Low-potential amperometric enzyme biosensor for xanthine and hypoxanthine

Article abstract of DOI:10.1021/ac3025027

The bacterial xanthine dehydrogenase (XDH) from Rhodobacter capsulatus was immobilized on an edge-plane pyrolytic graphite (EPG) electrode to construct a hypoxanthine/xanthine biosensor that functions at physiological pH. Phenazine methosulfate (PMS) was used as a mediator which acts as an artificial electron-transfer partner for XDH. The enzyme catalyzes the oxidation of hypoxanthine to xanthine and also xanthine to uric acid by an oxidative hydroxylation mechanism. The present electrochemical biosensor was optimized in terms of applied potential and pH. The electrocatalytic oxidation response showed a linear dependence on the xanthine concentration ranging from 1.0 × 10^-5 to 1.8 × 10^-3 M with a correlation coefficient of 0.994. The modified electrode shows a very low detection limit for xanthine of 0.25 nM (signal-to-noise ratio = 3) using controlled potential amperometry.

Full text of DOI:10.1021/ac3025027

Article
pubs.acs.org/ac
Low-Potential Amperometric Enzyme Biosensor for Xanthine and
Hypoxanthine
†
‡
,†
Palraj Kalimuthu, Silke Leimku
†
̈
hler, and Paul V. Bernhardt*
School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, 4072, Australia
‡
Institut fu
̈
̈
r Biochemie und Biologie, Universitat Potsdam, 14476 Potsdam, Germany
*
S Supporting Information
ABSTRACT: The bacterial xanthine dehydrogenase (XDH) from Rhodobacter capsulatus
was immobilized on an edge-plane pyrolytic graphite (EPG) electrode to construct a
hypoxanthine/xanthine biosensor that functions at physiological pH. Phenazine
methosulfate (PMS) was used as a mediator which acts as an artificial electron-transfer
partner for XDH. The enzyme catalyzes the oxidation of hypoxanthine to xanthine and
also xanthine to uric acid by an oxidative hydroxylation mechanism. The present
electrochemical biosensor was optimized in terms of applied potential and pH. The
electrocatalytic oxidation response showed a linear dependence on the xanthine
−
5
−3
concentration ranging from 1.0 × 10 to 1.8 × 10 M with a correlation coefficient
of 0.994. The modified electrode shows a very low detection limit for xanthine of 0.25 nM
(
signal-to-noise ratio = 3) using controlled potential amperometry.
anthine is an intermediate of the purine nucleotide and
applied to samples without any pretreatment. Several chemi-
X
deoxynucleotide metabolism and present in most of our
cally modified electrodes have been employed for the
1
20
body tissues and fluids. The normal concentration of xanthine
in human plasma ranges from 0.5 to 2.5 μM (40−160 μM in
determination of xanthine including polymer films, pretreated
1
2
21
carbon paste, nanoporous carbon fiber, preanodized
2
22
urine). As the metabolic precursor of uric acid, xanthine is the
first indicator of an abnormal purine profile and can serve as a
marker of many diseases including hyperuricemia, gout,
xanthinuria, perinatal asphyxia, cerebral ischemia, tumor
hyperthermia, and pre-eclampsia. Uric acid is typically present
at serum concentrations of ∼200−400 μM and is by far the
dominant purine of this class in biological fluids.
nontronite-coated carbon, and multiwall carbon nanotube
23
composite modified electrodes. Usually these chemically
modified electrodes oxidize xanthine above +600 mV versus
Ag/AgCl (Scheme 1), but at this potential, interference from
other electroactive compounds present in biological samples
limits the practicality of this approach.
3
4
5
6
7
8
9
1
0−15
Xanthine oxidoreductase, encompassing both xanthine
oxidase (XO) and xanthine dehydrogenase (XDH), is a
complex molybdo/iron−sulfur/flavoenzyme found in bacteria
Hypoxanthine (Scheme 1) is the precursor to xanthine and is
typically present at plasma concentrations of ∼10−30 μM in
24
1
5
and animals. The enzyme catalyzes the oxidative hydrox-
ylation of purines, pyrimidines, pterins, and aldehyde substrates
healthy humans. In xanthinuria, the concentrations of
xanthine are elevated and similar to that of hypoxanthine in
serum and in urine. The accumulation of xanthine is at the
+
using molecular oxygen (XO) or NAD (XDH) as electron
24,25
2
acceptor.
