# Microfluidic chip for electrochemical detection of circulating miRNAs: real sample testing of cardiac disease biomarkers

A Tarasova, I.M. Mbomekalle, S Banzet, H M Man, I Le Potier, J Gamby

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A Tarasova, I.M. Mbomekalle, S Banzet, H M Man, I Le Potier, et al.. Microfluidic chip for elec-
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Distributed under a Creative Commons Attribution 4.0 International License
Microfluidic chip for electrochemical detection of circulating miRNAs: real-sample testing of cardiac disease biomarkers

A. Tarasova<sup>a</sup>, I. Mbomekalle<sup>b</sup>, S. Banzet<sup>c</sup>, H.M. Man<sup>a</sup>, I. Le Potier<sup>a</sup>, J. Gamby<sup>a</sup>

<sup>a</sup>Laboratoire Centre de Nanosciences et de Nanotechnologies (C2N), Palaiseau, 91120, France

<sup>b</sup>Institut de Chimie Physique d'Orsay (ICP), Orsay, 91400, France

<sup>c</sup>INSERM UMR-MD-1197, Centre de transfusion sanguine des armées, Clamart, 92140, France

e-mail: anna.tarasova@universite-paris-saclay.fr

Keywords: microfluidics, miRNAs, electrochemical sensor, EIS, CV, biosensing, microfabrication

The quantification of circulating microRNAs (miRNAs), particularly miR-1 and miR-133a, has emerged as a powerful strategy for the early detection and monitoring of cardiac diseases as acute myocardial infarction (AMI) [1]. These muscle-enriched miRNAs are released into the bloodstream during cardiac injury and show strong biomarker potential due to their stability [2], disease specificity, and rapid response to physiological stress [3]. However, existing analytical methods such as real-time quantitative PCR (RT-qPCR) and sequencing remain time-consuming, expensive, and require complex sample processing, which limit their use in point-of-care (POC) or emergency clinical settings.

This work aims to develop and validate a miniaturized microfluidic electrochemical biosensor capable of simultaneous and label-free detection of miRNAs from real samples. The ultimate goal of the platform is to create a profiling map of miRNA expression patterns in different pathologies, allowing for real-time patient monitoring, early diagnosis and therapeutic decision-making.

Our approach builds upon the microelectrode-based reference-free electrochemical detection in portable analytical platforms. The device is shown in **Fig. 1**. [4] Embedding it into microfluidic channel allows uniform distribution of the sample over the detection surface, which minimizes losses due to diffusion limitations. [5] This enhancement results in higher sensitivity, faster response times, lower sample consumption (nanoliters) and more accurate quantification of low-abundance miRNAs.

The working electrodes are functionalized with DNA probes complementary to miR-1 and miR-133a. The hybridization event is detected by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) achieving detection limits in the low attomolar range (Fig. 2) with high specificity, even in the presence of similar miRNA sequences. [6]

The approach presented here uses 6-mercapto-1-hexanol (MCH) for a better orientation of immobilized probes and TCEP as a reducing agent for thiols attached to DNAS. [7, 8] The working principle is presented in Fig.3. Additionally, we are currently testing alternative strategies for electrode functionalization to improve sensitivity and prevent non-specific adsorption. [9]

To validate our method, we analyzed real plasma samples from mice obtained from our collaborator (INSERM). These samples were collected from mice following notexin-induced muscle injury. Our results were consistent with RT-qPCR findings, confirming that circulating miRNAs, particularly miR-133a, are upregulated in response to muscle damage. In the sample with the lowest degree of muscle lesion, miR-133a was detected at concentrations of 4.9 pM and 620 fM using CV and EIS (via charge transfer resistance, Rct), respectively, while 45 pM for the highest lesion. miR-1, which was detected at low levels by RT-qPCR, also showed lower expression in our measurements (29 fM for lowest and 112 fM for highest), compared to 3 fM in control samples. These findings are summarized in Fig. 4. EIS was used also to evaluate the surface coverage dependence.

Our results show a dynamic detection range from attomolar to micromolar concentrations for synthetic miR targets. The varying concentrations of miRNAs in real samples provide a preliminary validation of our approach without the need for PCR amplification. In summary, our device introduces multiple innovations: it operates in a label-free miR detection platform, supports real-time, and direct analysis of clinical samples without complex preparation steps. These features, together with its compact and low-power design, render the platform ideal for POC diagnostics and continuous patient monitoring.
[1] Y. Kuwabara, et al., Circ. Cardiovasc. Genet. 4 (2011) 446–454.

[2] P.S. Mitchell, et al., Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 10513–10518

[3] J. Siracusa, et al., The American Journal of Pathology, 186 (2016) 1313-1327.

[4] M. Lazerges, et al., Sensors and Actuators B 182 (2013) 510–513.

[5] C. Poujouly, et al., Electrochemistry Communications, 137 (2022) 107262

[6] C. Poujouly, et al., Frontiers in chemistry, 10 (2022).

[7] O. Choyab, et al., Langmuir 2024, 40, 50, 26616–26625

[8] H. H. Man, et al., ACS Electrochem, (2025)

[9] L. Meng, et al., Inorganic Chemistry 63 (46) (2024) 22149-22161

[10] M. Freisa, et al., Electrochimica Acta 505 (2024) 14500

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Illustration of the microfluidic device fabricated by UV lithography, platinum deposition and molded PDMS bonding; with dimensions reproduced from reference [10].</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**Figure 2.** A) Example of CV experiment for detection of miR-1 with 5 synthetics target samples at different concentrations; B) EIS experimental (dots) and fitting (lines) performed by Measurement Model software.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Example of experiments for detection of miR-133a in real sample with the lowest lesion: A) CV showing the current absolute value decreasing after hybridization. B) PEIS showing R<sub>ct</sub> increase suggesting non-linear mass diffusion due to surface coverage. [10]</figcaption></figure>

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<table><caption>**Figure 4.** CV results of detection miR-1 and miR-133a in real samples. Control (green), low lesion (orange), and highest lesion (blue). Table with values.</caption><thead><tr><th>Sample</th><th>Repetitions</th><th>I norm averaged</th><th>Error</th><th>Concentration (M)</th></tr></thead><tbody><tr><td>Control miR-133a</td><td>5</td><td>0.26</td><td>0.02</td><td>3.1 10<sup>-13</sup></td></tr><tr><td>Lowest lesion miR-133a</td><td>3</td><td>0.29</td><td>0.03</td><td>4.9 10<sup>-12</sup></td></tr><tr><td>Highest lesion miR-133a</td><td>3</td><td>0.32</td><td>0.02</td><td>4.5 10<sup>-11</sup></td></tr><tr><td>Control miR-1</td><td>4</td><td>0.25</td><td>0.04</td><td>3.79 10<sup>-15</sup></td></tr><tr><td>Lowest lesion miR-1</td><td>3</td><td>0.29</td><td>0.05</td><td>2.92 10<sup>-14</sup></td></tr><tr><td>Highest lesion miR-1</td><td>3</td><td>0.31</td><td>0.03</td><td>1.12 10<sup>-13</sup></td></tr></tbody></table>
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