is not straightforward that CDH from P.c. or from S.r. would be the better choice and therefore both enzymes were investigated. Operating the CDH-modified electrode at pH 6 and above will be the most suitable for detection of catecholamines with high amplification factors, which is shown in this paper, and in contrast to our previous report, where the CDH electrode was used for measuring diphenols at the optimum pH for intramolecular charge transfer of the enzyme (4.5).32 MATERIAL AND METHODS Reagents. The following chemicals were used: catechol (CAT) and hydroquinone (HQ) from Acros; 4-aminophenol (4AP), 6-hydroxydopamine hydrochloride (6OHDA), L-adrenaline (ADR), and L-noradrenaline (NOR) from Fluka; 3-hydroxytyramine hydrochloride (DA) from Janssen Chimica; 3,4-dihydroxybenzylamine hydrobromide (DHBA) from BAS; 3,4-dihydroxyphenylacetic acid (DOPAC) (Aldrich), 3,4-dihydroxyphenylalanine (DOPA), o-methylepinepherine hydrochloride (ME), normetanepherine hydrochloride (NME), and β-D(+)-cellobiose (S) from Sigma. All chemicals were of analytical grade (>97% purity) and used as received. All aqueous solutions were prepared using water (18 mΩ conductance) purified with a Milli-Q system (Millipore, Bedford, MA). Stock solutions of diphenols (10 mM) were prepared in water right before use and further diluted in carrier solution to the necessary concentrations. Different buffers were (27) Hallberg, B. M.; Henriksson, G.; Pettersson, G.; Divne, C. J. Mol. Biol. 2002, 315, 421-434. (28) Cameron, M. D.; Aust, S. D. Biochemistry 2000, 39, 13595-13601. (29) Baminger, U.; Subramaniam, S. S.; Renganathan, V.; Haltrich, D. Appl. Environ. Microbiol. 2001, 67, 1766-1774. (30) Lindgren, A.; Larsson, T.; Ruzgas, T.; Gorton, L. J. Electroanal. Chem. 2000, 494, 105-113. (31) Lindgren, A.; Gorton, L.; Ruzgas, T.; Baminger, U.; Haltrich, D.; Schu ¨ lein, M. J. Electroanal. Chem. 2001, 496, 76-81. (32) Lindgren, A.; Stoica, L.; Ruzgas, T.; Ciucu, A.; Gorton, L. Analyst 1999, 124, 527-532.

