A Label-Free Biosensor for Electrochemical Detection of Femtomolar MicroRNAs

Abstract

A simple and ultrasensitive label-free microRNA (miRNA) biosensor, based on hybridized miRNA-templated deposition of an insulating polymer film and electrochemical impedance spectroscopic detection, is described in this report. The biosensor is made of a monolayer of charge-neutral morpholino capture probes on an indium tin oxide (ITO)-coated glass slide. Upon hybridization, the neutral surface of the biosensor is converted to anionic by the hybridized miRNA strands. The deposition of the insulating polymer film, poly(3,3'-dimethoxybenzidine) (PDB), is then carried out by the horseradish peroxidase-catalyzed polymerization of 3,3'-dimethoxybenzidine in the presence of H(2)O(2). The cumulative nature of the PDB deposition process significantly enhances the sensitivity of the biosensor. Under optimized conditions, miRNA expression profiling can be performed label-freely from 5.0 fM to 2.0 pM with a detection limit of 2.0 fM. The biosensor is applied to the detection of circulating miRNAs in blood and miRNAs in total RNA extracted from cultured cells.

Electrochemical Detection of miRNAs

“…As a low cost alternative of PNA, antisense morpholino oligos (MCPs), which have higher affinity to complementary RNA than natural nucleic acid probes, were also reported [34]. Despite of the well-documented advantages of using synthetic nucleic acid analogues as selective miRNA probes [21] their implementation in electrochemical detection methodologies is still scarce [35,36]. It must be mentioned that miRNA detection, beside the availability of high affinity and selectivity hybridization probes, benefits also from the implementation of new signaling probes with superior properties.…”

“…2.0 fM [50]. To reduce the complexity of the assay, Gao et al [36] proposed a variation of this method, which eliminates the need for enzymatic cleavage of the unreacted CP while achieving the same detection limit of ca. 2 fM.…”

“…This results in the generation of a thin electrically insulating PDB film on the sensor surface which increases the charge transfer resistance. [36] Figure 7. Schematic of the three main steps of the miRNA assay with the MCP-based impedimetric biosensor: (a) formation of a monolayer of uncharged MCPs on silane activated ITO electrode; (b) capture of the target miRNA resulting in an increase of the negative surface charge density; and (c) electrostatic adsorption of DB on the negatively charged surface and its surface confined polymerization in the presence of HRP and H2O2.…”

“…Schematic of the three main steps of the miRNA assay with the MCP-based impedimetric biosensor: (a) formation of a monolayer of uncharged MCPs on silane activated ITO electrode; (b) capture of the target miRNA resulting in an increase of the negative surface charge density; and (c) electrostatic adsorption of DB on the negatively charged surface and its surface confined polymerization in the presence of HRP and H2O2. [36] Reprinted with permission from Ref. [36] Copyright (2013) American Chemical Society.…”

Engineering solutions for cancer

“…Other areas where there is obvious potential for engineering input include: novel biosensors and pointof-care diagnostics for early detection of disease via the capture and detection of circulating biomarkers (liquid biopsies) [24][25][26][27]; the use of smart wearable or implantable electrochemical sensors to characterise tumour response to chemotherapy in situ or to monitor disease progression and/or signs of relapse [28,29]; artificial intelligence (AI), such as machine learning to help physicians make better diagnoses and guide treatment decisions while minimising costs [30]; metabolic and proteomic profiling of body fluids for the identification of tumoural areas in situ [31][32][33]; and disease-detecting or disease-fighting nanotechnologies (e.g. DNA nanobots [34], quantum dots [35]) for reporting disease status and selectively delivering drugs to tumour cells whilst minimising systemic side effects [36].…”