- Email: [email protected]
Flow-injection determination of trace hydrogen peroxide or glucose utilizing an amperometric biosensor based on glucose oxidase bound to a reticulated vitreous carbon electrode
Talanta ELSEVIER
Talanta 43 (1996) 957-962
Flow-injection determination of trace hydrogen peroxide or glucose utilizing an amperometric biosensor based on glucose oxidase bound to a reticulated vitreous carbon electrode* Masoud Khayyami”.“, Gillis Johanssonb, Dario Kriz”, Bin Xie”, Per-Olof Larsson”, Bengt Danielsson” ~‘Pure und Applied hAnalytical
Biochemistry. Chemistry,
Chemical CXemicaI
Center,
Center.
Lund
Lund
Uniwrslty.
Unrrrr.Gty.
P.O.
P.O.
Bos
Box
124. S-221
124, S-221
00 Lund,
00 Lund.
Swrdrn
Swden
Received 2X December 1995: accepted 3 January 1996
Abstract An electron transfer mediator, 8-dimethylamino-2.3-benzophenoxazine (Meldola Blue), dissolved in the carrier solution in a flow-injection system, was found to reduce the oxidation potential for hydrogen peroxide from 600- 1200 mV without mediator to - 100 mV vs. AgiAgCI with the mediator present. The very low background current of reticulated vitreous carbon (RVC) at this potential makes it possible to detect very low levels of hydrogen peroxide or glucose. Glucose oxidase was covalently coupled with carbodiimide to RVC, and the RVC was formed into a column inserted in a flow-injection system. The calibration curve was linear from 30 nM to 10 PM glucose with 5 ,uM mediator. At higher mediator concentrations. the linear range was extended to 1000 PM, but with a much higher background current. The sample throughput was about 60 h- I. The current response decreased to 50% of the original response after 20 days. The coulometric yield was high because the sample was pumped through the pores of the RVC. It was 16% and 55% at a flow rate of 1 ml min ’ at mediator concentrations of 5 and 50 ,uM respectively. Keywords:
vitreous
Nanomolar glucose and hydrogen carbon; Amperometry
peroxide
detection;
Glucose
biosensor;
Enzyme
electrode;
Reticulated
1. Introduction
* Corresponding author. Fax: ( +46)46-46-10-46-l 1. ’ Presented at the Seventh International Conference on Flow Injection Analysis (ICFIA ‘95), held in Seattle, WA, USA, August 13-17. 1995.
A great number of biosensors for glucose determination have been described in the literature over the last two decades [l-3]. The major goal has been to develop sensors for determining glucase in blood. Some work has also been devoted
0039-9140~96~$15.00 8 1996 Elsevier Science B.V. All rights reserved PII SOO39-9140(95)01872-3
958
121. Khu~;vomi
et rd.
Tdunta
to determination of glucose in biotechnological processes. Very little emphasis has been put on developing glucose sensors for the submicromolar range. Hydrogen peroxide is a reaction product in a number of enzymatic reactions used in substrate determinations. It can be determined spectrophotometrically using peroxidase-catalyzed color reactions of the Trinder type [4]. It can also be determined electrochemically by oxidation at about + 1200 mV vs. SCE on glassy carbon [5] or at lower potentials on platinum or by using chemically-modified electrodes, e.g. + 400 mV at palladium sputtered on carbon [6]. With catalysts such as peroxidases on the electrode surface reduction starts at + 600 mV and reaches a constant value at - 200 mV [7]. A number of soluble or immobilized mediators have been used for lowering the necessary electrode potentials for detection of hydrogen peroxide produced by oxidases. Mediators
Counter electrode -I-
13 (1996)
957
962
for glucose determination include N-methylphenazinium salts [8] and Meldola Blue. Both mediator and enzymes were incorporated into carbon paste electrodes [9]. The superior electrochemical properties of reticulated vitreous carbon (RVC) [lo] have prompted a study of this material for use as a sensor at very low concentration levels. Previously glucose oxidase has been coupled covalently to RVC and the production of hydrogen peroxide was determined by oxidation at + 900 mV vs. SCE [l 11. In this paper we report on the use of a soluble electron transfer mediator, Meldola Blue, which can detect hydrogen peroxide or glucose down to - 100 mV vs. Ag/AgCl. Glucose oxidase was used in this study because this enzyme is very stable and has been comprehensively studied previously. It is thus a good model system although the use of other oxidases may provide even higher sensitivity from a practical point of view. The ability to select a potential at which the background currents are extremely low should be of great importance in making sensors for low substrate levels.