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used: type A, 20 mM phosphate-citrate buffer pH 5; type B, 20 mM phosphate-citrate buffer pH 6, containing 200 µM cellobiose; type C, 20 mM sodium acetate buffer pH 5; and type D, same as type C but containing 200 µM cellobiose. All buffers were filtered through a 0.22-µm porous membrane (Millipore) and followed by vacuum degassing. Two different CDH enzymes were used, viz. from P.c.27 and from S.r..29 For most cases, experiments were performed with CDH from P.c. or otherwise with S.r., where specified. CDH from P.c. (solution of 0.5 g‚L-1 in sodium acetate buffer, pH 5, purity 95%) was kindly provided by Dr. Gunnar Henriksson and Dr. Go¨ran Pettersson, Uppsala University, Sweden, and CDH from S.r. (solution of 0.55 g‚L-1 in sodium acetate buffer pH 5 and purity of >90%) kindly provided by Dr. Dietmar Haltrich and Dr. Ursula Baminger, University of Agricultural Sciences, Vienna, Austria. Preparation of the Enzyme Electrodes. Preparation of the enzyme electrode and the flow injection system was previously described in detail.32 Briefly, the enzyme was immobilized by simple chemophysical adsorption on solid spectroscopic graphite (Ringsdorff Werke GmbH, Bonn, Germany, Type RW001, 3.05mm diameter), which was previously polished on wet fine emery paper (Tufbak Durite, P1200). After that, the electrode was carefully rinsed with Milli-Q water. and 5 µL of CDH enzyme solution (2.5 µg) was added to the entire active surface of the electrode. The enzyme-modified electrode was then placed in a glass beaker covered with sealing film and stored overnight in a refrigerator (4 °C). Before utilization, the enzyme electrodes were thoroughly rinsed with Milli-Q water in order to remove any weakly adsorbed enzyme. The enzyme-modified electrode was inserted in an electrochemical flow-through cell and used as the working electrode, with an Ag|AgCl (0.1 M KCl) electrode as the reference electrode and a platinum wire as the auxiliary electrode. The electrodes were connected to a three-electrode potentiostat (Za¨ta Elektronik, Lund, Sweden), and the current was recorded on a strip chart recorder (Kipp and Zonen, Delft, The Netherlands). The electrochemical cell was connected to a single line flow injection system, in which the carrier flow was maintained with a peristaltic pump (Alitea, Stockholm, Sweden) at a flow rate of 0.5 mL‚min-1. An electrically controlled six-port valve (LabPRO, Rheodyne, Cotati, CA) with a 50-µL injection loop was used as injector. Mainly, the carrier solution was a 20 mM phosphate-citrate buffer at pH 6; otherwise the buffer and pH are specified for particular experiments. RESULTS AND DISCUSSION The electron transfer between the enzyme and the electrode can occur along different electron-transfer pathways and is classified according to the pathway the electrons flow to reach the electrode as either direct electron transfer (DET) or mediated electron transfer (MET) as outlined in Figure 1. The charge of the initial form of the reduced enzyme, CDHFADH2-hemeox, formed in the catalytic reaction, can be transferred in different directions depending on pH and on the availability of 2e-/2H+ or 1e- acceptors in the vicinity of the enzyme. At low pHs, i.e., below pH 5, DET is accomplished via an efficient internal electron transfer (IET) pathway (not necessarily through the linker region) and one e- at a time is transferred to the heme domain. The resulting CDH-hemered is able to 4692

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Figure 1. (A) Schematic representation of DET, including IET. (B) Schematic representation of MET, which represents the principle for recycling of CA. (C) Examples of real measurements at the CDHmodified electrode: (i) DET, by injection of 200 µM cellobiose; (ii) DO (direct oxidation), by injection of 1 µM hydroquinone; (iii) SS (steady-state) by addition of 200 µM cellobiose in carrier flow; (iv) MET, by injection of 1 µM, 500 nM, 250 nM, and 100 nM hydroquinone, respectively. Experimental conditions: buffer C, 20 mM phosphate-citrate, pH 6; buffer D, 20 mM phosphate-citrate, pH 6, including 200 µM cellobiose; applied potential, +300 mV vs Ag|AgCl; flow rate, 0.5 mL min-1; volume of injection loop, 50 µL.

directly release the electron to the electrode, thus acting as a 1eacceptor; see Figure 1A. On the other hand, MET does not imply the heme domain in the electron-transfer pathway; see Figure 1B. In MET, the electrons of CDH-FADH2 are transferred to the electrode with the help of a soluble 2e-/2H+ mediator (e.g., quinones), as shown in reaction 1. This represents one step of

CDH-FADH2-hemeox + quinone f CDH-FAD-hemeox + hydroquinone (1) the recycling process of the mediator at the electrode. The produced hydroquinone can then in turn be electrochemically oxidized at the electrode (Eappl > E°′ Q/HQ), and thus, reaction 1 in combination with electrochemical oxidation of the hydroquinone found the basis for a mediated electron transfer between the reduced enzyme and the electrode.

Figure 2. Influence of applied potential on DET (injection of 200 µM cellobiose in buffer type C (b)) and on MET (injection of 1 µM hydroquinone in buffer type D) (O) for CDH (P.c.); (in inset, DET for CDH (S.r.)).

