Preconcentration, Release and Electrochemical Detection of microRNA on Microfluidics

Introduction One of the challenges of the 21st in medical diagnosis lies in the identification of specific biomarkers for the detection of infectious or cancerous diseases before the first symptoms appear and thus treat the patients effectively. After the discovery of their implication in cancer pathologies [1] at early stage, micro ribonucleic acids (miRNA) became promising candidates for this application due to their rapid, precise and reliable diagnosis. These miRNAs, constituted by around 20 nucleotides, are highly involved in post-transcriptional regulation mechanisms capable of inducing the extinction or expression of certain gens and their presence in body fluids (plasma, urine, saliva...) is a sign of important perturbations [2]. However, in order to detect the in-vivo change of molecular expression levels (under femtomolar [3]) current techniques rely in chemical amplification stages of a specific target to be detected usually done by Polymerase Chain Reaction (PCR). The main drawbacks of PCR are an increase in the entire protocol time from miRNA extraction in biological sample to its detection (optical or electrical), as well as nonspecific sequence amplification, especially for short sequences as in miRNAs [4]. The approach developed and patented by our laboratory [5] proposes an alternative to the use of chemical amplification techniques. This alternative is based in the preconcentration of the circulating miRNA, their localized release and detection. During the preconcentration stage, functionalized magnetic nanoparticles (NPMs) capture the circulating miRNAs and then release them, via magnetic hyperthermia. Once released, the miRNAs are detected by functionalized gold microelectrodes embedded in a microfluidic chip [6]. In this communication we will present the results obtained combining the localized release of the miRNA and their electrochemical detection. Method The used nanoparticles have a diameter of 80 nm and they are formed by a maghemite core protected by a silica shell functionalized with –NH2 groups which allow a covalent coupling with modified RNA probe. The microelectrode pairs used to detect the hybridation of the previously released miRNA are formed by a 30 µm width by 300 µm length working gold microelectrode and a 30 µm width by 2 mm length gold counter-electrode patterned on a glass substrate by a lift-off process. The microfluidic channels are microfabricated by molding dimethylpolysiloxane (PDMS) onto a SU-8 (from MicroChem) mold 50-micron thickness with the pattern of the channels. PDMS is spin coated on the mold to obtain a thickness of 500 µm which allows to keep the total thickness of the microfluidic device under 1mm after the bonding of the glass substrate and the PDMS microchannels. Functionalization of the microelectrodes is done by introducing a solution of DNA probes modified with a thiol termination in 0.5 M NaCl. Then the channel is rinsed with 0.5 M NaCl to stabilize the probes. The magnetic field needed for the magnetic hyperthermia is generated by a power amplifier that provides the current to the tank circuit formed by a capacitor and an air-gapped ferrite with a certain number of turns. The microfluidic device is placed in the air-gap of the ferrite to maximize the amplitude of the applied alternating magnetic field Electrochemical measurements are performed using a commercial potentiostat connected to the microfluidic device through a dedicated chip holder. Electrochemical measurements are performed in 0.5 M NaCl as supporting electrolyte containing a 3 mM FeIIICN6 3-/ FeIICN6 4- redox couple, and 10-8 M methylene blue as redox intercalant. Results and Conclusions The measured impedance between a working microelectrode and the counter-electrode before and after the hybridation of the miRNA released by the magnetic nanoparticles after being subjected to an alternating magnetic field as previously described [5]. In this presentation we will present impedance measurements before hybridation that shows a high electron charge transfer resistance (Rct) since grafted DNA probe (single strands) inhibits the redox complex diffusion towards the electrodes. After the miRNA hybridization on microelectrodes (double strand), the electron charge transfer resistance decreases related to the intercalation of MB in DNA duplex which acts as an electron charge mediator between the redox and the microelectrode. Indeed, Rct decrease highlights the success of the hybridization step after it release from magnetic hyperthermia runs. The proposed methodology, based in the combination of magnetic hyperthermia and electrochemical detection, has shown to be effective to locally release the recruited miRNA and detect them by electrochemical means. In addition, the whole process is performed in a microfluidic device offering the advantages of this devices: batch processing, low sample volume, portability, etc. References [1] Swarup V, Rajeswari MR. Circulating (cell-free) nucleic acids. A promising, non-invasive tool for early detection of several human diseases. FEBS Lett. 2007, 581, 795-9. [2] Gilad S, Meiri E, Yogev Y, et al. Serum microRNAs are promising novel biomarkers. PLoS One 2008, 3, 3148 [3] Terry T. Goodrich, Hye Jin Lee, , and Robert M. Corn. Enzymatically Amplified Surface Plasmon Resonance Imaging Method Using RNase H and RNA Microarrays for the Ultrasensitive Detection of Nucleic Acids. Anal. Chem. 2004, 76, 21, 6173-6178. [4] Leni Moldovan, Kara E. Batte, Joanne Trgovcich, Jon Wisler, Clay B. Marsh, and Melissa Piper. Methodological challenges in utilizing miRNAs as circulating biomarkers. J. Cell. Mol. Med. , 18(3):371–390, 2014 [5] M. C. Horny, J.-M. Siaugue, V. Dupuis, M. Lazerges, J. Gamby. Patent CNRS, Mai 2017, FR demande 1759346, 5 octobre 2017. PCT 2018 n°0772080. [6] M. C. Horny, M. Lazerges, J. M. Siaugue, A. Pallandre,D. Rose, F. Bedioui, C. Deslouis, A. M. Haghiri-Gosnet and J. Gamby, Lab Chip, 2016,16, 4373–4381.