(Pt)
2. Experimental Reference
electrode
2.1. Clzemicu1.s
S~hcon rubber RVC (counter
electrode)
RVC (working
electrode)
Flanged tubing
Working
electrode
(Platinum)
Fig. I. hydrogen
Cell configuration peroxide.
for
determination
of glucose
or
Glucose oxidase (GOx) type S-X (Aspergilltls n&r). Meldola Blue, 8-dimethylamino-2,3-benzophenoxazine and 1-cyclohexyl-3-(2-morpholimetho-p-toluenesulfonate noethyl)-carbodiimide were obtained from Sigma. The RVC, porosity grade 100-S (pore size nominally 0.25 mm), was from Energy Research and Generation Inc. (Oakland, CA). The potentiostat (model MA 5410, Chemel AB, Lund, Sweden) was set at a voltage of- 100 mV vs. Ag/AgCl. The carrier buffer was 0.1 M sodium phosphate, pH 7.0, containing 1.9 mg Meldola Blue 1~ ’ (5.0 PM). Meldola Blue is unstable at higher pH, the half-life time at pH 7.0 is 100 h [12], and new buffer solutions were therefore prepared every day. The peristaltic pump was from Alitea AB (model C-4~). Sample injection was through a 100 ,~l loop.
&I. Kh~~~m~i Table 1 Peak currents at various buffer. pH 7.0. containing Applied voltage Peak current
applied voltages 5 /tM Meldola (mV) (nA)
-150 270
ci al.
Talunru
43 (1996)
(vs. Ag AgCI) observed when Blue. Flow rate I ml mini’ -100 230
2.2. Preparation of the column RVC cylinders (1.5 x 0.5 cm’) were cut (using eye and dust inhalation protection devices) from a block of material and left in 6 M HCl for 1 h. They were washed with distilled water until the solution reached pH 5, and then left in dry methanol for 2 h. Finally, the cylinders were dried in an oven at 110°C overnight. A cylinder was fixed in a glass tube as shown in Fig. 1 with a counter electrode of the same material. The reference electrode was mounted downstream, which should be acceptable due to the low currents. 2.3. Immobilization A 0.05 M acetate buffer (pH 5.1) containing carbodiimide (40 mg ml ‘) was pumped through the system at a flow rate of 0.2 ml min ’ for 150 min in order to activate the RVC surface. Then acetate buffer was circulated under an ice bath at a flow rate of 1.2 ml min ’ for 30 min. For immobilization of GOx, an enzyme solution (60 mg per 10 ml) in buffer was circulated in the column under an ice bath at a flow rate of 0.2 ml min’ for 3 h. The final rinsing procedure consisted of pumping a cold phosphate buffer (0.1 M, pH 7.5) for 5 min.
3. Results and discussion The RVC material is porous and can be shaped in the form of a column permitting a liquid to be pumped through, thus giving efficient mass transfer to the surface. The surface area will be very large but the background current will still be low because of the favourable properties of RVC. A large surface area is necessary in order to immobilize a sufficient amount of enzyme but it is also necessary to obtain a high coulometric yield. The
-50 230
injecting
957~-962
959
100 ,uI of I ,uM
0 230
50 250
glucose
100 250
samples
into
a phosphate
150 280
electrode was made in the form of a cylinder and inserted into a flow-injection system. 3.1. Reaction properties Injection of 10 ,uM glucose samples produced oxidation peaks which were almost independent of the applied voltage, see Table 1. The redox potential of adsorbed Meldola Blue is - 175 mV at pH 7.0 [12]. The applied voltage should therefore be sufficient to keep the mediator in the oxidized form. The potential was set to - 100 mV for all subsequent experiments. The mediator concentration has a pronounced effect on both the background current (Table 2) and the response factor and linearity of the calibration curve. Fig. 2. The lowest background was obtained with a mediator concentration of only 5 /i M. The current at 10 ,uM glucose for example was much lower than that at the higher mediator concentrations; compare the current scale in the inset of Fig. 2 with that of the main graph. A low mediator concentration decreased the linear range (not shown in Fig. 2) because the amount present in the sample zone becomes less than that of the hydrogen peroxide or glucose. The coulometric yield at a flow rate of 1 ml min ~ ’ was 16, 45 and 55% at mediator concentrations of 5, 20 and 50 jr M respectively. The current due to glucose oxidation at - 100 mV in the presence of mediator decreased when the flow rate increased. see Fig. 3. For compariTable 2 Observed background currents of the enzyme-covered RVC electrode in a phosphate buffer, pH 7.0. containing various concentrations of mediator. Applied voltage, - 100 mV vs. Ag:‘AgCl: flow rate, 1 ml mm’ Mediator concentration Background current
(nM) (nA)
5 IO
20 200
50 6000
960
.
