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This experiment was done with the aim of preparing a conducting polypyrrole film that contains an immobilised enzyme, characterizing the film, and then using the film as a biosensor.
Electrochemical oxidation of polyanaline, polypyrole or polythiophene molecules frequently result to the formation of an organic polymer film that is electrically active when used at the surface of an electrode. The polymer film is characterized by good adhesion in addition to electrical contact. One important feature of an electropolymerization reaction is that this type of reaction normally takes place in an electrochemical stoichiometry process, which allows for characterization of the reactions involved in the formation of the film.
These electro-active conducting polymers and can be converted to reducing state by applying an electrical potential. Conducting polymers are gaining popularity in making biosensors with the role of immobilizing active species at the electrode. They have also been used in immobilising biological species such as receptors, cells, DNA and enzymes, and also in electronic nose devices .
This lab experiment involves immobilisation of glucose oxidase in an electrical conducting polypyrole film on an electrode, then using the same as a glucose sensor. Cyclic voltammetry is the technique that has been used in testing electrical performance in these electrochemical reactions. The current produced from the reaction is dependent on the rate of diffusion from the bulk solution to the surface of the electrode. The anodic and the cathodic peak current are equal and depends on the square root of the rate of scan. Scanning starts where the current plot is zero and increases to the peak as the voltage sweeps from the left to the right.
Please refer to the lab manual, NANO8702 “Frontiers of Nanotechnology &GE 2011, PP. 24-29”.
Results and Discussion
Polypyrole is a soluble polymer that is frequently used as a conducing polymer and can be easily converted from oxidizing to reducing state when electrical potential is applied to it . Addition of functional groups on the surface of the polymer to control properties such as hydrophobicity. The conducting polymers in biosensors act as a matrix that immobilises the active species at the electrode to improve the accuracy and sensitivity of analyte detection, as well as the role of a signal transducer. Glucose oxidase was immobilised in a conducting film of polypyrole on an electrode, then using it as a biosensor. Cyclic voltammogram technique was used to test electrode performance in a ferricyanide solution. The figures 1 to 4 below show the cyclic voltammetry of a ferricyanide on a platinum electrode produced at 20mV/s, 50mV/s, 100mV/s and 200mV/s scan rates respectively.
The cyclic voltammogram was performed on ferricyanide in order to test the performance of the electrode. The Fe2+/Fe3+ is a single electron couple redox reaction as shown in the equation below:
Fe2+ Fe3+ + e-
The current is determined by the rate of transport of species from the ferricyanide solution to the surface of the electrodes, and not the rate of transfer of electrons between the electrode and the species. Therefore, the peak position does not depend on the scan rate. The difference between the anodic and cathodic peak potentials is equivalent to the potential produced for a single electron transfer.
It can be clearly observed from the plots of potential versus current that as the scan rate increases, the peak is approached.
On measuring the geometric area, it was found to be 0.0314. In order to determine the surface area of the electrode, the Randles-Sevcik equation was used. The equation states:
– peak current at 25 Co
A – surface area of the electrode (
n – the number of electrons in the reaction
C – bulk concentration of active species
D – the coefficient of diffusion (
V = 20 mv/s, 50 mv/s, 100 mv/s and 200 mv/s respectively
For 20 mv/s:
The Randles-Sevcik equation was repeated for the other values of scan rates (50mv/s, 100mv/s and 200mv/s) and the results recorded in the table below:
Square root of scan rate(mv/s) Peak current(A)
When the graph of peak current (Ip) was plotted against the square root of various points of scan rate.
From the equation of the linear line (y=0.000144807 + 2E-07), which is in the form , the area of the electrode can be determined. This area of electrode is the coefficient of =A= 0.0001448 cm2.
When oxidative potential is applied, the polymer is deposited on the surface of the electrode to form a layer. By varying the time of deposition, the quality of the layer can be suitably reproduced. The electrode appeared to be covered with a light brown film. The morphology of the polymer determine sensor characteristics such as limit of detection and selectivity.
The charge increases from nearly zero to the maximum value (1.158mC) when it was measured over time period of seconds. Figures 7 to 10 below show the cyclic voltammograms for an electrode with polypyrole coating at scan rates of 20mV/s, 50mV/s, 100mV/s and 200mV/s respectively.