The two principal substrates are xanthine and
expense of uric acid whose concentrations are very low.
hypoxanthine (Scheme 1). Both XO and XDH contain a Mo
active site comprising a single bidentate molybdopterin ligand,
an equatorial terminal sulfido, an axial oxido, and an equatorial
In the food industry xanthine is also an important biomarker.
Inosine monophosphate (present in fresh fish samples)
degrades (by microbial action or the action of other
endogenous enzymes) to inosine, then to hypoxanthine and
xanthine. Elevated levels of hypoxanthine and xanthine are a
sign of spoilage. Thus, developing a stable, sensitive, and
selective hypoxanthine/xanthine sensor may also have
applications in food quality control.
26−28
hydroxido/aqua ligand depending on pH.
The most intensively studied xanthine oxidoreductase is
bovine milk XO, which is commercially available. Similar to
other oxidase enzymes, when dioxygen is the electron acceptor,
1
6
29
H O is a product of XO turnover. The electroactivity of
2
2
H O enables its voltammetric detection and provides an
The most common methods for detecting and quantifying
purines from this class are electrophoresis, GC/MS,
HPLC, chemiluminescence, and UV spectrophotometry.
2
2
13
17
19
indirect method for monitoring XO turnover. Alternatively, the
enzyme horseradish peroxidase may be incorporated to
1
4
18
However, these methods are time-consuming, require expensive
equipment, and are labor-intensive in terms of sample
preparation. By comparison, electrochemical methods offer
simplicity, portability, high sensitivity, and selectivity and can be
Received: August 30, 2012
Accepted: November 7, 2012
Published: November 7, 2012
©
2012 American Chemical Society
10359
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Article
Scheme 1. Redox Reactions and Potentials (mV vs Ag/AgCl, pH 8) of Purines in This Work
catalytically reduce the product H O to water (mediated by a
EXPERIMENTAL SECTION
2
2
■
30
coimmobilized electron-transfer relay). Several XO-based
electrochemical biosensors have been reported where XO is
immobilized within various supporting matrixes including
Materials. Xanthine dehydrogenase was purified from a
heterogeneous expression system in E. coli as previously
28
described. Xanthine, hypoxanthine, uric acid, and phenazine
3
1
32
polypyrrole, graphite (mediated by ferricyanide), osmium
methosulfate were purchased from Aldrich and were used as
received. All other reagents used were of analytical grade purity
and used without any further purification. All solutions were
prepared in purified water (Millipore, resistivity 18.2 MΩ·cm).
Phosphate buffer solution (PBS) was prepared using 100 mM
Na HPO and NaH PO , and the desired pH was obtained
30
poly(vinylpyridine) gel polymer, silk fibroin/cellulose acetate
3
3
34
membranes, gold nanoparticles, multiwall carbon nano-
3
5
36
tubes, calcium carbonate nanoparticles, poly(mercapto-p-
39
3
7
38
benzoquinone), layered double hydroxides, and Nafion
modified electrodes. However, these sensors operate at high
overpotentials (>+0.20 V vs Ag/AgCl). In the detection of
xanthine, this is particularly important as the product of
xanthine oxidation, uric acid, is also oxidized at this potential
2
4
2
4
with dilute H PO or NaOH.
3
4
Electrochemical Measurements and Electrode Clean-
ing. Cyclic voltammetry (CV) and controlled potential
amperometry were carried out with a BAS 100B/W electro-
chemical workstation using a three-electrode system consisting
of an EPG working electrode, a platinum wire counter
electrode, and a Ag/AgCl reference electrode. Unless otherwise
stated, electrochemical solutions were purged with nitrogen for
at least 30 min prior to the series of experiments and all
experiments were performed under a blanket of nitrogen. The
EPG electrode surface was cleaned by cleaving several 1 μm
layers from the face of the electrode with a microtome followed
by sonication in Milli-Q water for 15 min. No abrasives were
used. No other electrode surface conditioning was necessary,
and no promoters were used. All CV measurements were made
with a cell that was protected from light to avoid photochemical
degradation of the PMS mediator.
