Clay Composite Modified Electrodes I: Voltammetric Method for Selective Analysis of Dopamine and Ascorbic Acid

Neurotransmitters aid in the transmission of signals from one neuron to another across synapses and play major roles in the stimulation of muscle fibers.  Dopamine (DA) is one of the neurotransmitters in the central nervous system.  Release of DA in the brain plays a key role in the regulations of pleasure and pain.  Abnormal DA levels are associated with serious diseases such as Schizophrenia and Parkinson’s.  Development of a highly sensitive and selective electrochemical sensor for dopamine detection in clinical and biological samples is essential since there is increasing demand for more reliable, rapid and simple methods.

Considering electrochemical detection, some interfering species co-exist in samples, of which ascorbic acid (AA) is the major one.  AA exists in relatively high concentrations as compared to DA.  The presence of high levels of AA in biological samples poses a problem for DA detection as both species have similar oxidation potentials.  Another problem is electrode fouling due to adsorption of species onto the electrode surface, which can be prevented by modifying the electrode surface with a polymeric membrane. 

This work aims to eliminate both problems by employing glassy carbon electrode that is modified with clay composite.  The goal is to use clay composite to exclude AA while enhancing DA detection.  Clay film serves as the polymeric membrane as clays are able to form membrane-like films.  Clays are naturally occurring and are abundant, nontoxic, much more stable and cheaper than synthetic membranes.  Clay film on an electrode surface provides important functions such as charge-exclusion, immobilization of species, preconcentration, catalysis of electrochemical reactions, and electron transfer enhancement. 

A suite of experiments were performed to analyze how DA and AA behave at the bare electrode (BE), clay modified electrode (CME), glycerol-clay modified electrode (GCME), glycerol-nafion-clay modified electrode (GNCME), and glycerol-octanohydroxamate-clay modified electrode (GOHATCME).  Four voltammetric techniques were used, namely Cyclic Voltammetry (CV), Square-Wave Voltammetry (SWV), Differential-Pulse Voltammetry (DPV), and Differential Normal-Pulse Voltammetry (DNPV).  Experiments were performed at the physiological pH of 7.4 and with an instrument sensitivity of 1.00 x 10^-6A/V.

Cracks are frequently observed in CME films after air drying, hence the addition of glycerol to make GCME, which are free of cracks.  Glycerol was chosen because it is inert and nonvolatile.  Different percentages (v/v) of glycerol in the clay suspension were investigated with 0.050 mM DA, using cyclic voltammetric potential sweep between -0.40 V and 0.40 V at 50 mV/s.  The highest oxidation peak current is observed with 5% (v/v) glycerol.  Comparing the 5% GCME to CME (with no cracks), no significant differences were observed in the cyclic voltammograms, implying that the addition of glycerol has no effect on DA signals.  Subsequent experiments were performed with 5% glycerol.  Similar studies with the addition of nafion reveal that oxidation peak current decreases with increasing percent nafion.  This implies that GCME performs best compared to all nafion percentages in GNCME.  Nafion was chosen as it is a well-known polymeric membrane. 

Calibration curves with CV signals produce linear ranges of 1.0 µM – 250 µM and 10 µM – 750 µM for DA and AA, respectively, at the stated sensitivity.  The DA oxidation peak potentials at BE and GCME are around 0.25 V and 0.26 V versus Ag/AgCl, respectively.  Similarly, the AA oxidation peak potentials at BE and GCME are around 0.33 V and 0.32 V versus Ag/AgCl, respectively.  For a mixture of DA and AA in solution, a single oxidation peak is observed around 0.27 V and 0.23 V at the BE and GCME, respectively.  A slightly positive potential is seen at the GCME, indicating a catalytic effect from the clay film.  The peak currents for DA at GCME are higher than those at BE.  On the other hand, the peak currents for AA at GCME are lower than those at BE.  This is evidenced by the fact that at pH of 7.4, AA exists in its anionic form (pK_a = 4.10) while DA exists in the cationic form (pK_a= 8.87).  Most clay layer surfaces and edges are negatively charged at this pH so are expected to readily attract the cationic DA into the interlayers and repel the anionic AA.  This enhances DA detection while diminishing AA detection.  However, the exclusion effect is not efficient using only clay, which calls for the incorporation of sodium octanohydroxamate to form clay composite.   

Analyses with the GOHATCME modified electrode were carried out with 25 µM DA and 250 µM AA, using all the four voltammetric techniques mentioned earlier.  Although the DA peak currents at GOHATCME are reduced compared to the peak currents at the GCME, they are still comparable to those of AA at the GCME (refer Table 1).  For AA, no peaks are seen at the GOHATCME.  This is true for all four voltammetric techniques.  This implies that the GOHATCME efficiently discriminates against AA detection.   

Mixtures of DA and AA exhibit relatively high peak currents at BE, indicating contributions from both DA and AA.  On the other hand, the GOHATCME exhibits peak currents that are comparable to those produced by only DA, confirming the exclusion of AA (refer Table 1).  SWV and DPV give very reproducible results.  Artificially high currents are, however, seen with CV in some cases, as seen in Table 1.  The cause is still under investigation.   

Stability and durability of the GOHATCME were investigated.  Films were left uncovered for one, two, three, and four weeks.  Afterwards, the films were scanned with DA daily for five days and then with AA on the sixth day.  Reproducible results are obtained for the five days of scanning with DA.  Interestingly, the films are still able to exclude AA despite five times of scanning with DA, indicating a highly stable and effective GOHATCME.  

 

 

Technique	25 µM DA		250 µM AA		25 µM DA + 250 µM AA
	GCME	GOHATCME	GCME	GOHATCME	GCME	GOHATCME
CV	1.083	0.742	1.140	-----	2.406	2.550
SWV	1.682	0.848	0.824	-----	2.483	0.947
DPV	0.867	0.496	0.422	-----	1.281	0.696
DNPV	2.811	1.623	1.115	-----	3.120	1.801

Table 1.Peak currents (µA) of DA and AA at GCME and GOHATCME. Results are averages of three replicates.

REFERENCES

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