The efficiency of MET versus DET is assured since immobilization of the enzyme on the electrode is based on random orientation, and as a result, not all of the adsorbed enzyme molecules are anticipated to be properly oriented for DET. The electron-transfer rate between the heme domain and graphite is expected to have a finite value,33 and at pH 6, the internal electron transfer between the FAD and the heme domains is slowed, resulting in a much decreased background current due to DET; thus the MET pathway caused by the action of quinone will be increasingly more dominant compared with the situation at a lower pH. In the current study, the following parameters have been investigated to optimize the amplification factor for hydroquinone as well as for a number of catecholamines, viz. applied potential, pH, choice of buffer, and concentration of cellobiose in the flow buffer, and the effect of these parameters will be treated separately below. Effect of the Applied Potential. It is essential here in order to investigate how to tune the Eappl to maximize the rate of the heterogeneous oxidation of hydroquinone at the electrode without negatively affecting the activity and stability of the adsorbed CDH on the graphite surface. This was investigated by monitoring DET (for P.c. and S.r.) in buffer C and MET (for P.c.) in buffer D, at increasing potentials. The results are shown in Figure 2, and the conclusion that can be drawn is that for a higher Eappl the enzyme catalytic activity (measured from MET) is not affected, but somehow an irreversible partial inactivation of DET is observed in case of S.r. This actually is beneficial for noise reduction, since DET is included in the background current. It is obvious that when Eappl increases, MET increases, and as a consequence of that, the rate of the heterogeneous oxidation reaction of hydroquinone increases. For all further experiments reported below, the Eappl was set to +300 mV versus Ag|AgCl, which is high enough to ensure the initial heterogeneous reaction of catechol to form the enzymatically active o-quinone will not become rate limiting but also low enough to reduce the risk for possible interfering electrochemical reactions in a tentative real sample. When increasing the pH, the E°′ of the hydroquinone/quinone redox couple will move to more negative values, and thus, a potential of

Figure 3. Influence of pH value on DET (9) (injection of 200 µM cellobiose in buffer type A) and on MET (1) (injection of 1 µM HQ in buffer type B) using CDH (P.c.)

+300 mV should be sufficiently high for any of the experiments run at other pHs reported on below. Effect of pH on DET and MET. pH represents an important parameter to modulate the relation between DET and MET and, in this way, the sensor performance. As mentioned above, the IET between the reduced FAD domain and the heme domain is largely affected by pH, and for both P.c. and S.r., it is known that its rate is faster at lower pHs;26 when increasing the pH, the IET rate goes down and reaches very low values at pH 6 and above. The dependence of the response signal of DET and MET on pH, within the pH interval between 4.5 and 6.5, is shown in Figure 3. From these results it can be concluded that even though previous studies34,35 have shown that the optimum pH of CDH from P.c. is ∼4.5 (DET decreases 50% when the pH is changed from 4.5 to 6), the catalytic properties of the enzyme to oxidize cellobiose seem to be largely unaffected by the pH change as the MET response increases 75% in the same pH interval. The carrier buffer used in our previous study of CDH was a 20 mM acetate buffer, at pH 5.32 Since in an acetate buffer there is no buffer capacity at pH 6, a 20 mM phosphate-citrate buffer, pH 6, was used instead for further work. The buffer concentration (20-100 mM) does not influence the sensor characteristics (data not shown). In addition, when the phosphatecitrate buffer was used, MET was 3 times higher than when the acetate buffer was used (cf. Figures 2 and 3), and thus, the phosphate-citrate buffer represents a better environment for the enzyme. Effect of the Concentration of Cellobiose at Steady State. To have a high reaction rate with the quinone form of the analyte, CDH must be present in its reduced form, from which it follows that the enzyme must be exposed to a “saturating” concentration of cellobiose, so that the turnover rate will become dependent only on the concentration of the secondary substrate (quinone) participating in the ping-pong reaction mechanism, as expected for CDH.25 However, in a separate electrochemical investigation (33) Larsson, T.; Lindgren, A.; Ruzgas, T.; Lindquist, S. E.; Gorton, L. J. Electroanal. Chem. 2000, 482, 1-10. (34) Samejima, M.; Phillips, R. S.; Eriksson, K. E. L. FEBS Lett. 1992, 306, 165168. (35) Henriksson, G.; Ander, P.; Pettersson, B.; Pettersson, G. Appl. Microbiol. Biotechnol. 1995, 42, 790-796.