(
Scheme 1). Other species such as catecholamines and ascorbic
acid are also electroactive in this region.
On the other hand, XDH from Rhodobacter capsulatus has
+
very low reactivity with O and preferentially uses NAD as an
2
4
0
electron acceptor. In a recent paper we showed that N-methyl
phenazinium methanesulfonate (phenazine methosulfate, PMS)
+
is an effective substitute for NAD and can mediate electrons
41
between XDH and a thiol-modified Au working electrode.
Moreover, its redox potential (ca. −160 mV vs Ag/AgCl, pH 8)
is very low, so interference from the oxidation of other
compounds at the electrode is avoided. In the presence of
either xanthine or hypoxanthine, pronounced catalytic currents
4
1
were observed at the redox potential of PMS.
In earlier papers,
4
2,43
we observed that XDH adsorbed on an
edge-plane pyrolytic graphite (EPG) electrode shows pro-
nounced electrocatalytic activity (for the oxidation of xanthine
to uric acid) even in the absence of an added mediator. It is
Enzyme Electrode Preparation. A 6 μL droplet of XDH
66 μM) was transferred to a freshly prepared, inverted EPG
electrode, and this was allowed to dry to a film at 4 °C. To
prevent protein loss the electrode surface was carefully covered
with a perm-selective dialysis membrane presoaked in water
(
4
3
now understood that uric acid (the product of this catalytic
reaction) acts as a mediator of catalysis, being oxidized at ca.
+
250 mV versus Ag/AgCl (pH 8, Scheme 1) in an unusual
(
MW cutoff 12 kDa). The dialysis membrane was pressed onto
enzyme electroautocatalytic mechanism. In terms of a sensing
application, the simultaneous oxidation of both xanthine
the electrode using a Teflon cap and fastened to the electrode
with a rubber O-ring to prevent leakage of the internal
membrane solution. The resulting modified electrode was
stored at 4 °C in 100 mM phosphate buffer solution (pH 7.0)
when not in use.
(
enzymatically) and uric acid at the electrode in this system
makes unique determination of xanthine impractical as the
observed current is a composite of both processes. Indeed
similar behavior was seen for the XDH-catalyzed oxidation of
hypoxanthine to xanthine, so the catalytic current in that case
was due to the oxidation of three molecules concurrently:
hypoxanthine and xanthine by XDH and uric acid at the
electrode. On this point, uric acid is present in biological fluids
at concentrations ∼10−100 times higher than the xanthine.
Therefore, interference from uric acid oxidation is a chronic
issue in any practical electrochemical hypoxanthine/xanthine
sensor unless the operating potential can be lower than that of
the uric acid redox couple.
In the present study, we have developed an electrochemical
hypoxanthine/xanthine biosensor based on R. capsulatus XDH
immobilized on an EPG electrode. To avoid uric acid
interference, which is oxidized at +250 mV versus Ag/AgCl,
we have employed the mediator PMS. The present electro-
chemical biosensor has been optimized in terms of pH and
applied potential.
Electrochemical Measurements and Data Processing.
The variation of the observed limiting catalytic current (i ) as
lim
a function of substrate concentration ([S]) followed
Michaelis−Menten kinetics, and the data were fit to the eq 1
which is applicable to steady-state enzyme kinetics and where
the concentration of substrate [S] is constant. The presence of
a membrane covering the electrode inhibits diffusion of the
substrate to the enzyme, so steady-state conditions are not
strictly met, but the catalytic current does in fact saturate at
high substrate concentrations (vide infra Figures 3B and 4B).
For these reasons though, the apparent Michaelis constant
(
KM,app) will reflect the diffusion-limited mass transport of
substrate to the enzyme:
imax[S]
ilim = [
S] + KM,app
(1)
1
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where imax is the saturation limiting current and KM,app is the
apparent Michaelis constant.