a
s c .P z j$
:
2000 -
o.c
,-’ 0
A’ 2
4
6
8
PM /
.’
I’
,, IO ,’ /’
A
/’
/’
1000 Non-enzymatic
* . .._..._
oc / 1
I##, 0
.,,I,,,,, 250
1 500
Glucose,
750
1
pM
son, Fig. 3 also gives the currents for hydrogen peroxide oxidation at + 600 mV in the absence of mediator. This is the potential where the oxidation of hydrogen peroxide begins. The curves for hydrogen peroxide and glucose follow each other closely, indicating that similar rate-limiting steps are involved. The fast decrease in response with flow rate is due to a reduced residence time at the RVC electrode giving more time for the reactions.
-. l
0.5
1.0
1.5 2.0 Flow rate (mllmin)
2.5
GIUCOS~ and hydrogen
peroxidellrM
1000
Fig. 2. Influence of mediator concentration on the response to glucose injections (100 111) with various concentrations of Meldola Blue: (A) 50 /IM; (0) 20 PM. The inset shows the low range response with 5 HIM mediator. Phosphate butfer containing mediator. pH 7.0: applied potential, - 100 mV: flow rate. I ml min ‘.
0' 0.0
_I
n -’
3.0
Fig. 3. Response variation with flow rate for injections (100 /II) of IO PM glucose (0). Phosphate butfer, pH 7.0. containing 5 ELM Meldola Biue; applied potential. - 100 mV vs. Ag/AgCl. The Figure also shows the response to hydrogen peroxide at + 600 mV in a buffer without any added mediator (A).
Fig. 4. Peak currents for hydrogen peroxide (n) and glucose (e) injections (100 111) with a carrier of phosphate buffer, pH 7.0. containing 5 bM Meldola Blue. The non-enzymatic oxidation of glucose ( n ) is also shown. Flow rate, I.0 ml min-‘; applied potential. - 100 mV vs. Ag:AgC).
Diffusion within the pores also plays an important role in the mass transfer because less substrate reaches the pore walls at shorter residence times. Kulys et al. [9] suggest that Meldola Blue (MB) shuttles electrons from the redox center of glucose oxidase (GOx) to the graphite surface according to the following scheme (corrected by us): glucose + GOx( FAD) --f GOx(FADH,)
+ gluconolactone
(1)
GOx( FADH,)
+ MB +
--t GOx(FAD)
+ MBH + H +
(2)
MBH--+MB+
+H+
(3)
+2eP
In our studies, we observed that the response to hydrogen peroxide closely followed that of glucose. The following observations have to be accounted for in a complete description of the reaction mechanism. Hydrogen peroxide injections into a system with an RVC electrode without enzyme but with mediator produced only negligible responses at - 100 mV. This proves that the enzyme is necessary for the hydrogen peroxide reaction. A mixture of equal concentrations of mediator and hydrogen peroxide (5 PM) was put in a spectrophotometer cuvette and monitored at 568 nm. The absorbance decreased linearly with time from 0.81 to 0.75 AU in 15 min and continued to decrease after 2 h. Oxygen will react with
M. Klq:rumi
rl ul.