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of CDH from P.c. adsorbed on graphite, reported elsewhere,36 it was shown previously37 that cellobiose at concentrations higher than 10 mM causes inhibition of the enzyme. The substrate inhibition effect was also concluded from spectrophotometric investigations of CDH from P.c.38 Equation 2 characterizes the reaction rate for an enzyme obeying this kind of reaction mechanism, and the rate expression differs from a normal ping-pong rate equation by the term [S]/Ki, which takes into account the substrate inhibition effect.

I)

KM

(

Imax

)

KS [S] 1+ 1+ + K [Q] [S] i

(2)

where Imax ) nFAkcat[ET] represents the current corresponding to the maximal enzyme velocity, kcat the catalytic rate constant, [ET] is the total amount of active enzyme immobilized on the electrode surface (A), I is the current at steady-state velocity of the catalytic process, [S] and [Q] are the concentrations of cellobiose and mediator (quinone), respectively, KS and KM are the Michaelis-Menten constants of the substrate (cellobiose) and of the mediator, respectively, and Ki is the substrate inhibition constant (affinity of the substrate for the reduced state of the enzyme). Ki equals the [S], which inhibits half the amount of active enzyme molecules involved in the catalytic reaction in the absence of the mediator. From our previous studies,36 the value of Ki is 900 µM. The inhibition effect seems to be a blockage of the pathway to the active site for the mediator at very high concentrations of cellobiose. A similar inhibition effect was also observed for glucose PQQ-dehydrogenase.12 The substrate inhibition process can be used to design the optimum parameters for the sensor in relation to [Q] in the sample. From eq 2 it follows that a high [S] is necessary for an extended linear response range for Q, but with the acceptance of a decreased sensitivity. In contrast, when measuring very low [Q], the highest sensitivity (or amplification factor) is achieved by choosing a lower [S], but then, the extent of the linear range is compromised. In fact, the optimum concentration of cellobiose, [S]opt, when d(I)/d([S])[Q])[Q]lim ) 0, for a target concentration of mediator, [Q]lim, which becomes also the upper limit of the linear range, can be estimated from eq 3, which is derived from eq 2 to

[S]opt )

x

K iK S [Q]lim KM

(3)

Amplification Factor (AF). AF is defined as the ratio between the response currents obtained in the presence and in the absence of cellobiose in the flow buffer, i.e., with and with no enzymatic recycling of the analyte (quinone). Equation 4 can be used to predict the performance of the system, see Figure 4, which shows (36) Stoica, L.; Ruzgas, T.; Gorton, L., in manuscript. (37) Stoica, L.; Ruzgas, T.; Gorton, L. Swedish National Conference on Analytical Chemistry, June 11-14, Stockholm, Sweden, The Swedish Chemical Society, 2001; pp 173-174. (38) Igarashi, K.; Momohara, I.; Nishino, T.; Samejima, M. Biochem. J. 2002, 365, 521-526.

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Figure 4. Representation of nonlinear dependence of the amplification factor (AF) of the CDH biosensor versus concentration of hydroquinone and concentration of cellobiose, based on eq 3 and determined values of parameters.36

Figure 5. Calibration plot for hydroquinone in the presence (b) and in the absence (9) of 200 µM cellobiose based on CDH (P.c.) biosensor. Inset shows the real signal for hydroquinone in the lownanomolar range with a limit of detection of 1 nM HQ (injection volume 50 µL) equivalent to 50 fmol of HQ). Carrier solution: buffer type B and Eappl +300 mV.

that AF does not obey a linear dependence for different [S] and for each [Q] there exists a unique [S] to reach a maximum AF.

AF )

I([Q])withCDH I([Q])noCDH

)

[

Imax

(

)

[Q] [S] b KS + 1 + [Q] + KM Ki [S]

]

(4)

where I([Q])noCDH is expressed by b[Q] and b is the sensitivity for catecholamine detection in the absence of the amplification cycle and dependent on the type of electrode/detection. For our basic investigations, a concentration of cellobiose of 200 µM was chosen.32 For this cellobiose concentration, a linear range for the most sensitive analyte, noradrenaline, will be extended up to 500 nM, but in the case of catecholamines with lower sensitivities, the linear range can be extended up to 1 µM; see below. Detection of Catecholamines. Figure 5 illustrates the drastic increase in the sensitivity for hydroquinone in the presence and

Table 1. Sensitivities and Limits of Detection at the Biosensor Based on CDH from P.c. and S.r.a sensitivity (A‚M-1‚cm-2)

LOD (nM)

analyte

P.c.

