A Novel Copper(I) Oxide Based Creatinine Biosensor
Meng Shan Lin* and Hoang Jyh Leu
Department of Chemistry, Tamkang University, Tamsui 251, Taipei, Taiwan
A novel creatinine biosensor was based on the measurement of ammonia by flow
injection analysis(FIA). Using the immobilization of creatinine deiminase onto a
copper(I) oxide mixed carbon ink composite electrode which effectively senses the
ammonium ion in aqueous solution, a very sensitive creatinine biosensor was
developed. All parameters, such as applied potential, buffer pH value, flow rate of
carrier, that may affect the performance of the creatinine biosensor were carefully
studied. For the optimum conditions of the creatinine biosensor, the detection limit as
low as 25 μM and the response time of 45 s were obtained in this flow-injection
system. A relative standard deviation of 3.2% (n =20) was obtained for the successive
analysis of standard creatinine solution. And the linear range of this system was upto
5mM.
Keywords: creatinine deiminase, copper(I) oxide, carbon ink, flow-injection system
1. Introduction
It is an increasing requirement for easy, selective, accurate and reliable
measurements for the important metabolites to be used as an index for the state of
health in these years. Creatinine is a final product of creatine metabolism in mammals.
Therefore, it is an important diagnostic substance in the biological fluids of renal,
thyroid and muscle function and is very important in treatment with external dialysis.
The physiologically normal concentration range for serum creatinine is 45-140μM
and creatinine is 0.8-2.0g per day in urine. But the concentrations may raise to a value
higher than 1000μM in serum during the case of kidney dysfunction or muscle
disorder.
Most creatinine determinations in clinical biochemical laboratories and
commercially available analysers are based on spectrophotometric detection of Jaffe
reaction(1). Creatinine reacts with alkaline picric acid to form orange–red complex
which is a colored species that absorbs at 500 nm. However, there are many
interferences may interfere with the formation of complex and lack specificity, such
as glucose, pyruvate, acetoacetate, bilirubin, dopamine and others(2-3). Although
several kinetic methods and different pre-treatments of the samples had been
developed, the accurate determination of creatinine concentration in the biological
samples remained a problem(4).
Because of specificity requirements, biosensors with the inherent specificity from
enzyme are seemed suitable tools for such analysis, and various types of the
biosensors for creatinine detection have been developed(5-7). They are amperometric
biosensors using mono or tri-enzyme system, potentiometric biosensors utilizing
ISFET and ion or gas sensitive electrodes, optic fluorescent biosensors, impedimetric
biosensors employing screen-printed carbon electrodes, and capacitimetric biosensors
using Ir-metal oxide semiconducting capacitors(8-10). However, all these biosensors
have some disadvantages such as low selectivity, unfavorable sensitivity, poor
stability, bad reproducibility and low storage stability for parts of those biosensors.
But enzyme based amperometric biosensors have the advantages of reducing time and
cost than other methods, the rapid, simple and promising methods are more feasible
than others.
Most of the publications showed the amperometric creatinine biosensors which
were based on the tri-enzyme catalytic sequence through creatininase, creatinase and
sarcosine oxidase(11). They were coupled to convert creatinine to electrochemically
detectable hydrogen peroxide. As the three enzymes were integrated, responses
resulted from the sequencing catalytic reactions were decreased, and the sensitivity
and detection limit of these methods were affected(12).
Mono-enzyme based amperometric and potentiometric biosensors for creatinine
determination had been also published(13-14), the enzyme were used creatinine
deiminase to transform creatinine into NH4+ and N-Methylhydantoin below pH 9.2
buffer solution. By this way, the potentiometric methods were mainly based on the
detection of NH4+ by ion selective electrode or ISFET, but the sensitivity and linear
range were not always satisfied for biological real sample requirements.
In order to manufacture the effectively chemical sensors, several redox-active metal
oxide materials have shown considerable promise for application in the areas of
electrochemistry and photochemistry. Well known materials used in metal oxide
sensors are SnO2, ZnO, TiO2, Ga2O3 and WO3, but these oxides are mostly used for
gas sensors. For the liquid sample determination, the pH value, oxygen and hydrogen
peroxide are commonly used Ag, Pt, Pd, Ru, Ir, Cu and Rh oxide as the electrode
modifiers to promote the electrochemical reaction. Regarding copper(I) oxide(Cu2O),
it is a metal-oxide p-type semiconductor that has potential applications in solar energy
conversion and catalysis, but there are still a few application for sensor. A lot of effort
has been devoted to the synthesis of Cu2O thin films, nanoparticles, nanowires and
whiskers.