the reduced mediator until it is finally colorless and no quantitative data can be drawn from the experiment. The mean residence time in the flow system is 28 s at 1 ml min ‘. i.e. the direct reaction between mediator and hydrogen peroxide is far too slow to explain the observation that hydrogen peroxide produces peaks at - 100 mV. The peak heights for glucose injections decreased substantially when the carrier was deoxygenated. Although the oxygen was not completely removed the experiment nonetheless proves that oxygen is important. This indicates that the glucose first reacts with oxygen to produce hydrogen peroxide which is then oxidized enzymatically. This path does not exclude a parallel reaction according to Eq. (2). The mediator is reduced and reoxidized electrochemically. The reaction scheme will then be glucose + 0, d
H,O, + gluconolactone Ci0-X H,O,+MB+---tMBH+O,+H+
(4) (5)
followed by Eq. (3). The proposed scheme is tentative and has to be supported by a mechanistic study. Fig. 4 shows calibration curves for three decades. The current reached a plateau at 100 /IM and did not increase when the glucose or hydrogen peroxide concentration was increased to 1
Stability 0.05 1
Tulunru
4-i (1996)
957-962
961
or 10 mM (not shown). The plateau is due to small amounts of mediator in comparison with the concentrations of glucose or hydrogen peroxide. The two curves are almost identical (even at 1 and 10 mM) which indicates that Eq. (5) is the rate-limiting step. Thus the rate is highly dependent on the mediator concentration (see Fig. 2). The current observed with glucose as a sample decreased only slightly over the pH range 6-8. Hydrogen peroxide generated by the enzymatic reaction of glucose is produced at the pore walls within the diffusion layer. It reacts with the enzyme and mediator which are also present within the diffusion layer. The mediator is regenerated electrochemically and immediately available for a new oxidation cycle. The fact that the calibration curves for glucose fall very close to those for hydrogen peroxide demonstrates that very little hydrogen peroxide is lost to the bulk solution during glucose oxidation. A blank electrode without enzyme was also prepared and its response to various levels of glucose is shown in Fig. 4. The oxidation current is concentration dependent and l-3 orders of magnitude lower than the currents in the presence of enzyme. It is, however, much higher than the background current obtained in the absence of glucose. A possible explanation for this non-enzymatic reaction is a direct oxidation of some of the six tautomers of glucose. The aldehyde, and possibly also the gem-dial. which are present at 0.0024% and 0.0077% respectively (27°C) [13], could be oxidized by the mediator or directly at the electrode. The electrode stability can be seen in Fig. 5. It can be seen that the response remains almost constant initially because the enzyme is in excess and some deactivation can occur without affecting the results. Subsequently, enzyme or electrode degradation reduces the response.
4. Conclusions Time/days
Fig. 5. Stability tests made by injecting 100 /tl of I /rM glucose. Phosphate buffer, pH 7.0. containing 5 /rM Meldola Blue: applied potential, - 100 mV; flow rate. I ml min ‘.
The low level calibration curve is linear up to about 10 ,DM and the detection limit is at present around 30 nM glucose but could perhaps be
962
M. Khur;vami
rf al.
lowered with further optimization. The electrode performs well in a flow-injection system and a throughput of 60 samples h ’ was readily obtained. The detection system is very attractive for low level measurements. The porous electrode material results in a high coulometric yield combined with a low inherent background current. Another advantage is the possibility of covalently immobilizing enzymes directly onto the RVC electrode.
Acknowledgements This work was supported by the Swedish Research Council for Engineering Sciences and by the Swedish Natural Research Council. Thanks are due to Mrs Maj-Britt Larsson for skilful technical assistance. The authors also thank Mr. Michael Mecklenburg for linguistic advice.
