In-flow real-time detection of spectrally encoded microgels for miRNA absolute quantification.

David Dannhauser,Paolo A Netti,Domenico Rossi,Edmondo Battista,Filippo Causa,Angela M Cusano

doi:10.1063/1.4967489

Abstract

We present an in-flow ultrasensitive fluorescence detection of microRNAs (miRNAs) using spectrally encoded microgels. We researched and employed a viscoelastic fluid to achieve an optimal alignment of microgels in a straight measurement channel and applied a simple and inexpensive microfluidic layout, allowing continuous fluorescence signal acquisitions with several emission wavelengths. In particular, we chose microgels endowed with fluorescent emitting molecules designed for multiplex spectral analysis of specific miRNA types. We analysed in a quasi-real-time manner circa 80 microgel particles a minute at sample volumes down to a few microliters, achieving a miRNA detection limit of 202 fM in microfluidic flow conditions. Such performance opens up new routes for biosensing applications of particles within microfluidic devices.

Highlights

MicroRNAs are a class of short, singlestranded, non-coding endogenous RNAs that play important roles in regulating gene expression via target miRNA degradation or translational repression (Bartel, 2004)
We investigated miRNA 21 as a proof of principle since it has been related to the pathogenesis of various malignant tumours, including prostate, gastric, colon, breast, and lung cancers (Di Girolamo et al, 2016)
Afterwards, two subsequent shells were added: first, the Rhodamine B (Rh) labelled cores were used as seeds for polymerization of 0.5% PEGDMA; a second fluorescent shell was added on top by copolymerizing PEGDMA, Acrylic Acid (AAc), and a different amount of Fluorescein O-methacrylate (Fluo), namely, 0.2 mM, 0.1 mM, and 0.05 mM

Summary

Introduction

MicroRNAs (miRNAs) are a class of short (approximately 18–23 nucleotides), singlestranded, non-coding endogenous RNAs that play important roles in regulating gene expression via target miRNA degradation or translational repression (Bartel, 2004). It has been demonstrated that the expression levels of circulating miRNAs are correlated with the different states of various diseases including cancer, neurodegenerative disorders, and cardiovascular diseases (Schwarzenbach et al, 2014; Grasso et al, 2014; and Sayed et al, 2014). An accurate and robust quantification of circulating miRNA biomarkers in blood for early stage, metastatic or recurrent diseases is still a major challenge due to high sequence homology, complex secondary structures, and low concentration levels. Due to the low amount of target miRNAs, large volumes of clinical samples—from a hundred to several millilitres—are typically required, which only few conventional detection systems can directly handle without sample preparation and volume reduction. Even though the quantitative reverse transcription polymerase chain reaction (qRT-PCR) method shows high sensitivity (down to 10 fM) and high-throughput ability, it requires miRNA extraction, amplification, and calibration steps (Chen et al, 2011). All of them require pre-treatments as well as amplification steps of the sample

Methods

Results

Conclusion