S.r.

P.c.

S.r.

CAT HQ DA DHBA ADR NOR

9.50 ( 0.11 11.14 ( 0.24 9.16 ( 0.09 13.44 ( 0.32 1.14 ( 0.01 15.81 ( 0.52

6.75 ( 0.04 2.07 ( 0.02 8.93 ( 0.20 5.60 ( 0.13 0.93 ( 0.01 8.28 ( 0.22

1 0.75 2.5 1 5 1

1 10 5 1 25 3

a The conditions are the same as those in Figure 5. Each calibration curve was obtained by injection of nine different concentrations and three injections per concentration. LODs were calculated at S/N ) 3.

in the absence of 200 µM cellobiose. In the presence of 200 µM cellobiose, the response signal for 500 nM hydroquinone is equivalent to that of 31 µM hydroquinone in the absence of cellobiose (AF ) 62, determined as the ratio of the slopes of the linear part of the calibration curves). The sensitivities and the detection limits for the different catecholamines and related compounds investigated are presented in Table 1. Theoretically, a higher sensitivity for Q means a more restricted linear range. It can be observed that the electrodes modified with CDH from P.c. yield higher sensitivities than those modified with CDH from S.r. For the P.c.-modified electrodes. the highest sensitivity was obtained for noradrenaline, whereas for the S.r.-modified electrodes, the highest response was obtained for dopamine. S.r. seems to be able to discriminate between the para (hydroquinone) and ortho (catechol) position of the hydroxyl groups, showing a lower sensitivity for hydroquinone. In separate investigations (unpublished results), it was shown that CDH immobilized at the surface of the electrode effectively prevents the polymerization of any cation radicals produced by oxidation of catecholamines at the electrode. In this way, no fouling of the active electrode surface occurs. The relative response (RR) of the CDH (P.c.) biosensor corresponding to injections of 1 µM concentrations of the model compounds or catecholamines was evaluated using the same electrode and taking the response for catechol as 100%. Thus, 4-AP (RR ) 28%), ADR (55%), L-DOPA (35%), and DOPAC (45%) have lower sensitivities (an AF of ∼5) but with an extended linear range. In comparison to DA (137%), for analyte molecules with a structure including a secondary amino group (the case of ADR) or the primary amino group in closer proximity to a carboxyl group (the case of L-DOPA), the efficiency of recycling decreases. ADR has an AF of 5 and this could be explained by the formation of an unprotonated open-chain quinone that can suffer a cyclization reaction to give leukoadrenochrome39 and in this way be excluded from the recycling. However, since the working pH is 6 (decreasing the amount of unprotonated open-chain quinone formed) and GDH biosensors present very efficient recycling for ADR, it can be concluded that ADR has a much higher KM for CDH. Similarly, DOPAC has also a high KM, since the molecule does not present any amino group. The low relative responses for L-DOPA, DOPAC, and ADR compared to NOR (142%), suggest a role of the amino (39) Dryhurst, G.; Kadish, K. M.; Scheller, F.; Renneberg, R. Biological Electrochemistry; Academic Press: New York, 1982.