Ammonia is also a critical parameter in food and pharmacological analysis as well
as in clinical chemistry. Furthermore, there is a need for an alternative measuring
method for the rapid, easy, low-cost and ecologically beneficial determination of
ammonia, which could be fulfilled by an amperometric determination. The
conventional analytical technique for the detection of ammonia was developed using
Pt electrode at a high overpotential of >1V vs. SCE. In order to reduce the
overpotential for interferences avoided, a variety of electrode materials and
configurations, such as polypyrrole, polyaniline, polyaniline-Nafion, and zeolite
clinoptilolite have been reported. Nevertheless, a serious problem in using these
electrodes is the stability and the interference.
In this work, we utilized a new catalyst, copper(I) oxide, to develop a ammonia
chemical sensor and apply to creatinine biosensor. This purpose was to construct a
high sensitivity, less interferences, rapid and inexpensive analytical method by flow
injection analysis. In order to improve the sensitivity and detection limit of the
amperometric method, this copper(I) oxide modified electrode was used to catalyze
the oxidation and reduction of the ammonia in aqueous solution. And the creatinine
deiminase was paired for above ammonia sensor to construct this creatinine biosensor.
The cross-link type enzyme immobilization methods for this system were also been
discussed for the system. Characteristic of the proposed ammonia sensor and
creatinine biosensor for this work were studied.
2. Materials and methods
2.1. Chemicals and reagents
Creatinine deiminase (E.C. 3.5.4.21, Grade III from Microorganism), with specific
activity of 13.7Umg-1, was obtained from TOYOBO (Osaka, Japan). Bovine Serum
Albumin (96-99%), Creatinine (anhydrous) and Glutaraldehyde (50% Aqueous
Solution) were obtained from Sigma (St. Louis, USA). Copper(I) Oxide (97%),
Tetraethyl orthosilicate (98%) and Chitosan (medium molecular weight) were
obtained from Aldrich (Milwaukee, USA). All other chemicals and solvents used were
of analytical-reagent grade and were used as received. The buffer solutions were
prepared from sodium salts aqueous solution with the addition of an appropriate
amount of NaOH solution. Deionization-RO water prepared from Barnstead Easypure
Ro and Easypure UV/UF (Dubuque, IA, USA) with a resistance of 18.3MΩ-cm was
used for preparing the solution.
2.2. Apparatus
A three electrode system was used for the flow injection analysis and the cyclic
voltammetric(CV) studies. A piece of Pt wire or stainless flow cell body was used as
the counter electrode. A commercial Ag/AgCl (with 3M NaCl solution) electrode was
used as the reference electrode. And the glassy carbon electrodes with catalyst
modifiers were used as the working electrode. CV measurements and amperometric
FIA experiments were carried out with a model 750A electrochemical workstation
(CH Instruments, Austin, TX, USA) and a model CC-5 flow-through thin-layer
electrochemical cell (Bioanalytical System, West Lafayette, IN, USA) connected with
the above electrochemical analyzer. The FIA measurements were conducted with a
Cole-Parmer microprocessor pump drive (Vernon Hills, IN, USA), a Rheodyne 7125
injector with a 100 μL sample loop on the CC-5 flow device and the enzyme based
electrochemical detection system.
2.3. Design and fabrication of ammonia chemical sensor and creatinine biosensor
Design of the ammonia chemical sensor was carried out according to our
experiences in the past. The Copper(I) Oxide modified electrode was fabricated by the
weight ratio of 3:2:5 for Cu2O:carbon ink:cyclohexanone. A 3mg mixture was applied
onto a glassy carbon disk electrode (3mm diameter) and 40 oC air–dried in convection
oven to allow the solvent to evaporate.
The first part of creatinine biosensor fabrications was followed the same procedures
of ammonia chemical sensor on the down series partition of dual working electrode.