The pH dependence of the catalytic current was modeled by
eq 2 which is applicable for an active form of the enzyme that is
summarizes the overall coupled electrochemical and chemical
reactions that are involved in this process. The membrane
shown in Scheme 2 traps the enzyme XDH but permits
diffusion of xanthine and PMS from the bulk solution to the
electrode and also allows uric acid to diffuse away from the
enzyme. The kinetics of these various steps have been discussed
reversibly deactivated by either deprotonation (at pK ) or
a1
protonation (at pKa2).
41
in detail in a recent publication.
iopt
ilim(pH) = 1
Catalytic Voltammetry. The response of bare EPG and
XDH-modified EPG electrodes toward 800 μM xanthine in the
presence of 5 μM PMS was investigated, and the results are
displayed in Figure 1. In the control experiment, at a bare EPG
electrode, PMS (5 μM, pH 7) shows a reversible redox wave at
(
pH−pKa1)
+ 10(pKa2−pH)
+ 10
(2)
RESULTS AND DISCUSSION
Mechanism. The catalytic electrochemical reaction is a
ternary system comprising XDH, substrate (xanthine or
hypoxanthine), and mediator (PMS). In the absence of any
of these three components no catalysis can occur. Scheme 2
■
−
139 mV with a peak-to-peak separation of 47 mV along with a
weaker response at −268 mV (Figure 1A, red curve a). Two
redox waves at −139 and −268 mV were attributed to the
diffusional and adsorbed PMS responses, respectively, at the
44
EPG electrode. This was established by the sweep rate
dependence of these responses where the lower potential peak
currents increased linearly with sweep rate while the higher
Scheme 2
45
potential peaks increased with the square root of sweep rate.
No change was found upon addition of 800 μM xanthine to the
same solution (Figure 1A, blue curve b), illustrating that
xanthine is electroinactive within this range and does not react
with PMS . The EPG/XDH electrode also shows a similar
ox
response for 5 μM PMS in the absence of xanthine (Figure 1B,
red curve a; note different vertical scale). In this case the
capacitive current is somewhat smaller.
On addition of xanthine (800 μM) to the cell the EPG/XDH
electrode shows a pronounced anodic current and loss of the
corresponding cathodic peak (Figure 1B, blue curve b). The
enhancement of anodic current and asymmetry of the
waveform are indicative of a catalytic (EC ) mechanism.
cat
The potential coincides with that of the PMSox/red couple as this
compound is the only electroactive species present within this
potential window. Briefly, the anodic sweep switches on
catalysis as the mediator PMSred is converted to its active
PMSox form which may accept electrons from XDHred after
turnover (Scheme 2) regenerating PMSred for further electro-
chemical oxidation. The XDH chemical reaction comprises
hydroxylation of xanthine to uric acid. The XDH /PMS
red
ox
reaction is an outer-sphere electron-transfer reaction.
We have also investigated the response of EPG/XDH toward
uric acid in the presence of 5 μM PMS (Supporting
Information Figure S1). This is relevant because uric acid is
the product of XDH turnover and also a potential electroactive
Figure 1. CVs obtained for 5 μM PMS in the (a) absence and (b) presence of 800 μM xanthine at (A) bare EPG (no XDH) and (B) the EPG/XDH
−1
electrode [100 mM phosphate buffer (pH 7), sweep rate of 5 mV s ].
1
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interfering species due to its high concentrations in biological
fluids and low redox potential. However, the EPG/XDH
electrode does not show any electrochemical response toward
uric acid within the potential window of −350 to +180 mV
versus Ag/AgCl, so the observed catalytic current is exclusively
due to the PMS-mediated XDH/xanthine reaction.