group to decrease the KM for CDH. Comparing DHBA (150%) and DA (137%), it seems as though the shorter the alkyl chain of the amino group, the higher is the efficiency for recycling. The CDH biosensor discriminates the catecholamines from their methoxylated metabolites as NME (2%) and ME (2%), which have one blocked hydroxyl group and are not involved in the recycling process. These compounds can in principle be involved in a recycling process by increasing the Eappl in order to demethoxylate the molecule and thus form the corresponding quinone40 but then at the expense of a decreased activity and stability of the adsorbed CDH. 6OHDA (4.11%) supports the cyclization reaction and does not undergo the recycling process. Other monophenolic compounds (e.g., phenol) show almost no amplified signal.32 In contrast, hydroquinone (144%) undergoes a very efficient recycling. Operational Stability and Addition of Organic Modifiers. The operational stability of the P.c. CDH-modified electrodes was tested for buffer type B with no organic modifier, addition of 5% methanol, and addition of 5% acetonitrile by injection of 1 µM hydroquinone (14 times) during 4 h, for each buffer composition. The CDH biosensor showed absolute stability (100%) in the plain aqueous buffer, and its sensitivity was considered 100% for comparison with the other carriers. The sensor sensitivities decreased by 12% for methanol and by 28% for acetonitrile; however, the stability of the sensor is maintained in the case of acetonitrile (only a 2% decrease), whereas for methanol the signal decreased by 7% after 14 injections during 4 h. Since the most common mobile phases used for HPLC separation of catecholamines contain 3-8% methanol or acetonitrile,41,42 in order to use the CDH biosensor for detection, one must choose a compromise between stability and sensitivity in organic media, but it will still gain in AF compared to a plain glassy carbon electrode. Comparison of CDH Biosensor with Other (Bio)electrochemical Systems for Catecholamine Detection Based on the Recycling. Amplification of the electrochemical signal for CA can be brought about according to two different principles: (i) using a biocomponent to sustain the reaction complementary to the reaction at the electrode (e.g., GDH, CDH, etc.); or (ii) employing a designed IDA, where two different potentials (oxidative and reductive potentials) are applied to sustain the recycling. Among all previous biosensors developed for detection of CA, GDH-based electrodes have shown the best analytical performances.13,15 However, when referring to AFs of 100013 or of 5000,15 previously reported for GDH-based electrodes, one must take into consideration the major differences in relation to work reported for CDH-based electrodes. The systems are not comparable since the response time for the GDH-based electrode is extended beyond a flow injection-type analysis permitting diffusion limitations to be overcome (i) by the time spent by the sensor in the batch analyte solution13 (AF of 1000) and (ii) and by the utilization of an increased amount of enzyme (1 mg of GDH) immobilized in a polymeric membrane13 at the surface of the electrode. The AF can be brought to increase especially due to diffusion limitations of the analyte through a polymer layer or multilayers (40) Ueda, C.; Tse, D. C.-S.; Kuwana, T. Anal. Chem. 1982, 54, 850-856. (41) Cheng, F. C.; Yang, L. L.; Chang, F. M.; Chia, L. G.; Kuo, J. S. J. Chromatogr. 1992, 582, 19-27. (42) Niwa, O.; Morita, M.; Solomon, B. P.; Kissinger, P. T. Electroanalysis 1996, 8, 427-433.

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of the enzymes and also due to entrapment of a small fraction of the analyte in the recycling region leading to a prolonged recycling process. This produces smaller currents in the absence of recycling (smaller denominator in eq 4). Utilization of a membrane to entrap a large amount of enzyme dramatically decreases the possibility of using the biosensor in a flow system, in which the sample plug rapidly bypasses the biosensor and thus relies on as low diffusion restrictions as possible. When the GDH-based electrode was designed for a flow-through setup14 (with a flow rate of 0.5 mL/min and an injection volume of 125 µL), thus decreasing the response time, the GDH biosensor lost its characteristics in AF (unspecified in the reference) and in sensitivity (only 50 nA/µM DA). A few advantages of using CDH in a biosensor construction can be pointed out in comparison to GDH or other biosensors for CA: simple construction (enzyme immobilized on a graphite electrode by simply physical adsorption); comparable sensitivities (even if the systems are not comparable, i.e., flow/batch) but at a lower amount of enzyme (2.5 µg of CDH instead of 1 mg of GDH); the signal is independent of the amount of oxygen in the carrier buffer (cf. laccase and tyrosinase); CDH activity does not depend on any leaking cofactor (cf. GDH for which PQQ leaks out); real-time response (complies with flow analysis requests, instead of batch analysis for GDH); the enzyme prevents the process of electrode fouling (resulting in good operational stability); CDH can distinguish between monophenolic and diphenolic compounds in contrast to laccase and tyrosinase; biocompatibility with real sample (using cellobiose as substrate instead of glucose, which can be found in the sample of interest). Another positive aspect of the CDH biosensor is that the applied potential of the sensing electrode in this way is decreased to +300 mV, thus reducing the potential window for possible interfering signals from other easily oxidable compounds in the sample. The higher KM for ADR in the case of CDH compared with GDH can offer the possibility for an electrode array, i.e., including GDH and CDH to identify the presence of specific catecholamines, using a chemometric evaluation.43 Under these conditions, critically evaluating the characteristics of the various biosensors, the CDH sensor presented here would turn out to be the most sensitive biosensor used in flow-through setup for detection of CA developed until now. When comparing the stability of the different enzyme-modified electrodes used for catecholamine detection, it can be stated that for GDH the stability is a real problem as the PQQ cofactor is loosely bound and will slowly leach out from the enzyme.10-15 Tyrosinase-modified electrodes are notoriously known for their decrease in stability even though much work has been undertaken trying to stabilize the enzyme on the electrode surface or in a membrane held close to the electrode.18-20 Compared with laccasemodified electrodes, the stability of the CDH-modified electrode is much superior.11,15-17 The second principle of amplification of the signal for CA, using IDA, has been continuously improved, primarily focusing on

