Microrna Detection by Electrochemical Impedance Spectroscopy on Platinum Microelectrodes Integrated in a Microfluidic Chip
Authors: Freisa Martina , Dinh Thi Hong Nhung , Omar Choayb , Kechkeche Djamila , Bouville David , Le Potier Isabelle , Gamby Jean 
Published: September 2023

Herein, we present a microfluidic device for performing microRNA (miRNA) detection by Electrochemical Impedance Spectroscopy (EIS) on platinum electrodes.

miRNAs are noncoding RNAs composed of approximately 19-24 nucleobases; recent research demonstrated their potential as biomarkers for inflammations, cancers, and other diseases diagnosis [1]. Electrochemical biosensors for short nucleic acid detection, compared with other detection techniques, are promising due to low-cost production, fast response, and direct electronic signal transduction.

Our biosensor principle is based on the functionalization of capture probes at the electrodes, and successive target recognition via hybridization. For this reason, the immobilization and good alignment of the single-stranded DNA (ssDNA) probes at the electrode is the first delicate step, and it is necessary for facilitating the hybridization with complementary targets. Previous studies demonstrated a strong affinity between Pt surfaces and oligonucleotide backbones [2]; as a result, providing suitable probes orientation on Pt electrodes is challenging.

The EIS provides high sensitivity for characterizing surface modifications. It consists of measuring the equivalent impedance of the electrochemical system by applying a small AC voltage to the electrodes. The well-known Randles circuit represents the equivalent circuit of an electrode/electrolyte system, and the resulting equivalent impedance is translated into the formula expressed in Figure A [3]. The solution resistance element (Rs
) depends on the electrolyte conductivity. The charge transfer resistance (Rct
) is related to the electroactive electrode surface. The double-layer capacitive component (Qdl
) is the electrical translation of the solution-free ions arranging form when a potential is applied to the electrodes; it represents a constant

phase element, which depends on the exponent α related to the surface uniformity. Finally, we have the diffusion-convection complex impedance (Zdc
).

The microfluidic chip is composed of a glass substrate and a polydimethylsiloxane cover which contains a 60 µm heigh channel pattern. After a photolithography step, we evaporated 10 nm of Titanium as an adhesion layer, followed by 100 nm of platinum for our working and counter electrodes (WE and CE) on the glass substrate. The surface area of the WE is 90·10-6cm2, 60 times bigger than the CE.

We successfully completed an electrochemical pretreatment of the Pt electrodes in microfluidics with Potassium hydroxide (100mM), performing 10 cycles of cyclic voltammetry between -0.2 V and -1.2 V.

The EIS measurements employ a 20 mmol/L [Fe(III)(CN)6] 3- / [Fe(II)(CN)6] 4- electrolyte under a 0.5 µL/s flow. The applied AC voltage amplitude is 10 mV, and its frequency is swept down from 1 MHz to 100 mHz.

In this study, we compare the immobilization of ssDNA with and without the introduction of MCH (Figure B). Our 21-base thiol-labeled ssDNA has a concentration of 10-6 mol/L. The functionalization is held for 1 hour and 30 minutes in static for both cases, but, in the second case, 10-5 mol/L of MCH is introduced before the probes for 30 minutes. We performed a static hybridization for 30 min of 10-14, 10-10, and 10-6 mol/L complementary target concentrations.

We collected the data for bare electrodes, probes-functionalized electrodes, and after the introduction of the targets at the three concentrations in the case with and without MCH, and we fitted with the equation in Figure 1A. Figure C represents the electrode fractional coverage (θ) with respect to the concentration of the targets, in the case without (black line), and with MCH (red line). It is possible to observe that the θ parameter increases at each target concentration introduction in the case with MCH. In fact, the DNA-monolayer is negatively charged, and it attenuates the electrode/electrolyte redox exchange; consequently, the charge transfer resistance significantly increases. On the other hand, the fractional coverage in the case without MCH introduction is stable next to zero. We suppose this is because the ssDNAs are not well oriented (as depicted in Figure B-left ); therefore, the hybridization does not occur.

In this study, we presented our microfluidic chip and a new protocol for DNA probes functionalization on Pt electrodes. In particular, we succeeded in providing suitable ssDNAs orientation that avoids DNA bases-Pt interactions. Current studies include coupling our electrochemical biosensor with a microcalorimeter to increase the specificity of the detector and testing in biological samples.

The authors would like to thank the DIMELEC project for funding and RENATECH clean room facilities at C2N, Palaiseau, France.

[1] G. A. Calin and C. M. Croce, Nat. Rev. Cancer, vol. 6, no. 11, pp. 857-866, 2006.

[2] W. Zhou, et al. Chem. Commun., vol. 51, pp. 12084-12087, 2015.

[3] C. Poujouly, et al. Electrochemistry Communications, vol. 137, 107262, 2022.

Figure 1