Sample Preparation-Free, Real-Time Detection of microRNA in Human Serum Using Piezoelectric Cantilever Biosensors at Attomole Level

Abstract

A sensitive, selective, sample preparation-free method for near real-time detection of microRNA in buffer and human serum is given using gold (Au)-coated dynamic piezoelectric cantilever sensors. Sensor response to thiolated DNA probe chemisorption, hsa-let-7a hybridization, labeled-DNA hybridization, and Au nanoparticle-functionalized DNA hybridization was monitored continuously in flowing liquid samples using custom flow-cells. The assay showed successful detection of target let-7a with a dynamic range spanning 6 orders of magnitude (10 fM-1 nM) with a limit of detection of less than 10 attomoles (∼4 fM). The serum background had negligible effect on sensitivity relative to the results obtained in the buffer due to reduction in nonspecific binding caused by continuous sensor vibration. Both hybridization and nonspecific binding reduction were confirmed using fluorescence-based assays to support sensor-based results. The sensor-based method demonstrated excellent selectivity for the microRNA target in comparison with similar microRNA differing by only a single nucleotide (hsa-let-7c) and random microRNA sequences. Au nanoparticle-based amplification of sensor response was investigated and led to an order of magnitude improvement in the detection limit and a 128% amplification of sensor response over the entire dynamic range. Au nanoparticle amplification was verified by scanning electron microscopy. The cantilever sensor-based microRNA assay provides competitive sensitivity with current microRNA detection methods and has the advantage of requiring no sample preparation, even when working with biological samples that contain a complex background.

An Exonuclease I-Based Quencher-Free Fluorescent Method Using DNA Hairpin Probes for Rapid Detection of MicroRNA

“…Recently, a variety of device-based analytical strategies and methods-based approaches have been developed to quantitatively analyze miRNAs. Device-based analytical strategies mainly include electrochemical sensors and MEMS [14,15]. Methods-based approaches mainly include fluorescence, colorimetric, and luminescence assays [16,17,18,19,20,21,22,23,24,25,26,27,28].…”

miRNA assays in the clinical laboratory: workflow, detection technologies and automation aspects

“…Although methods exist that can measure miRNA without further sample preparation steps [43], most subsequent detection methods require that the miRNAs are purified by methods that are specialized to this substance class [44]. miRNAs are usually purified using a solid-phase extraction procedure on silica columns or beads, or glassfibre filters, respectively [45].…”

Recent Developments in Optical Detection Technologies in Lab-on-a-Chip Devices for Biosensing Applications

“…The binding events cause changes in the resonant frequency of the devices, which are proportional to the amount of bound analyte. Using frequency shift measurements, detection limits in the attogram range [33] can be achieved with nanocantilevers when operated in vacuum. Another unique characteristic of resonant cantilevers is the availability of various techniques used to actuate and detect resonance.…”

“…This cantilever system was coupled to a flow cell, and chemical detection was demonstrated in ethanol solution. Further, gold was sputtered onto the tip of the PZT cantilever to detect DNA hybridization [33]. A decrease in the resonant frequency of the PZT was related to an increase of cantilever mass due to the interaction between biochemical targets and DNA probes immobilized onto gold-modified PZT surface.…”