Abstract
The self-assembly of cyclohexylamine dithiocarbamate (C6DTC) on gold (Au) and gold nanoparticles (AuNPs) was studied electrochemically using cyclic voltammetry (CV). Cyclic voltammetry was used to study the system at the electrode surface of (C6DTC/Au) and (C6DTC/AuNPs). The application of the resulting chemically modified surface as a selective sensor for cysteine, among other amino acids, was investigated. Linear oxidative desorption technique was used to characterize the modified electrode that consists of the self-assembled monolayer of cyclohexylamine dithiocarbamate on gold nanoparticles deposited on Au electrode (C6DTC/AuNPs/Au). The results showed an enhancement in the oxidation peak of cysteine on the modified electrode and hence a greater sensitivity.
1. Introduction
Dithiocarbamate derivatives have been extensively studied due to their antimicrobial activity [1] and the various applications in industrial and chemical processes, such as vulcanization accelerators [2], flotation agents, fungicides [3], pesticides [4], and corrosion inhibitors [5]. One of the most interesting properties of dithiocarbamates is their strong metal-binding ability and the ease to form self-assembled monolayers (SAMs) on gold surfaces making them useful to many applications. The structural features of SAMs on gold surfaces are dictated by two major factors: the sulfur gold interaction and the organic interchain interaction. It has been shown that both sulfur atoms in the headgroup of a dithiocarbamate chemically bond to the surface of gold [6].
Owing to their large surface area and biocompatibility, many nanostructured materials represent a powerful detection platform for a broad range of sensors including biological, electrochemical, gas, optical, pH, and molecular orientation. The sensing devices may include individual nanostructured sensors, multi-nanostructured sensors, nanostructured film sensors, and others. These nanosensors have a number of key features, including high sensitivity, exquisite selectivity, fast response and recovery, and potential for integration of addressable arrays on a massive scale, which set them apart from other sensors technologies [7, 8]. Many researchers agree that more development is needed at the molecular level of detection to improve miniature and nanosensor devices.
Cysteine (2-amino-3-mercaptopropoinic acid) is a biologically important substance in living systems and its oxidized form (cysteine) is utilized as a model of the thiol group in proteins. Cysteine, mainly the L-enantiomer, is a precursor in the food, pharmaceutical, and personal care industries. One of the largest applications is the production of flavors. For example, L-cysteine is used as a processing aid for baking while the reaction of cysteine with sugars yields meat flavors [9].
Very little research targeted the selective sensing of cysteine due to difficulties arising from the structural similarity with α-amino acids and peptides (carboxylic and amino groups). The need to improve the sensing selectivity of cysteine is therefore obvious. Dithiocarbamates attached to gold nanoparticles have already been tested in the electrochemical sensing for dopamine and ascorbic acid [10].
In this paper, cyclohexylamine dithiocarbamate is self-assembled onto gold (Au) and nanogold (gold electrode with adsorbed gold nanoparticles; AuNPs/Au) and the chemically modified electrode was tested as a sensor for several aminoacids including cysteine.
2. Experimental
2.1. Chemicals
Cyclohexylamine was purchased from Aldrich (99%), sodium hydroxide NaOH (Aldrich), carbon disulfide CS2 (97%) and ethanol, absolute (99%), sulfuric acid H2SO4 (98%), potassium ferrocyanide (K3Fe(CN)6) (99.0%), potassium chloride (99.9%), HAuCl4 (99.9%) and ammonium peroxydisulfate (NH4)2S2O8 (99%) were all of analytical grade and used as received. All solutions were prepared with triply distilled water.
2.2. Instrumentation
Cyclic voltammetry experiments were carried out with a VoltaLab PGZ 100 potentiostat in a double-wall three-electrode glass cell under nitrogen (Figure 1). Solutions were degassed with ultrapure nitrogen for about 20 minutes before electrochemical measurements. The working electrode (WE) was a gold polycrystalline electrode embedded in a glass tube with a surface diameter of 3.0 mm. Prior to measurements, the surface of the working electrode was carefully polished with alumina slurry, rinsed several times, and then sonicated for about 1 minute. Afterwards, the electrode was electrochemically cleaned in 0.5 M H2SO4 until a stable and acceptable gold oxidation/reduction CV was obtained.
All reported potential values are versus the SCE reference electrode and all measurements were carried out at around 20°C. A platinum wire was used as the auxiliary electrode. Solutions’ pH values were measured with a Hanna calibrated digital pH meter (Switzerland).
All glassware for electrochemical experiments were carefully cleaned by immersion in a solution of concentrated sulfuric acid containing ammonium peroxydisulfate (NH4)2S2O8 for 10 hours followed by copious rinsing with deionised and triply-distilled water.