PMS Concentration Dependence. Figure 2 illustrates the
CVs obtained for various PMS concentrations in the presence
Figure 3. (A) CVs obtained for 5 μM PMS at the EPG/XDH
electrode at varying xanthine concentrations of (a) 0, (b) 20, (c) 50,
(d) 100, (e) 200, (f) 400, (g) 800, (h) 1600, (i) 3200, (j) 4800, and
(
k) 6400 μM in 100 mM phosphate buffer solution at a scan rate of 5
−1
mV s . (B) Plot of the electrocatalytic anodic current at −90 mV vs
Ag/AgCl as a function of xanthine concentration in panel A. The curve
is from data fit to eq 1 [KM,app (xanthine) 1.0 ± 0.1 mM].
Figure 2. CVs obtained for the EPG/XDH electrode with 1 mM
xanthine at varying PMS concentrations of (a) 0, (b) 2, (c) 4, (d) 6,
and (e) 8 μM in 100 mM phosphate buffer solution at a scan rate of 5
−
1
mV s .
of 1 mM xanthine at the EPG/XDH electrode. In the absence
of PMS, no faradaic response is seen within the potential
window of −300 to +180 mV (Figure 2, broken curve a). As
PMS is added a pronounced sigmoidal oxidation wave emerges
Figure 4. (A) CVs obtained at the EPG/XDH electrode for 5 μM
PMS at varying hypoxanthine concentrations of (a) 0, (b) 20, (c) 50,
(d) 100, (e) 200, (f) 400, (g) 800, (h) 1600, (i) 3200, (j) 4800, and
(
Figure 2, curves b and c) which is indicative of an
electrochemical steady state where PMSox is consumed by
XDHred at the same rate as it is replenished at the electrode
(
k) 6400 μM in 100 mM phosphate buffer solution at a scan rate of 5
−1
mV s . (B) Plot of the electrocatalytic anodic currents at −90 mV vs
Ag/AgCl as a function of both hypoxanthine and xanthine
concentration. The curves are from data fit to eq 1 [KM,app
(
Scheme 2). At higher bulk concentrations of PMS (Figure 2,
curves d and e) the waveform becomes peak-shaped (transient
voltammetry) because the XDH /PMS reaction overruns
red
ox
(hypoxanthine) 630 ± 70 μM and KM,app (xanthine) 1.0 ± 0.1
the XDH /xanthine reaction and the concentration of PMS
ox
ox
mM]. Note: CV data were acquired for both substrates using the same
EPG/XDH electrode after washing all previous substrate out with
fresh buffer.
at the electrode surface accumulates thus establishing a time-
dependent diffusion layer.
Xanthine Concentration Dependence. The xanthine
concentration dependence of the voltammetry in the presence
of 5 μM PMS is shown in Figure 3. The main catalytic anodic
peak current at −70 mV versus Ag/AgCl increases linearly with
xanthine concentration within the range of 25 μM to 1 mM and
then nonlinearly at higher concentrations before saturating at
around 7.3 mM as predicted by Michaelis−Menten kinetics
Figure 3) the same electrode was removed from the solution
and placed in fresh buffer containing only PMS (5 μM). The
original (noncatalytic) PMS redox response was again
observed; then additional amounts of hypoxanthine were
added to produce the responses in Figure 4. The EPG/XDH
electrode shows very similar catalytic voltammetry in the
presence of hypoxanthine, but the current response to
hypoxanthine is ∼50% greater than that from xanthine (Figure
4B). This higher current may be attributed to a contribution
from the consecutive hypoxanthine → xanthine → uric acid
(four-electron) process in addition to the simple hypoxanthine
→ xanthine (two-electron) process. We have noted similar
behavior previously where the consecutive oxidation processes
hypoxanthine → xanthine and xanthine → uric acid were
(K
1.0 ± 0.1 mM, Figure 3B). This value is much higher
M,app
than the previously reported value (K = 64.5 μM) for an XDH
M
46
solution assay at pH 7.8 in 50 mM Tris buffer solution. This
increase is most likely a consequence of inhibited mass
transport of xanthine from the bulk solution across the
membrane to the reaction layer and restricted access to the
enzyme active site within a protein film at the working
electrode surface.