Tuianru
4.3 (I 996)
957- 962
References PI
F. Scheller and F. Schubert, Biosensors, Elsevier, Amsterdam, 1992. Prog. RePI P.N. Bartlett. P. Tebbutt and R.G. Whitaker. act. Kin&, 16 (1991) 55. T. Nwosu. P.-O. Larsson and B. PI B. Xie, M. Khayyami. Danielsson, Analyst. I 18 (1993) 845. Ann. Clin. Biochem.. 6 (1969) 24. [41 P. Trinder, and G. Johansson, Anal. Chim. Acta, 155 [51 H. LundbPck (1983) 47. PI L. Gorton. Anal. Chim. Acta. 178 (1985) 247. G. Jonsson-Pettersson, E. Csoregi. K. Jo[71 L. Gorton, hansson, E. Dominguez and G. Marko-Varga. Analyst, 117 (1992) 1235. PI G. Jonsson and L. Gorton. Biosensors, I (1985) 355. J. Wang and [91 J. Kulys, H.E. Hansen, T. Buch-Rasmussen, M. Ozsoz. Anal. Chim. Acta. 288 (1994) 193. Acta, 26 (1981). [lOI J. Wang, Electrochim. Anal. [Ill H.J. Wieck. G.H. Heider. Jr. and A. Yacynych, Chim. Acta, 158 (1984) 137. .4. Torstensson, H. Jaegfeldt and G. JoU21 L. Gorton, hansson, J. Electroanal. Chem.. 161 (1984) 103. J. Am. Chem. Sot., 109 [I31 S.R. Maple and A. Allerhand. (1987) 3168.
Talanta 43 (1996) 957-962
Flow-injection determination of trace hydrogen peroxide or glucose utilizing an amperometric biosensor based on glucose oxidase bound to a reticulated vitreous carbon electrode* Masoud Khayyami”.“, Gillis Johanssonb, Dario Kriz”, Bin Xie”, Per-Olof Larsson”, Bengt Danielsson” ~‘Pure und Applied hAnalytical
Biochemistry. Chemistry,
Chemical CXemicaI
Center,
Center.
Lund
Lund
Uniwrslty.
Unrrrr.Gty.
P.O.
P.O.
Bos
Box
124. S-221
124, S-221
00 Lund,
00 Lund.
Swrdrn
Swden
Received 2X December 1995: accepted 3 January 1996
Abstract An electron transfer mediator, 8-dimethylamino-2.3-benzophenoxazine (Meldola Blue), dissolved in the carrier solution in a flow-injection system, was found to reduce the oxidation potential for hydrogen peroxide from 600- 1200 mV without mediator to - 100 mV vs. AgiAgCI with the mediator present. The very low background current of reticulated vitreous carbon (RVC) at this potential makes it possible to detect very low levels of hydrogen peroxide or glucose. Glucose oxidase was covalently coupled with carbodiimide to RVC, and the RVC was formed into a column inserted in a flow-injection system. The calibration curve was linear from 30 nM to 10 PM glucose with 5 ,uM mediator. At higher mediator concentrations. the linear range was extended to 1000 PM, but with a much higher background current. The sample throughput was about 60 h- I. The current response decreased to 50% of the original response after 20 days. The coulometric yield was high because the sample was pumped through the pores of the RVC. It was 16% and 55% at a flow rate of 1 ml min ’ at mediator concentrations of 5 and 50 ,uM respectively. Keywords:
vitreous
Nanomolar glucose and hydrogen carbon; Amperometry
peroxide
detection;
Glucose
biosensor;
Enzyme
electrode;
Reticulated
1. Introduction
* Corresponding author. Fax: ( +46)46-46-10-46-l 1. ’ Presented at the Seventh International Conference on Flow Injection Analysis (ICFIA ‘95), held in Seattle, WA, USA, August 13-17. 1995.
A great number of biosensors for glucose determination have been described in the literature over the last two decades [l-3]. The major goal has been to develop sensors for determining glucase in blood. Some work has also been devoted
0039-9140~96~$15.00 8 1996 Elsevier Science B.V. All rights reserved PII SOO39-9140(95)01872-3
958
121. Khu~;vomi
et rd.
