Electrochemical detection of miRNA-222 by use of a magnetic bead-based bioassay

Abstract

MicroRNAs (miRNAs, miRs) are naturally occurring small RNAs (approximately 22 nucleotides in length) that have critical functions in a variety of biological processes, including tumorigenesis. They are an important target for detection technology for future medical diagnostics. In this paper we report an electrochemical method for miRNA detection based on paramagnetic beads and enzyme amplification. In particular, miR 222 was chosen as model sequence, because of its involvement in brain, lung, and liver cancers. The proposed bioassay is based on biotinylated DNA capture probes immobilized on streptavidin-coated paramagnetic beads. Total RNA was extracted from the cell sample, enriched for small RNA, biotinylated, and then hybridized with the capture probe on the beads. The beads were then incubated with streptavidin-alkaline phosphatase and exposed to the appropriate enzymatic substrate. The product of the enzymatic reaction was electrochemically monitored. The assay was finally tested with a compact microfluidic device which enables multiplexed analysis of eight different samples with a detection limit of 7 pmol L(-1) and RSD = 15 %. RNA samples from non-small-cell lung cancer and glioblastoma cell lines were also analyzed.

Electrochemical Liposome-Based Biosensors for Nucleic Acid Detection

“…In the previous years, researchers have been challenged to push the sensitivity of analytical bioassays down to subnanomolar values while keeping these procedures as simple, reliable, and cost-effective as possible. In particular, the formation of supramolecular assemblies and the use of enzymes to enhance the sensitivity of genoassays have been the subject of many research efforts [ 6 ] [ 7 , 8 ]. Outstandingly low detection limits were achieved.…”

Microfluidic Platform for Enzyme-Linked and Magnetic Particle-Based Immunoassay

“…Previously magnetic beads have been used as immobilization surface for various components of complex biological reactions on microfluidic chips [18][19][20][21][22]; however, their use as a label has been explored in many studies. Use of magnetic beads as a label for immunoassays is, indeed, better as it avoids reaction with various chemical tags, which further reduces the assay time and increases sensitivity of the measurement system.…”

Adsorptive Transfer Stripping for Quick Electrochemical Determination of microRNAs in Total RNA Samples

“…This renders miRNAs promising candidates not only as cancer biomarkers, but also as potential therapeutic targets. Simple and inexpensive methods are currently being developed for their sensitive detection not only in tissues, but ideally in body fluids such as blood, urine or saliva, including methods based on electrochemical (EC) detection [3][4][5][6][7].Recently, we have reported that miRNAs can be easily and quickly labeled with electroactive osmium(VI)-based complexes [8]. In the first step, we took an advantage of high kinetics of reaction between terminal ribose at the 3'-end and Os(VI)py, followed by an exchange of pyridine (py) for 2,2'-bipyridine (bipy) to achieve higher stability of the adducts, more convenient for the EC analysis.…”