An Integrated Multiple Electrochemical miRNA Sensing System Embedded into a Microfluidic Chip

Abstract

In this article, we present the design, fabrication and characterization of a microfluidic device and a dedicated electronic system to perform 8 multiplexed electrochemical measurements of synthetic miRNA strands, as well as the biochemical protocols developed for the functionalization of the electrodes and the quantification experiments. The outcomes of this work highlight that the parallelization of eight microchannels containing 2-electrode cells driven by the dedicated electronics offers a solution as robust as a conventional 3-electrode cell and commercially available potentiostats. In addition, this solution presents the advantage of simultaneously reduce the microfabrication complexity, as well as offering an integrated; multiplexed and portable system for the quantification of miRNA. The results presented demonstrate that the system shows a linear response on concentrations down to 10^-18 mol/L of perfect matched reporter and capture sequences of synthetic miRNA.

Keywords: cyclic voltammetry; electrochemical detection; microRNA; microfabrication; microfluidics; readout electronics.

Figure 1

Fabrication process for the microfluidic device realization. (A1). Glass substrate. (A2). Lithography step. (A3). Metal sputtering (50 nm of TiW and 200 nm Au). (B1). Silicon substrate. (B2). Lithography with 2 µm SU8-2002 and 50 µm SU8-2050. (B3). PDMS pouring on silicon mold. (B4). PDMS peeling-off. (C1,C2). PDMS and glass substrate alignment and plasma bonding.

Figure 2

(A) Architecture of the 8-channel readout electronics system and schematic of an individual channel. (B) Frequency response of the TIA for different capacitor values. (C) Summary of the cutoff frequency obtained for the three different capacitors.

Figure 3

(A). Figure representing the final microfluidic device on the chip holder. A zoom on the embedded electrochemical cell is reported. (B). Characterization response of the embedded electrochemical cells (using a BioLogic VSP 300) at different flow rates after its fabrication. (C). Dedicated electronic circuit developed for the multiplexed readout of up to 8 different sensors. (D). Frequency response characterization of the analog stage comprising the current−voltage conversion, amplification and filtering for different values of gain: maximum gain (blue), medium gain (red) and minimum gain (yellow). The dotted vertical line represents the theoretical cut off frequency, and the red stars the found cut off frequency for each measurement.

Figure 4

(A). CV curves for each channel of the device. Characterization of bare gold electrodes. (B) Absolute maximum (blue) and minimum (red) currents histogram for each device channel, values from Figure 4A. The black lines are for the mean values. (C) Fitted slope parameters histogram CV curves in Figure 4A, the red line is for the mean value.

Figure 5

(A) Example of CV experiments for the hybridization in channel A (see Figure 4B). Blue curve for the bare electrodes, red curve for functionalized electrodes with ssDNA probes, green curve for a miRNA target concentration of 10⁻¹⁸ mol/L, purple curve for a miRNA target concentration of 10⁻¹² mol/L, yellow curve for a miRNA target concentration of 10⁻⁶ mol/L. (B) Histogram representing the extracted slope by MatLab for each CV curve in Figure 5A. (C) Graph representing the three slopes for the hybridization process respect to target concentration in log scale representation. (D). Table for the five measurements in which the important values are extracted by an implemented MatLab code. The first column, m, represents the slope parameter, q the intercept of the fit, abs max the CV curve absolute maximum and abs min the CV curve absolute minimum.