Tdunta
to determination of glucose in biotechnological processes. Very little emphasis has been put on developing glucose sensors for the submicromolar range. Hydrogen peroxide is a reaction product in a number of enzymatic reactions used in substrate determinations. It can be determined spectrophotometrically using peroxidase-catalyzed color reactions of the Trinder type [4]. It can also be determined electrochemically by oxidation at about + 1200 mV vs. SCE on glassy carbon [5] or at lower potentials on platinum or by using chemically-modified electrodes, e.g. + 400 mV at palladium sputtered on carbon [6]. With catalysts such as peroxidases on the electrode surface reduction starts at + 600 mV and reaches a constant value at - 200 mV [7]. A number of soluble or immobilized mediators have been used for lowering the necessary electrode potentials for detection of hydrogen peroxide produced by oxidases. Mediators
Counter electrode -I-
13 (1996)
957
962
for glucose determination include N-methylphenazinium salts [8] and Meldola Blue. Both mediator and enzymes were incorporated into carbon paste electrodes [9]. The superior electrochemical properties of reticulated vitreous carbon (RVC) [lo] have prompted a study of this material for use as a sensor at very low concentration levels. Previously glucose oxidase has been coupled covalently to RVC and the production of hydrogen peroxide was determined by oxidation at + 900 mV vs. SCE [l 11. In this paper we report on the use of a soluble electron transfer mediator, Meldola Blue, which can detect hydrogen peroxide or glucose down to - 100 mV vs. Ag/AgCl. Glucose oxidase was used in this study because this enzyme is very stable and has been comprehensively studied previously. It is thus a good model system although the use of other oxidases may provide even higher sensitivity from a practical point of view. The ability to select a potential at which the background currents are extremely low should be of great importance in making sensors for low substrate levels.
(Pt)
2. Experimental Reference
electrode
2.1. Clzemicu1.s
S~hcon rubber RVC (counter
electrode)
RVC (working
electrode)
Flanged tubing
Working
electrode
(Platinum)
Fig. I. hydrogen
Cell configuration peroxide.
for
determination
of glucose
or
Glucose oxidase (GOx) type S-X (Aspergilltls n&r). Meldola Blue, 8-dimethylamino-2,3-benzophenoxazine and 1-cyclohexyl-3-(2-morpholimetho-p-toluenesulfonate noethyl)-carbodiimide were obtained from Sigma. The RVC, porosity grade 100-S (pore size nominally 0.25 mm), was from Energy Research and Generation Inc. (Oakland, CA). The potentiostat (model MA 5410, Chemel AB, Lund, Sweden) was set at a voltage of- 100 mV vs. Ag/AgCl. The carrier buffer was 0.1 M sodium phosphate, pH 7.0, containing 1.9 mg Meldola Blue 1~ ’ (5.0 PM). Meldola Blue is unstable at higher pH, the half-life time at pH 7.0 is 100 h [12], and new buffer solutions were therefore prepared every day. The peristaltic pump was from Alitea AB (model C-4~). Sample injection was through a 100 ,~l loop.
&I. Kh~~~m~i Table 1 Peak currents at various buffer. pH 7.0. containing Applied voltage Peak current
applied voltages 5 /tM Meldola (mV) (nA)
-150 270
ci al.
Talunru
43 (1996)
(vs. Ag AgCI) observed when Blue. Flow rate I ml mini’ -100 230
2.2. Preparation of the column RVC cylinders (1.5 x 0.5 cm’) were cut (using eye and dust inhalation protection devices) from a block of material and left in 6 M HCl for 1 h. They were washed with distilled water until the solution reached pH 5, and then left in dry methanol for 2 h. Finally, the cylinders were dried in an oven at 110°C overnight. A cylinder was fixed in a glass tube as shown in Fig. 1 with a counter electrode of the same material. The reference electrode was mounted downstream, which should be acceptable due to the low currents. 2.3. Immobilization A 0.05 M acetate buffer (pH 5.1) containing carbodiimide (40 mg ml ‘) was pumped through the system at a flow rate of 0.2 ml min ’ for 150 min in order to activate the RVC surface. Then acetate buffer was circulated under an ice bath at a flow rate of 1.2 ml min ’ for 30 min. For immobilization of GOx, an enzyme solution (60 mg per 10 ml) in buffer was circulated in the column under an ice bath at a flow rate of 0.2 ml min’ for 3 h. The final rinsing procedure consisted of pumping a cold phosphate buffer (0.1 M, pH 7.5) for 5 min.