2.3. Synthesis of Cyclohexyl Dithiocarbamate (C6DTC)
The sodium salt of cyclohexyl dithiocarbamate (C6DTC) was synthesized as reported in the literature with modification [11]. To summarize, the C6DTC was prepared by reacting cyclohexylamine with an equimolar ratio of CS2 and NaOH solutions in an ice bath. To 0.1 M of NaOH, dissolved in minimum water, cyclohexylamine in 50 mL of ethanol (0.1 M) was added. After thorough stirring, an ethanolic solution of 0.1 M CS2 was added drop wise. The reaction was stirred for 1 hour at 0–5°C followed by another hour at ambient temperature (~22°C). The solid product was filtered and washed with cold water/ethanol solution giving a white product which is then recrystallised from ethanol (yield = 60%, m.p ~ 130°C). IR/cm−1: 3350 (–OH), 3200 (–NH), 1480 (C–N), and 990 (C=S).
2.4. Preparation of the Nanogold Electrode (AuNPs/Au)
The Au electrode surface was freshly polished prior to use with 0.05 μm α-Al2O3 slurry, and rigorously rinsed with doubly distilled water. The electrode was successively ultrasonically rinsed with 98% H2SO4 and 95% ethanol and distilled water for 10minutes, dried with nitrogen gas, and then electrochemically cycled in 1.0 M H2SO4 until a stable gold oxide formation/reduction CV was obtained (Figure 2(a)). The electrode was then immersed into 3 mM HAuCl4 solution containing 1.0 M H2SO4 solution as an electrolyte, and electrochemical deposition of gold nanoparticles was conducted by the electrochemical scanning the electrode between a potential of −200 mV and −190 mV (versus SCE) for a selected number of times at a scan rate of 10 mV/s. The electrode surface showed a very visible light red color indicating the presence of gold nanoparticles as shown in the photograph in Figure 1. We call the resulting surface the nanogold electrode (AuNPs/Au). After experiments, the electrode was regenerated by soaking it in piranha solution (a mixture of 3 : 7, v/v of 30% H2O2 and 98% H2SO4) overnight and then rinsing it with 95% ethanol and distilled water followed by drying in a stream of nitrogen gas.
2.5. Preparation of C6DTC-SAMs on the Gold Electrode (C6DTC/Au)
The electrode was initially cleaned in the same way described in 2.4 above. The C6DTC/Au were prepared by immersing the gold electrode in freshly prepared aqueous solutions of 1.0 mM of cyclohexyl dithiocarbamate (C6DTC) solution at ambient temperature for 6 hours followed by another 6 hours in triply distilled water to remove any physically adsorbed molecules. The electrode was then rinsed with water and dried in a stream of nitrogen gas.
2.6. Preparation of the C6DTC-Modified Nanogold Electrode (C6DTC/AuNPs/Au)
The electrode was cleaned and nanogold was deposited as described in 2.4 above. The self-assembly of C6DTC was accomplished with the immersion of the electrode into a freshly prepared 1 mM C6DTC aqueous solution for 24h in the dark at room temperature. The modified electrode was then taken out, soaked in triply distilled water for 6 h, and finally dried in a stream of nitrogen gas.
3. Results and Discussion
This section is divided into three parts. In the first part, the (C6DTC/Au) and (C6DTC/AuNPs/Au) are investigated using cyclic voltammetry. The redox system of is studied on both electrodes using cyclic voltammetry in the second part. Finally, the C6DTC-modified nanogold electrode, C6DTC/AuNPs/Au, is tested for the electrooxidation of cysteine.
3.1. Cyclic Voltammetry of the Electrode Surface of (C6DTC/Au) and (C6DTC/AuNPs/Au)
Figure 2 shows cyclic voltammograms (CVs) of the bare gold electrode and nanogold electrodes in deoxygenated 1.0 M H2SO4 solution. The nanogold particles were deposited at 5, 10, and 15 minutes. We noticed that further deposition (over 15 minutes) resulted in CVs different from that for the polycrystalline gold mainly with extra and sharper peaks. We chose the 5-minute modification time in all experiments to eliminate any complications that may result from producing different surfaces at different deposition times. We also noticed that more deposition deepens the color of the electrode and makes it more difficult to regenerate to a clean gold electrode afterwards. Previous studies showed that small deposition times give reasonable and acceptable modification of the gold electrode with gold nanoparticles [12]. The relative surface area of the electrode could be estimated from the coulombic integration of the reductive waves of gold oxide [13]. By analyzing Figure 2, the ratio of the surface area of the nanogold electrode to that of the bare gold electrode is around 1.1 : 1 for the 5-minute deposition time and 1.8 : 1 for the 15-minute deposition time. Studying the electrochemical behavior of the electrode as a function of gold nanoparticles deposition time, we conclude that there is no significant change in the main reduction peak potential as a function of gold nanoparticles deposition time.
