CHI 600B Electrochemical Analyzer (USA) with a three-electrode cell assembly was used for electrochemical studies. Electrochemical experiments were carried out under a blanket of Nitrogen gas after passing the gas through the solution for 10 minutes. The electrode potential values were reported with respect to the normal hydrogen electrode, NHE. The working electrode is Glassy Carbon (GC) disc, reference electrode is Ag-AgCl (3M NaCl) and Sodium Nitrate (0.1M) is the supporting electrolyte. In the Osteryoung Square Wave Voltammetry (OSWV) experiments, the square wave amplitude is 25 mV, the frequency is 15 Hz and the potential height for base staircase wave front is 4 mV. Prior to every experiment the GC electrode was polished firmly on micro- cloth using fine 0.05 μM alumina powders followed by sonication for 2-3 minutes in double distilled water and then rinsed thoroughly with water.

5 g of the NaMMT was dissolved in 500 mL hot water to obtain a uniform dispersion. 2 g of CTAB was dissolved in 200 mL double distilled water. The two solutions were mixed together and stirred vigorously for 6 h at 60°C using a hot plate magnetic stirrer. The solution was centrifuged using high speed REMI 24 centrifuge at 6000 rpm for 5 min. The modified MMT suspension was washed repeatedly using double distilled water and filtrated, until no precipitate appeared when the filtrate was titrated with a 0.1 mol/L AgNO₃ solution. The filtered cakes were dried in a vacuum oven at 80°C for 12 h, and ground using mortar to obtain fine powder of the organo-modified nanoclay 1. Then, a CTAB-NaMMT clay suspension was prepared by taking 0.135g clay in 40 ml MeOH. Added 0.9g (6 mmol) 4NA to the suspension and stirred well for 2 hours. Allowed another 12 hours to diffuse 4NA. Centrifuge the mixture at 6000 rpm for 5 min, washed repeatedly with MeOH and dried in oven at 50oC. The intercalated clay is now designated as 4NA-CTAB-NaMMT. The modified clay was then characterized by XRD, FTIR and TGA (Supplementary data).

A suspension of the modified clay was prepared by mixing 0.1g of the clay in 10mL dichloromethane. 1.0 μL of the suspension was placed on the tip of a pre-cleaned GC electrode surface using a Hamilton micro-syringe and allowed to dry under nitrogen environment. 1.0 μL of a styrene solution (prepared by dissolving 0.1 g styrene in 10 mL of dichloromethane) was dropped over the above modified electrode and again dried under the blanket of nitrogen for 10 minutes. The modified electrode now has been designated as 4NACME.

It is clear that DA sensor in physiological application must have high sensitivity at pH 7.4. Therefore, we investigated the effect of pH on the modified electrode. The experimental results showed that both peak current and peak potential of the 4NACME changed with the pH value of the bulk solution (Supplementary data).

By comparing both the curve it may be concluded that within pH range 7-8 the peak current reaches maximum value with minimum and consistent Ep value. But when the pH of the immediate environment was changed to strong basic condition of pH 9 and 10, the peak potential shifted negatively a lot from the consistent value of -0.096 to approximately -0.25V. Hence it is proved that the 4NACME showed the highest sensitivity at the lowest oxidation potential value in physiological environment (pH 7.4). It is promising as a sensor for real in vivo measurement of DA. The pH dependency of the peak potential indicates that protonation/deprotonation is taking part in the charge transfer process.

Square wave voltammogram (SWV) of the 4NACME in PBS of pH 7.4 is demonstrated in supplementary data.

It shows an oxidation peak at potential -0.076V with peak current 1.925 × 10^-6 A. Cyclic voltammogram of the modified electrode also shows a pair of redox peaks with redox potential value -0.090 V ± 0.005 V (not shown). The redox peak currents found to increase linearly with scan rates (Supplementary inset). This linearity tells the electrochemical reversibility of the modified electrode.

SWV of 4NACME were also conducted for 200nM DA, 360nM UA and 960nM AA separately in PBS of pH 7.4 with 6 s preconcentration time at open circuit (Supplementary data).

As shown in supplementary data the peak potential for DA was found at 0.620V with peak current 0.879 × 10^-6­­­A, peak potential of UA was found at +0.024V with peak current 2.764 × 10^-6A and peak potential of AA was found at -0.320V with peak current 1.073 × 10^-6A.

It can now be expected that the problem for the electrochemical detection of DA due to overlapped voltammetric response of coexisted species AA and UA can be solved with the 4NACME. We tried to record the SWV response of 200 nM DA in presence of 360 nM UA and 360 nM UA in presence of 960 nM AA (Figure 1). Two peaks were observed with peak potential 0.595 V and 0.020 V which were confirmed to be for DA and UA by comparing with individual DA & UA peak as shown in supplementary data. Similarly in Figure 1 also two peaks observed at 0.034V and at -0.380V were confirmed for UA and AA respectively by comparing with individual UA & AA peaks as shown in supplementary data.

It is well known that AA widely coexists with DA and UA in biological sample. Therefore, eliminating AA interference is an important target for any DA and UA analytical method. A comparison on the performance of the 4NACME to a mixture of 200nM DA, 360nM UA and 960nM AA in PBS of pH 7.4; over the bare GCE are shown in Figure 2.

As evident from the Figure 2, in case of 4NACME three separated peaks were observed at potential value +0.604, +0.060 and -0.375V with peak current value 2.81×10^-6A, 6.049×10^-6A and 2.622×10^-6A respectively (I_UA/I_DA = 2.15, I_UA/I_AA = 2.30). The peaks at +0.604V, +0.060V and -0.375V was confirmed to be for DA, UA and AA by comparing their individual peaks (figures in supplementary data). After modification of the bare GCE, the oxidation potential for UA and AA shifted to negative potential and three well-resolved anodic peaks were present at the 4NACME. These results were again confirmed by taking CV of 4NACME in PBS of pH 7.4 containing mixture of 200nM DA, 360nM UA and 960nM AA (Figure 3). It is well known that the reversibility of the electrochemical reaction of DA, UA and AA at bare GCE is slow, indicating the sluggish electron transfer kinetics of these compounds at bare GCE, which may be related to the electrode fouling caused by the deposition of these compounds and their oxidation products on the electrode surface 22.

Figure 3

In Figure 3 three couples are observed at E_1/2 values +0.600V, +0.045V and -0.365V which were confirmed to be for DA, UA and AA respectively by comparing their individual peaks (supplementary data). The anodic and cathodic peak potentials of DA appear at 0.613V and 0.587V respectively; with potential peak separation 0.026V which indicate the reversibility of DA at 4NACME (It also indicates the involvement of two electrons in DA oxidation at 4NACME). The peak potentials for oxidation of UA occur at 0.073V and 0.017V with peak separation value 0.056V which again indicate the reversibility of the reaction. Similarly for AA oxidation at 4NACME surface we observe anodic and cathodic peak potentials at -0.328V and -0.401V respectively with peak separation value 0.073V indicating reversibility.

The reversibility of electrochemical reaction of DA, UA and AA was also demonstrated by plotting peak current versus scan rate in their respective individual CV experiments (results not shown). These results demonstrate that 4NACME not only catalyses the oxidation of DA, UA and AA, but also dramatically enlarge the peak separation among DA, UA and AA in their mixture. The increase in separation of peak potentials, and the enhanced sensitivity, allows simultaneous determination of DA, UA and AA.