The ability to detect cancer at early stage using biomarkers, e.g. microRNA (miRNA), could be the difference between life and death of a patient, especially in a metastasis case. Here we present a biosensor that can detect miRNA based on four-way junction (4WJ) platform. This platform consists of a universal stem-loop probe immobilized on the gold electrode, along with two adaptor strands (m and f), that form a 4WJ structure only in the presence of a specific miRNA. The sensor response is obtained by methylene blue redox marker conjugated to m-strand and monitored by square wave voltammetry (SWV). This work is aimed to amplify the electrochemical signal for early detection of miRNA 200c, an important cancer biomarker. Chemometric tools were used to optimize physical (SWV parameters) and chemical (adaptor strands concentration and hybridization time) factors. The SWV parameters typically used for 4WJ biosensor usually are frequency (f) = 100 Hz; amplitude (A) = 70 mV; step potential (Es) = 3 mV; while typical chemical parameters are m- and f-strand at concentrations of 0.25 and 0.50 µM, respectively, and hybridization time of 90 min. With these parameters, the average current density is 23 ± 2 µA/cm2 for miRNA target at concentration of 50 nM. For amplification, a 3-factor 2-level full factorial design with center point was used to optimize physical and chemical factors. First, the SWV parameters were studied: f = 100 and 400 Hz, A = 35 and 105 mV, Es = 1 and 5 mV. From Pareto plot, f and A were the parameters with the best influence on the response. A trend plot demonstrated the higher level of all parameters (f = 400 Hz, A = 105 mV, Es = 5 mV) obtained the highest signal, 73 ± 6 µA/cm2, an increase of 3.2 times (217%). Subsequently, using the optimal SWV parameters, the chemical factors were evaluated with 0.1 and 1.0 µM as m- and f-strands concentrations and hybridization time of 30 and 90 min. Pareto plot showed the m-strand is the parameter with the most influence on the sensor response. According to trend plot, the highest current density was obtained with the center point (m- and f- concentration = 0.55 µM and hybridization time = 60 min), whose current density was 97 ± 4 µA/cm2, which means an increase of 4.2 times (322%) of the initial response. Therefore, the chemometric studies were sufficient to amplify the electrochemical response of the biosensor for miRNA 200c, using just few experiments.

Chemometric analyses for electrochemical signal amplification for the detection of cancer biomarker

2019 Spring National Meeting

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