Figure 3 shows the cyclic voltammograms of bare Au and C6DTC/Au electrodes in 0.5 M H2SO4 solution at a potential scan rate of 50 mV/s. A more diluted H2SO4 solution was used here and onwards to increase the pH value and therefore avoid any interference with the organic molecule: C6DTC. When the potential was scanned above +800 mV, a C6DTC desorption peak was noticed. Therefore, all following experiments involving the C6DTC molecule were limited to a maximum potential of +600 mV. We found that when the scanning is continued to higher potentials (>+800 mV), the peaks’ intensity decreased and the CV became very similar to the bare gold electrode CV (not shown here).
Figure 4 shows the cyclic voltammograms of AuNPs and C6DTC/AuNPs electrodes in 0.5 M H2SO4 at a potential scan rate of 50 mV/s. The oxidation behavior is similar to that of the bare polycrystalline gold except that the C6DTC desorption peak is much broader. In a similar manner to the bare surface, all following experiments were performed at potential values under +600 mV.
3.2. Cyclic Voltammetry of Fe/Fe at the Electrode Surface of (C6DTC/Au) and (C6DTC/AuNPs)
Figure 5 shows the cyclic voltammograms of bare Au and C6DTC/Au electrodes in 5 mM potassium ferrocyanide with 0.1 M KCl as the supporting electrolyte at a potential scan rate of 50 mV/s. We notice from the figure that the bare Au electrode (Figure 5(a)) shows a reversible voltammogram for the redox couple indicating that the electron transfer reaction is mainly diffusion controlled. On the other hand, the absence of major peaks in the CVs of the C6DTC/Au electrode (Figure 5(b)) shows that the redox reaction has been somehow inhibited.
As illustrated in Figure 5(b), the CV exhibits perfect blocking behavior which means that a highly ordered, compact monolayer is formed on the Au surface. This is also proven by the EIS measurements where the modified interface displays a much higher resistance compared to that of the bare surface [14].
Figure 6 shows the cyclic voltammograms obtained for the nanogold modified electrode in 5 mM potassium ferrocyanide in 0.1 M KCl as the supporting electrolyte at a potential scan rate of 50 mV/s.
The bare Au electrode witnessed a pair of well-defined redox peaks with the anodic and cathodic peak potential of 0.236 V and 0.124 V, respectively, and a peak potential difference of 112 mV (curve a). These peaks are attributed to the redox behavior of the [Fe] electrochemical system. The self-assembly of C6DTC monolayer on electrode surface induced a small decrease of peak current, which was invoked by the diffusion inhibition of [Fe] to the electrode surface (curve c). The modification with gold nanoparticles increased the effective electrode surface area and enhanced the rate of electron transfer, which was evidenced by a slight increase in the voltammetric responses of [Fe] compared to that of the bare electrode (curve b).
3.3. Linear Oxidative Desorption of Cysteine on the C6DTC-Modified Nanogold Electrode
The linear oxidative desorption of an aqueous solution of 1 mM cysteine was performed on bare gold electrode (Figure 7(a)). No oxidation peaks were detected under a potential of 600 mV. Upon the deposition of gold nanoparticles on Au producing the AuNPs electrode, a slight increase in the measured current was seen in the region of 300 mV and above (Figure 7(b)) compared to the bare Au electrode (Figure 7(a)). C6DTC was assembled on both bare Au and AuNPs electrodes. The linear oxidative desorption curve was measured. For the C6DTC/Au, a desorption broad peak started to appear below 200 mV and peaked at around 500 mV (Figure 7(c)). Some C6DTC desorption might be attributed to this curve in addition to the oxidation of cysteine. The curve in Figure 7(d) is a linear desorption of 1 mM cysteine on the C6DTC-modified nanogold electrode (C6DTC/AuNPs/Au). The curve shows the presence of two major peaks in the regions −100 to −50 mV and 300 to 400 mV. It is clear that the modified electrode has better sensitivity compared to the other electrodes. The 400 mV peak potential is more negative than that in the C6DTC/Au electrode which indicates that nanogold plays an important role in lowering the peak potential. The two peaks in curve d can be employed as a basis in the fabrication of a selective sensor for cysteine in aqueous solutions due to their high current values. In addition, the combination of gold nanoparticle and C6DTC modification clearly increased the bonding of C6DTC to the gold surface and enhanced the self-assembly stability (compare curve d with c). When a C6DTC-modified nanogold electrode was stored in a buffer solution (pH 7.0) for at least 1month at ~5°C, the electrode retained about 95% of its initial response provided that the potential is kept below 600 mV.
4. Conclusions
An electrochemical method for sensing cysteine has been tested. It consists of a self-assembled monolayer of cyclohexylamine dithiocarbamate on gold nanoparticles deposited on Au electrode. Cyclic voltammetry (CV) and linear oxidative desorption were used to characterize the modified electrode. We also conclude that gold nanoparticle modification increased the bonding of C6DTC to the gold surface and enhanced its stability.
Acknowledgments
The authors thank the Faculty of Scientific Research at the University of Jordan for their financial support.