The second part of creatinine deiminase (CD) immobilization was cross-linked on the
up series partition of dual working electrode by a procedure similar to that reported in
the literature. The mixed solution for creatinine deiminase immobilization was
prepared by dissolving 5 units of CD in 10μL of pure water. A 2 % glutaraldehyde
(GA) solution was used as the cross-linking agent. A portion of 1μL of the prepared
CD solution was then mixed with 0.5μL of 2 % GA and 1μL of 1 % bovine serum
albumin (BSA) solution. After applying the prepared CD immobilization solution on
the bare glassy carbon electrode, it was allowed to 10 oC air–dried in incubator to
form a creatinine biosensor. The finished biosensor was stored at 4 oC in run buffer
solutionwhen not in use.
2.4. Analytical measurements
The chemical sensor and biosensor were first washed thoroughly with de-ionized
water, then set the electrodes in normal three electrode cell or flow-through thin-layer
electrochemical cell. For FIA, the sensors were equilibrated in blank buffer solution at
an optimized condition until the current became lower and constant. The
quantification of ammonia or creatinine was achieved by measuring the reduction
current from amperometric signals. All experiments were performed at room
temperature (25 oC).
3. Results and discussion
3.1. Electrochemical oxidation and reduction behaviors of ammonia on the Cu2O
modified electrode
Cyclic voltammograms of the Cu2O modified electrode under optimum static
condition in pH 10 phosphate buffer is shown in Fig. 1. An obvious peak at -0.4V (vs.
Ag/AgCl) in a cathodic sweep with one anodic shoulder at 0.15V were observed.
These two peaks indicate the redox transition of Cu(I)/Cu(0) and Cu(I)/Cu(II) when
the scan rate was higher than 50mV. Based on earlier reports, the cathodic peak
correspond to Cu2(I)O → Cu(0) and the anodic shoulder correspond to Cu2(I)O →
Cu(II)O. It is worth noting that Cu2O is a semiconductor in nature because Cu(I) has a
filled electronic configuration and is the key oxide of Cu in most electrocatalytic
applications.
The feasibility of using the Cu2O modified chemical sensor to measure ammonia
ion was investigated. The increasing responses, from 0 to -0.6V and -0.2 to 0.3V
showing five successive additions of 1mM ammonia chloride in the cyclic
voltammogram in 0.05M pH 10 phosphate buffer, are demonstrated in Fig. 2. These
successive cyclic voltammetric responses indicate the feasibility of utilizing this Cu2O
catalyst to develop an effective electrochemical scheme for ammonia ion
determination at potentials from 0.3 to -0.6V.
Due to the ratio of sensitivity over background, we optimized the oxidation
processes for the ammonia chemical sensor. The exact ammonia ion oxidation
processes on the Cu2O modified electrode could be demonstrated with a serial
electrochemical type reaction mechanism as shown in scheme1.
3.2. Optimizations for ammonia chemical sensor in FIA system
Hydrodynamic voltammograms for 5mM ammonia chloride under various poised
potentials (0.5 to -0.3 V), flow rate (0.15 to 1.5 mL/min) and sample loop (10 to 500
μL) are shown in Fig3. The optimized values were chosen based on the peak
resolution (calaulated by dividing the peak current by the peak width) and current
sensitivity. The poised potential of 0.15 V, flow rate of 0.5 mL/min and sample loop
of 20 μL gave the best results and these conditions were therefore used to construct
the calibration curve. It is important that the optimized poised potential of 0.15 V
matches exactly the redox potential of the Cu(I)/Cu(II) transition and thus
corresponds the reaction mechanism.
Fig.4 shows the calibration plot for ammonia ion under the optimized conditions.
The linear range is up to 10 mM and the sensitivity is 1334.2 nA mM-1 (r =0.997) with
a detection limit of 18.7 μM (S/N = 3). The RSD obtained for continuous injection (n
= 20) of 5mM ammonia chloride was 1.59 %, demonstrating the high precision of the
present method.
3.3. Optimizations for creatinine biosensor in FIA system
In order to understand the various immobilization method for this biosensor,
several cross-linked methods were studied and evaluated. In this experiment, we
chose BSA-glutaraldhyde, chitosan-glutaraldhyde and TEOS based sol-gel materials
for the immobilization of creatinine deiminase. Table 1 shows the results of these
three methods, we found the BSA-glutaraldhyde method had the best sensitivity and
reproducibility. In the following optimization, we used this cross-linked method for
the creatinine determination.