enhancement of the coulometric efficiency.8,9,44 For this principle, a sensitivity closer to the one of the CDH-based sensor can be achieved8 with the benefits of the same approach used for the GDH-based electrode: (i) a longer time spent by the sample in the region of the electrodes (flow rate of 2 µL‚min-1) and (ii) by continuous pumping (steady state) of analyte, which overcomes the diffusion limitations. The flow profile in the amplification region seams to be an advantage of the enzyme monolayer electrode (i.e., CDH-based electrode), instead of an IDA. In the case of the CDHmodified electrode, the product of the electrode reaction must diffuse through the enzyme layer, where it is efficiently converted. On the other hand, the product of the enzymatic reaction of the analyte is forced to diffuse against the electrode by the concentration gradient of the injected analyte. This results in the face-toface recycling. In the case of the IDA system, the flow profile of the analyte in the recycling region must be a forced semiparabola, ideally with dimensions as small as possible. This represents the case of longitudinal recycling (a parabola with one dimension equal to the distance between electrodes and the other dimension depending on flow rate), which in our opinion seems to have a lack of efficiency compared to face-to face recycling (one dimension equal to 0 and the other equal to the distance between the electrode and the active site of the enzyme). Additional advantages of the CDH-based electrode compared to the IDA systems represent the use of a lower applied potential (+300 mV, instead of +800 mV) and the prevention of electrode fouling.

(43) Solna´, R.; Sapelnikova, S.; Skla´dal, P.; Winther-Nielsen, M.; Carlsson, C.; Emne´us, J.; Ruzgas, T. Talanta, in press. (44) Niwa, O.; Tabei, H.; Solomon, B. P.; Xie, F.; Kissinger, P. T. J. Chromatogr., B 1995, 670, 21-28.

Received for review March 18, 2004. Accepted May 21, 2004.

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CONCLUSIONS The optimization of a CDH-based biosensor for ultrasensitive detection of catecholamines is described. The improvement of the response for MET takes into account the pH and the composition of the carrier buffer and the applied potential. Moreover, the cellobiose concentration can be used as a parameter to vary the sensitivity and the linear range of the sensor. CDH presents the best recycling efficiency for catecholamines among other enzymes described or IDA used for the same application. A combination of an efficient hydrodynamic system (higher coulometric oxidation efficiency) and modification of the electrode with CDH for analyte recycling can lead to a considerable improvement of the present sensitivities. By implementation of a CDH biosensor into a separation system (microbore-HPLC or capillary electrophoresis), further applications for real analysis are envisaged. ACKNOWLEDGMENT The authors thank the Swedish Agency for Innovation Systems (VINNOVA) and The Swedish Research Council (VR) for financial support. The authors thank Dr. Gunnar Henriksson and Dr. Go¨ran Pettersson (Uppsala University, Uppsala, Sweden) for donating CDH from P.c., and Dr. Ursula Baminger and Dr. Dietmar Haltrich (University of Agricultural Sciences Vienna, Austria) for donating CDH from S.r.

AC049582J