3. Results and discussion The RVC material is porous and can be shaped in the form of a column permitting a liquid to be pumped through, thus giving efficient mass transfer to the surface. The surface area will be very large but the background current will still be low because of the favourable properties of RVC. A large surface area is necessary in order to immobilize a sufficient amount of enzyme but it is also necessary to obtain a high coulometric yield. The
-50 230
injecting
957~-962
959
100 ,uI of I ,uM
0 230
50 250
glucose
100 250
samples
into
a phosphate
150 280
electrode was made in the form of a cylinder and inserted into a flow-injection system. 3.1. Reaction properties Injection of 10 ,uM glucose samples produced oxidation peaks which were almost independent of the applied voltage, see Table 1. The redox potential of adsorbed Meldola Blue is - 175 mV at pH 7.0 [12]. The applied voltage should therefore be sufficient to keep the mediator in the oxidized form. The potential was set to - 100 mV for all subsequent experiments. The mediator concentration has a pronounced effect on both the background current (Table 2) and the response factor and linearity of the calibration curve. Fig. 2. The lowest background was obtained with a mediator concentration of only 5 /i M. The current at 10 ,uM glucose for example was much lower than that at the higher mediator concentrations; compare the current scale in the inset of Fig. 2 with that of the main graph. A low mediator concentration decreased the linear range (not shown in Fig. 2) because the amount present in the sample zone becomes less than that of the hydrogen peroxide or glucose. The coulometric yield at a flow rate of 1 ml min ~ ’ was 16, 45 and 55% at mediator concentrations of 5, 20 and 50 jr M respectively. The current due to glucose oxidation at - 100 mV in the presence of mediator decreased when the flow rate increased. see Fig. 3. For compariTable 2 Observed background currents of the enzyme-covered RVC electrode in a phosphate buffer, pH 7.0. containing various concentrations of mediator. Applied voltage, - 100 mV vs. Ag:‘AgCl: flow rate, 1 ml mm’ Mediator concentration Background current
(nM) (nA)
5 IO
20 200
50 6000
960
.
a
s c .P z j$
:
2000 -
o.c
,-’ 0
A’ 2
4
6
8
PM /
.’
I’
,, IO ,’ /’
A
/’
/’
1000 Non-enzymatic
* . .._..._
oc / 1
I##, 0
.,,I,,,,, 250
1 500
Glucose,
750
1
pM
son, Fig. 3 also gives the currents for hydrogen peroxide oxidation at + 600 mV in the absence of mediator. This is the potential where the oxidation of hydrogen peroxide begins. The curves for hydrogen peroxide and glucose follow each other closely, indicating that similar rate-limiting steps are involved. The fast decrease in response with flow rate is due to a reduced residence time at the RVC electrode giving more time for the reactions.
-. l
0.5
1.0
1.5 2.0 Flow rate (mllmin)
2.5
GIUCOS~ and hydrogen
peroxidellrM
1000
Fig. 2. Influence of mediator concentration on the response to glucose injections (100 111) with various concentrations of Meldola Blue: (A) 50 /IM; (0) 20 PM. The inset shows the low range response with 5 HIM mediator. Phosphate butfer containing mediator. pH 7.0: applied potential, - 100 mV: flow rate. I ml min ‘.
0' 0.0
_I
n -’
3.0
Fig. 3. Response variation with flow rate for injections (100 /II) of IO PM glucose (0). Phosphate butfer, pH 7.0. containing 5 ELM Meldola Biue; applied potential. - 100 mV vs. Ag/AgCl. The Figure also shows the response to hydrogen peroxide at + 600 mV in a buffer without any added mediator (A).
Fig. 4. Peak currents for hydrogen peroxide (n) and glucose (e) injections (100 111) with a carrier of phosphate buffer, pH 7.0. containing 5 bM Meldola Blue. The non-enzymatic oxidation of glucose ( n ) is also shown. Flow rate, I.0 ml min-‘; applied potential. - 100 mV vs. Ag:AgC).
Diffusion within the pores also plays an important role in the mass transfer because less substrate reaches the pore walls at shorter residence times. Kulys et al. [9] suggest that Meldola Blue (MB) shuttles electrons from the redox center of glucose oxidase (GOx) to the graphite surface according to the following scheme (corrected by us): glucose + GOx( FAD) --f GOx(FADH,)
+ gluconolactone
(1)
GOx( FADH,)
+ MB +
--t GOx(FAD)
+ MBH + H +
(2)
MBH--+MB+
+H+
(3)
+2eP
In our studies, we observed that the response to hydrogen peroxide closely followed that of glucose. The following observations have to be accounted for in a complete description of the reaction mechanism. Hydrogen peroxide injections into a system with an RVC electrode without enzyme but with mediator produced only negligible responses at - 100 mV. This proves that the enzyme is necessary for the hydrogen peroxide reaction. A mixture of equal concentrations of mediator and hydrogen peroxide (5 PM) was put in a spectrophotometer cuvette and monitored at 568 nm. The absorbance decreased linearly with time from 0.81 to 0.75 AU in 15 min and continued to decrease after 2 h. Oxygen will react with
M. Klq:rumi
rl ul.
