Electrochemical Detection of Micro-RNAs on an Amorphous Carbon Nitride a-CNx Working Electrode in a Microfluidic Chip
Authors: Poujouly Claire , Horny Marie-Charlotte , González-Losada Pedro , Billon Florence , Pailleret Alain , Gamby Jean 
Published: May 2020

Introduction

Polymerase Chain Reaction (PCR) is the most common method for DNA detection. Based on DNA amplification at high temperature, it is hour lasting and not reliable for short DNA strands. Recently, on-chip electrochemistry has been used for direct quantification of nucleic acids, peptides and proteins, without chemical amplification unlike PCR protocol. Instead of integrating gold electrodes in microfluidic devices, carbon materials have demonstrated their performances such as carbon nanotubes or graphene. For instance, amorphous carbon nitride (a-CNx) films are widely used as electrode materials due to a large potential window (3V-4V) [1]. The a-CNx is a polarizable material with a chemical surface composition dependent on the atomic percentage of nitrogen incorporated during the deposition [2][3]. These properties are interesting for a microfluidic chip integrating a-CNx as working microelectrode for an electrochemical detection of microRNAs. In this communication, we will present the integration, activation and functionalization of a-CNx as a working microelectrode in a microfluidic chip for a 30 minutes detection of a 23 basis RNA sequence with a limit of detection of 10-18 M.

Microfluidic Chip Fabrication

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 (Ptot=0.4 Pa and PN2/Ptot=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 O2 plasma bonding.

Method

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 10-6 M. The COOH-modified probe sequence is activated by a 2.10-7 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 (10-18 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 10-8 M methylene blue solution.

Results and Conclusions

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.

References

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