The pH effect on the response of the biosensor was studied by varying the pH of
the 0.05M phosphate buffer solutions from 6.00 to 11.00. Fig. 5 shows the plot of the
responses of this enzyme based biosensor to the injection of solutions of 0.5mM
creatinine at different pH values. As the highest current response was found for
solutions with a pH of 10.00, the 0.05M phosphate buffer solution with a pH of 10.00
was chosen as the optimum carrier solution for creatinine analysis. The effect of
sample loop of sample on the current response was also studied. In a sample loop
ranging from 10 to 500 μL, the current response decreased gradually as the sample
loop increased (from 100 to 500 μL). The optimized values were also chosen based on
the peak resolution and current sensitivity, a compromised value of 100 μL was
selected as the optimum sample loop volume for this experiment.
Fig.6 shows the calibration plot for creatinine under the optimized conditions. The
linear range is up to 5 mM and the sensitivity is 557 nA mM-1 (r =0.998) with a
detection limit of 25.0 μM (S/N = 3) and the response time of 45 seconds. The RSD
obtained for continuous injection (n = 20) of 0.5mM creatinine was 3.2 %,
demonstrating the high precision of the present method.
4. Conclusions
The creatinine deiminase based biosensor combined with FIA was successfully
demonstrated to be suitable for creatinine detection by effective catalytic oxidation
through Cu2O catalyst. The oxidation reaction mechanism was also be characterized.
Since the oxidation process on this biosensor is diffusion-controlled, the electrode
renewal is fast (45 s) with good repeatability (3.2%) and low signal-to-noise ratio.
The detection limit is 0.025 mM (S/N = 3). It is worth noting that the most
applications in clinical and pharmacological practice require ammonia ion
determination at neutral pH, and this FIA method can obviously fit this requirement
well. Furthermore, this biosensor can be constructed simply and inexpensively, and
thus offers an easy route to extension to real application.
Acknowledgement
The authors gratefully acknowledge financial support from the National Science
Council of the Republic of China and Department of Chemistry in Tamkang
University.
Fig. 1. Cu2O modified electrode under optimum condition in pH 10 phosphate buffer.
Fig. 2. Cyclic voltammograms of Cu2O modified electrode under optimum condition
in pH 10 phosphate buffer. There are five successive additions of 1mM
ammonia chloride in the in the optimum conditions.
Creatinine + H2O
Creatinine Deiminase
N-Methylhydantoin
Electrode
Cu2O (ox)
NH3
0.15V
-
e
Cu2O (red)
NH3 (ox)
vs. Ag/AgCl
Scheme 1. A serial electrochemical type reaction mechanism for creatinine biosensor.
(b)
11000
4000
10000
3000
9000
Current (nA)
5000
2000
1000
250
240
230
8000
220
7000
210
6000
0
200
5000
-1000
-400
-200
0
200
Operation Potential (mV)
400
600
-0.25
0.00
0.25
0.50
0.75
1.00
Flow Rate (mL/min)
1.25
1.50
1.75
Peak Resolution (nA/sec)
Current (nA)
(a)
(c)
280
14000
260
Current (nA)
220
12000
200
180
11000
160
140
10000
120
Peak Resolution (nA/sec)
240
13000
100
9000
80
0
100
200
300
400
500
Sample Loop (L)
Fig. 3. The optimized study of operation potential, flow rate and sample loop in
ammonia chemical sensor.
18000
16000
14000
Current (nA)
12000
10000
8000
Up to 10mM (R = 0.997)
6000
4000
2000
0
-2000
0
10
20
30
40
50
Ammonia Conc. (mM)
Fig. 4. The calibration plot for ammonia ion chemical sensor under the optimized
conditions.
Modified Method
Sensitivity
RSD
BSA
Cross-link
550 nA/mM
0.9 %
Chitosan
Cross-link
490 nA/mM
1.5 %
Sol-Gel
410 nA/mM
1.7 %
Table. 1. The results of the three cross-linked methods for creatinine biosensor in the
optimized conditions.
600
18
600
550
16
Current (nA)
Current (nA)
500
400
300
14
450
12
400
10
350
200
8
300
100
6
7
8
9
10
11
0
100
200
300
400
500
Sample Loop (L)
Buffer pH
Fig. 5. The optimized study of buffer pH and sample loop in creatinine biosensor.
4000
3500
Current (nA)
3000
2500
Up to 5 mM (R = 0.998)
2000
1500
1000
500
0
0
5
10
15
20
25
Creatinine Conc. (mM)
Fig. 6. The calibration plot for creatinine biosensor under the optimized conditions.
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