Hypoxanthine Concentration Dependence. We also
investigated the electrocatalytic response of the EPG/XDH
electrode toward hypoxanthine under identical experimental
conditions as those in Figure 3, in fact using the exact same
EPG/XDH electrode to eliminate any variations in XDH
concentration or electrode surface area, and the results are
shown in Figure 4A. After the xanthine data were acquired (see
4
1,43
observed.
The response to hypoxanthine does not simply
double relative to xanthine as evidently not all of the xanthine
intermediate is recaptured by the enzyme in competition with
hypoxanthine for the active site.
pH Dependence. To optimize the electrochemical
biosensor, the catalytic response of the EPG/XDH electrode
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toward xanthine was studied at different pH values. Figure 5
shows the pH dependence of the EPG/XDH electrode
maximum anodic current over the pH range of 6−10 in 100
mM phosphate buffer solution containing 1 mM xanthine and 5
μM PMS.
current response (Figure 6B) was observed in the presence of
10 μM xanthine, and further increments of 10 μM xanthine at
intervals of 100 s led to a step in the current, with a steady-state
current (plateau) being reached within 3 s. The current
increased linearly with each xanthine addition from 10 to 120
μM, and the detection limit calculated from the standard
49
deviation of the baseline current was found to be 0.25 nM
signal-to-noise ratio (S/N) = 3] (Figure 6). In separate
experiments, we found that the amperometric current was
[
linear from 1.0 × 10− to 1.8 × 10 M xanthine with a
regression coefficient of 0.994. The sensitivity of the EPG/
XDH electrode for xanthine was found to be 0.31 nA/μM.
Analytical Performance. The analytical performance of
the xanthine biosensor was compared with other reported
xanthine oxidase modified electrodes based on oxidation of
enzymatically generated H O , as summarized in Table 1.
5
−3
2
2
Evidently the EPG/XDH electrode exhibits an excellent lower
detection limit and very wide linear range compared to the
bovine XO immobilized on different substrates. To the best of
our knowledge, the present electrochemical biosensor showed
the lowest detection limit for xanthine [0.25 nM (S/N = 3)]
compared with XO-based modified electrodes. Most impor-
tantly, the present biosensor operates at very low potential (ca.
Figure 5. pH dependence of the maximum catalytic current for 1 mM
xanthine in the presence of 5 μM PMS at the EPG/XDH electrode in
−1
100 mM phosphate buffer at a scan rate of 5 mV s . The curve is a fit
to a dibasic acid model (eq 2) (ref 47) where the intermediate
protonated form is active (pKa1 = 9.0 and pKa2 = 6.4).
A classic “bell-shaped” profile was obtained which is
+
50 mV vs Ag/AgCl) and does not suffer from interferences
consistent with a dibasic acid model (eq 2) where the
from other electroactive biologically important molecules such
as uric acid, ascorbic acid, and catecholamines which are
oxidized around +300 mV.
Reproducibility and Stability. The stability of the EPG/
XDH electrode toward xanthine was assessed in two ways, first
for repeated use and second with regards to long-term storage.
The CVs for 500 μM xanthine in the presence of 5 μM PMS in
47
intermediate (monoprotonated) form is active. This model
gave approximate pK values of 9.0 and 6.4. On the basis of
a
2
8,46
48
previous studies,
xanthine deprotonation (pKa 7.7)
inhibits catalysis. Similarly, protonation of a catalytically
46
essential glutamate residue (E730), which acts as a base to
accept a proton from the oxidized substrate, switches off
catalysis. Optimal catalytic current was found at pH 7. These
results are comparable with the known activity profile of XDH
1
00 mM phosphate buffer solution were recorded every 3 min
28
to evaluate the stability of the biosensor. It was found that the
anodic peak current was constant with a relative standard
deviation of <2% for 10 repeat measurements. To check the
long-term storage stability of the biosensor, the EPG/XDH
electrode was kept in 100 mM phosphate buffer solution in a
refrigerator (4 °C). No apparent decrease in the catalytic
current response of xanthine was observed over the first 4 days
while the current decreased by 12% after 2 weeks and 22% after
4 weeks. The lowering in electrocatalytic activity could be due
in solution assays and reflect the pH dependence of the
enzyme and substrate but not the mediator PMS.