the reduced mediator until it is finally colorless and no quantitative data can be drawn from the experiment. The mean residence time in the flow system is 28 s at 1 ml min ‘. i.e. the direct reaction between mediator and hydrogen peroxide is far too slow to explain the observation that hydrogen peroxide produces peaks at - 100 mV. The peak heights for glucose injections decreased substantially when the carrier was deoxygenated. Although the oxygen was not completely removed the experiment nonetheless proves that oxygen is important. This indicates that the glucose first reacts with oxygen to produce hydrogen peroxide which is then oxidized enzymatically. This path does not exclude a parallel reaction according to Eq. (2). The mediator is reduced and reoxidized electrochemically. The reaction scheme will then be glucose + 0, d
H,O, + gluconolactone Ci0-X H,O,+MB+---tMBH+O,+H+
(4) (5)
followed by Eq. (3). The proposed scheme is tentative and has to be supported by a mechanistic study. Fig. 4 shows calibration curves for three decades. The current reached a plateau at 100 /IM and did not increase when the glucose or hydrogen peroxide concentration was increased to 1
Stability 0.05 1
Tulunru
4-i (1996)
957-962
961
or 10 mM (not shown). The plateau is due to small amounts of mediator in comparison with the concentrations of glucose or hydrogen peroxide. The two curves are almost identical (even at 1 and 10 mM) which indicates that Eq. (5) is the rate-limiting step. Thus the rate is highly dependent on the mediator concentration (see Fig. 2). The current observed with glucose as a sample decreased only slightly over the pH range 6-8. Hydrogen peroxide generated by the enzymatic reaction of glucose is produced at the pore walls within the diffusion layer. It reacts with the enzyme and mediator which are also present within the diffusion layer. The mediator is regenerated electrochemically and immediately available for a new oxidation cycle. The fact that the calibration curves for glucose fall very close to those for hydrogen peroxide demonstrates that very little hydrogen peroxide is lost to the bulk solution during glucose oxidation. A blank electrode without enzyme was also prepared and its response to various levels of glucose is shown in Fig. 4. The oxidation current is concentration dependent and l-3 orders of magnitude lower than the currents in the presence of enzyme. It is, however, much higher than the background current obtained in the absence of glucose. A possible explanation for this non-enzymatic reaction is a direct oxidation of some of the six tautomers of glucose. The aldehyde, and possibly also the gem-dial. which are present at 0.0024% and 0.0077% respectively (27°C) [13], could be oxidized by the mediator or directly at the electrode. The electrode stability can be seen in Fig. 5. It can be seen that the response remains almost constant initially because the enzyme is in excess and some deactivation can occur without affecting the results. Subsequently, enzyme or electrode degradation reduces the response.
4. Conclusions Time/days
Fig. 5. Stability tests made by injecting 100 /tl of I /rM glucose. Phosphate buffer, pH 7.0. containing 5 /rM Meldola Blue: applied potential, - 100 mV; flow rate. I ml min ‘.
The low level calibration curve is linear up to about 10 ,DM and the detection limit is at present around 30 nM glucose but could perhaps be
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lowered with further optimization. The electrode performs well in a flow-injection system and a throughput of 60 samples h ’ was readily obtained. The detection system is very attractive for low level measurements. The porous electrode material results in a high coulometric yield combined with a low inherent background current. Another advantage is the possibility of covalently immobilizing enzymes directly onto the RVC electrode.
Acknowledgements This work was supported by the Swedish Research Council for Engineering Sciences and by the Swedish Natural Research Council. Thanks are due to Mrs Maj-Britt Larsson for skilful technical assistance. The authors also thank Mr. Michael Mecklenburg for linguistic advice.
Tuianru
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References PI
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