One important observation that can be made is that the peak current (Ip) and the potential values for the electrode coated with polypyrole are higher compared to those of uncoated electrode. This can be attributed to the larger surface area provided by the polypyrole coat that forms a thick layer on the electrode, protecting it from reaction effects, mainly corrosion, by increasing its sensitivity .
In the analysis of glucose, no proper results were obtained from the lab and therefore, no analysis can be done.
Analysis of Results
Question 1: How do the geometric and experimentally determined electrode’s area compare and what other factors may affect the apparent electrode surface area?
Geometric area as measured by ruler = 0.0314 cm2
Geometric area determined experimentally = 0.0001448 cm2
The geometric electrode area of measured and experimentally determined differ slightly (figure 1). The geometric area is larger than experimentally determined area. This difference can be explained by the effect of reaction on the electrode. Factors that may affect the appearance of the electrode surface area include the concentration of the polymers and enzymes, and the roughness of the surface.
Question 2: Which area did you use for the subsequent calculations and why?
The measured electrode area was used for all scan rates. This is because experimentally determined electrode area is propagated with experimental errors.
Question 3, 4 & 7:
For question 3, 4 & 7, the results obtained for the part of glucose analysis were not correct and therefore, not able to correctly answer these parts.
Question 5: If a different amount of enzyme were incorporated into the polymer electrode, how would the results differ?
The concentration of enzyme in the polymer would affect the rate of reaction, which would be slower if the amount of enzyme is reduced, and increase if the amount of enzyme is increased. This would affect the amount of current produced. The higher the rate of reaction, the more current is produced.
Question 6: If a lower potential were chosen for the TB analysis, what effect would it have on the plot? Would a lower potential be advantageous? Explain why/why not.
If a lower potential was considered, less current would be produced. The advantage of a lower potential is that the sensitivity of the biosensor would improve, leading to further improvement in analyte detection .
Question 8: Calculate Km and Vmax by performing a Michaelis-Menten analysis (hint: start by inverting equation 4).
Km and Vmax are constants that can be obtained from the calibration curve of the rate of reaction vs. concentration or calculated from the curve using Michaelis-Menten equation:
From the literature, the values provided for these constants are:
Km=1.5 mM, and Vmax= 1 nm/s.
Question 9: How does this biosensor compare to other you have used or researched?
The biosensor is simple and less expensive to make and its performance can be easily tested compared to TNT biosensor. The TNT biosensor has a limited detection with a catalytic response that depends on the concentration of the solution. It also has high flexibility and therefore, highly adaptable compared to silicon sensors.
Question 10: If you were able to repeat the experiment several times what variables would change to optimise the sensor?
The concentration of glucose oxidase affects the performance of the sensor, and therefore, by varying its concentration, the performance of the biosensor can be optimized. Another variable would be the area of the electrode.
Question 11: Discuss the factors that limit the sensors dynamic range and relative sensitivity, the concentration range and errors associate with the biosensor. Also discuss the reproducibility of the technique?
The sensitivity of the biosensor is affected by the background current in the buffer solution. To eliminate this effect, the buffer solution was preconditioned to drain the background current. Other factors include the surface area of the electrode and concentration of the glucose. These factors affect the rate of reaction on the electrode and the amount of current produced, and therefore, the dynamic range and the relative sensitivity. The CV technique is simple to adapt and can be easily used in the production of conducting polymers with a wide applications in electronics and electrochemistry .
Polymer films in an oxidized state may be used to drive oxidation reactions. The redox reactions only occur at the surface of the electrode with moderate rate of transfer of electrons, and no absorption or adsorption. Conductive polymers are suitable for making biosensors since they are highly flexible and adaptable, and simple to make compared to silicon sensors.
Buer, Mark Anthony. Surface and Near Surface Analysis of a Glucose Biosensor. Australia: Flinders University of South Australia, School of Chemistry, Physics and Earth Sciences 2003,pp.51-76.
Inzelt, György. Conducting Polymers: A New Era in Electrochemistry. Amsterdam: Springer Science & Business Media, 2012.pp.151-160.
Serge Cosnier, Arkady Karyakin. Electropolymerization: Concepts, Materials and Applications. U.K: John Wiley & Sons 2011,pp.157-163.
Sadik, Omowunmi A., et al. "Electropolymerized Conducting Polymers as Glucose Sensors." Journal of Chemical Education 76.7 (1999): 967-970. journal. 25 August 2015.
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