Amperometric Determination of Xanthine. Constant
potential amperometry was performed to quantify the
sensitivity of the EPG/XDH electrode toward xanthine. Figure
6
A shows the amperometric i−t curve for the catalytic xanthine
oxidation at the EPG/XDH electrode in a constantly stirred
00 mM phosphate buffer solution (pH 7) at an applied
potential of +50 mV versus Ag/AgCl. An initial steady-state
1
Figure 6. (A) Amperometric i−t curve obtained for the determination of xanthine at an EPG/XDH electrode in stirred 100 mM phosphate buffer
solution. Each step comprises a 10 μM increment in added xanthine concentration at an applied potential of +50 mV vs Ag/AgCl. (B) Linear
response range for xanthine.
1
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Table 1. Comparison of Analytical Performance of Reported Xanthine-Based Biosensors
electrode
Eapp (mV vs Ag/AgCl)
pH
linear range (M)
detection limit (M)
a
−6
−4
−4
−5
−5
−4
−4
−5
−3
−6
XO/calcium carbonate nanoparticles
+590
+550
+390
+380
+400
+350
+900
+50
7.5
7.5
7.5
7.0
7.0
7.5
7.0
7.0
2.0 × 10 −2.5 × 10
2.0 × 10
b
−6
−7
XO/layered double hydroxide
1.0 × 10 −2.0 × 10
1.0 × 10
c
−8
−8
XO/laponite nanoparticles
3.9 × 10 −2.1 × 10
1.0 × 10
d
−7
−7
XO/ZnOx/polypyrrole
8.0 × 10 −4.0 × 10
8.0 × 10
e
−6
−6
XO/carbon nanotubes
5.0 × 10 −6.0 × 10
2.0 × 10
f
−6
−6
XO/poly(terthiophine-3-carboxylic acid)
5.0 × 10 −1.0 × 10
1.0 × 10
g
−7
−7
XO/glassy carbon paste
5.0 × 10 −4.0 × 10
1.0 × 10
h
−5
−10
EPG/XDH
1.0 × 10 −1.8 × 10
2.5 × 10
a
b
c
d
e
f
g
h
Ref 36. Ref 38. Ref 50. Ref 51. Ref 52. Ref 53. Ref 54. This work.
to degradation of XDH or leakage of XDH from the inner
membrane solution.
to hypoxanthine due to a subsequent oxidation of the product
xanthine by XDH to uric acid.
To ascertain the reproducibility of the results further, three
different EPG electrodes were modified with XDH and their
response toward the oxidation of 500 μM xanthine in the
presence of 5 μM PMS was tested by 10 repeat measurements.
The peak current obtained in the 10 repeat measurements of
three independent electrodes showed a relative standard
deviation (RSD) of 2.3%.
ASSOCIATED CONTENT
Supporting Information
Voltammetry of PMS in the presence of uric acid. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
*
S
AUTHOR INFORMATION
Corresponding Author
E-mail: p.bernhardt@uq.edu.au.
■
*
Limitations of the Biosensor. The present electro-
chemical xanthine biosensor is capable of accurately determin-
ing either xanthine or hypoxanthine across a wide range of
concentrations in buffered solution. However, as XDH oxidizes
both hypoxanthine and xanthine, this method cannot
discriminate between the two in a mixture of both purines,
which would require separation (HPLC) techniques to be
included. In biological fluids (plasma, urine) of healthy humans
the concentration of hypoxanthine greatly exceeds that of
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge financial support from the
Australian Research Council Discovery Grants DP0880288
and DP12014665 (to P.V.B.) and from the Deutsche
Forschungsgemeinschaft Cluster of Excellence “Unifying
concepts in catalysis” (to S.L.).
2
,10,13
xanthine (by at least a factor of 5).
So under normal
circumstances the major current response will be due to
hypoxanthine, but the proportion of hypoxanthine and xanthine
cannot, as such, be determined. This limitation has been
overlooked in previous studies that have employed xanthine
oxidase biosensors for hypoxanthine or xanthine determination
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