• Pailleret, Alain
• Gamby, Jean
• González-Losada, Pedro
• Horny, Marie-Charlotte
• Poujouly, Claire
• Billon, Florence

Abstract

<jats:p><jats:bold>Introduction</jats:bold></jats:p><jats:p><jats:bold>Microfluidic Chip Fabrication</jats:bold></jats:p><jats:p> The microfluidic chip is composed of an a-CNx working microelectrode (30 μm x 300 μm) and a platinum counter electrode (2 mm x 300 μm). To optimize the adherence and the conductivity of the a-CNx, a titanium/ platinum (20 nm/200 nm) underlayer is evaporated on a glass wafer. Then, the a-CNx (x=0.12) layer is deposited on the working microelectrode by DC magnetron sputtering (time: 20 min, power: 200 W, thickness: 200 nm) with a graphite target under a nitrogen flow (P<jats:sub>tot</jats:sub>=0.4 Pa and P<jats:sub>N2</jats:sub>/P<jats:sub>tot</jats:sub>=3 %). The microelectrodes were patterned by photolithography and lift-off processes in cleanroom. Then, the microfluidic chip is closed off with PDMS (ratio 1:10), patterned with a SU-8 mold and sealed by O<jats:sub>2</jats:sub> plasma bonding.</jats:p><jats:p><jats:bold>Method</jats:bold></jats:p><jats:p>The a-CNx working microelectrodes in the microfluidic chip are electrochemically pre-treated by cyclic voltammetry between -1V and 0V with a scan rate of 50 mV/s during 7 minutes, in a 0.5 M sulfuric acid solution at a flow of 0.5 μL/s. The microelectrodes can then be individually functionalized by loading a DNA probe at 10<jats:sup>-6 </jats:sup>M. The COOH-modified probe sequence is activated by a 2.10<jats:sup>-7 </jats:sup>M EDC and NHS protocol in the microfluidic chip. Then, to stabilize the self-assembled monolayer a 0.5 M NaCl solution is loaded in the chip for 30 minutes. The targeted microRNA can then be loaded in the microfluidic chip at ultra-low concentration (10<jats:sup>-18 </jats:sup>M) at a low 0.02 μL/s flow rate for 30 minutes. The detection of the hybridization is done electrochemically by cyclic voltammetry around 0V at a 50 mV/s scan rate and electrochemical impedance spectroscopy between 1 MHz and 100 mHz in an equimolar 3 mM ferro/ferricyanide as the redox probe and 10<jats:sup>-8 </jats:sup>M methylene blue solution.</jats:p><jats:p><jats:bold>Results and Conclusions</jats:bold></jats:p><jats:p> The methylene blue acts as an intercalator in between the strands of the DNA duplex [4]. The electrons move from the a-CNx working electrode to the intercalated methylene blue before to be reduced by the ferrocyanate in solution. It implies a current level measurements more important for a double stranded DNA than a single stranded DNA. For a non-complementary microRNA target, the same protocol shows a decreased current. Thus, the electrochemical properties of a-CNx films are interesting for the development of a microfluidic chip for an electrochemical detection of microRNAs. Indeed, an attomolar detection in 30 minutes of a microRNA has been reached, as well as a specific recognition of the microRNA hybridized on the microelectrode surface.</jats:p><jats:p><jats:bold>References </jats:bold></jats:p><jats:p> [1] G. Adamopoulos, C. Godet, C. Deslouis, H. Cachet, A. Lagrini, B. Saidani, The electrochemical reactivity of amorphous hydrogenated carbon nitrides for varying nitrogen contents : the role of the substrate, <jats:italic>Diam. Relat. Mater</jats:italic>., 12 (3) (2003) 613-617. Doi: 10.1016/S0925-9635(03)00038-4.</jats:p><jats:p> [2] R. Medeiros, R. Matos, A. Benchikh, B. Saidini, C. Debiemme-Chouvy, C. Deslouis, R. C. Rocha-Filho, O. Fatibello-Filho, Amorphous carbon nitride as an alternative electrode material in electroanalysis : Simultaneous determination of dopamine and ascorbic acid, <jats:italic>Analytica Chimica Acta</jats:italic>, 797 (2013) 30-39. Doi: 10.1016/j.aca.2013.08.018.</jats:p><jats:p> [3] M. Faure, F. Billon, A.-M. Haghiri-Gosnet, B. Tribollet, C. Deslouis, A. Pailleret, J. Gamby, Influence of the atomic nitrogen content in amorphous carbon nitride thin films on the modulation of their polarizable interfaces properties, Electrochimica Acta, 280 (2018) 238-247. Doi: 10.1016/j.electacta.2018.05.116.</jats:p><jats:p> [4] E. Tuite, J. M. Kelly, The interaction of methylene blue, azure B, and thionine with DNA: Formation of complexes with polynucleotides and mononucleotides as model systems, Biopolymers, 35 (1995) 419-433. Doi: 10.1002/bip.360350502.</jats:p>

Topics

• impedance spectroscopy
• surface
• amorphous
• Carbon
• thin film
• Platinum
• glass
• glass
• Nitrogen
• nitride
• titanium
• cyclic voltammetry