Carbon composite micro- and nano-tubes-based electrodes for detection of nucleic acids
© Prasek et al; licensee Springer. 2011
Received: 27 October 2010
Accepted: 16 May 2011
Published: 16 May 2011
The first aim of this study was to fabricate vertically aligned multiwalled carbon nanotubes (MWCNTs). MWCNTs were successfully prepared by using plasma enhanced chemical vapour deposition. Further, three carbon composite electrodes with different content of carbon particles with various shapes and sizes were prepared and tested on measuring of nucleic acids. The dependences of adenine peak height on the concentration of nucleic acid sample were measured. Carbon composite electrode prepared from a mixture of glassy and spherical carbon powder and MWCNTs had the highest sensitivity to nucleic acids. Other interesting result is the fact that we were able to distinguish signals for all bases using this electrode.
In the last two decades, nanomaterials in the form of nanotubes and nanowires have begun to be reported as promising materials for wide field of applications [1, 2]. Such materials could be also used for fabrication of miniaturized electrodes. The nanostructured electrodes could be fabricated using several techniques. The easiest fabrication technique is to use a mixture of nanomaterial as filler with a suitable vehicle, which could be deposited on the electrode substrate using screen-printing, drop-coating, dip-coating, spraying, etc. [3, 4]. The disadvantage of these nanocomposition-based electrodes is the irreproducible electrode surface with undefined active electrode area. The reproducible nanostructured electrode surface could be fabricated using lithography as a common tool for microelectronics devices implementation , anodization process for nanorods or nanotubes creation [6, 7]. One of these techniques is the creation of vertically aligned multiwalled carbon nanotubes (MWCNTs) grown directly on the surface using chemical vapour deposition (CVD) . The aim of this study was to fabricate MWCNTs and further to test the particles as a part of carbon composite electrodes with commercial carbon particles on detection of nucleic acids.
Results and discussion
Fabrication of vertically aligned MWCNTs
Detection of nucleic acids
Based on these promising milestones of electroanalysis of nucleic acids together with the fact that electrochemistry is still one of the most sensitive analytical technique voltammetric methods can be considered as a suitable tool for detection of nucleic acids. We show the successful application of modern nano-technologies not only for detecting of nucleic acids but also for distinguishing of all bases signals.
All chemicals of ACS purity used and parafilm were purchased from Sigma-Aldrich Chemical Corp. (USA) unless noted otherwise. Salmon sperm DNA was bought from Applied Biosystems (USA). Synthetic oligonucleotides, which were purified using high performance liquid chromatography, were obtained from Sigma-Aldrich with following sequence: influenza HPI 5'-CAG TCG CAA GGA CTA ATC TGT TTG-3'. Stock standard solutions of the oligonucleotides (100 μg/mL) were prepared with water of ACS purity (Sigma-Aldrich) and stored in dark at -20°C. The concentrations of oligonucleotides and DNA were determined spectrophotometrically at 260 nm using spectrometer Specord 210 (Analytic Jena, Germany). Deionised water underwent demineralization by reverse osmosis using the instrument Aqua Osmotic 02 (Aqua Osmotic, Czech Republic), and then it was subsequently purified using Millipore RG (Millipore Corp., USA, 18 MΩ) - MiliQ water. The pH value was measured using inoLab controlled by the personal computer program (MultiLab Pilot; WTW, Germany).
Electrochemical measurements were performed using AUTOLAB PGS30 Analyzer (EcoChemie, Netherlands) connected to VA-Stand 663 (Metrohm, Switzerland), using a standard cell with three electrodes. Carbon composite electrodes were employed as the working electrode. An Ag/AgCl/3 M KCl electrode served as the reference electrode. Glassy carbon electrode was used as the auxiliary electrode. For smoothing and baseline correction, the software GPES 4.9 supplied by EcoChemie was employed. Cyclic voltammetric parameters were as follows: potential step 5 mV; scan rates: 50, 100, 200, 400, 600 and 800 mV/s. Cyclic and square wave voltammetric measurements were carried out in the presence of acetate buffer pH 5. Square wave voltammetry parameters: potential step 5 mV, frequency 280 Hz. The samples measured by square wave voltammetry were deoxygenated before measurements by purging with argon (99.999%) saturated with water for 120 s. The temperature of supporting electrolyte was maintained by the flow electrochemical cell coupled with thermostat JULABO F12/ED (Labortechnik GmbH, Germany) and was 25°C .
chemical vapour deposition
multiwalled carbon nanotubes.
The study was supported by the Czech grant projects GACR 102/09/P640, NANIMEL GACR 102/08/1546 and GACR P205/10/1374.
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