Label-free and reagentless electrochemical detection of microRNAs using a conducting polymer nanostructured by carbon nanotubes: Application to prostate cancer biomarker miR-141

Highlights

• MicroRNA detection.

• Label-free and direct electrochemical detection.

• Conducting polymer nanostructured by carbon nanotubes.

• Prostate cancer biomarker miR-141.

Abstract

In this paper, a label-free and reagentless microRNA sensor based on an interpenetrated network of carbon nanotubes and electroactive polymer is described. The nanostructured polymer film presents very well-defined electroactivity in neutral aqueous medium in the cathodic potential domain from the quinone group embedded in the polymer backbone. Addition of microRNA miR-141 target (prostate cancer biomarker) gives a “signal-on” response, i.e. a current increase due to enhancement of the polymer electroactivity. On the contrary, non-complementary miRNAs such as miR-103 and miR-29b-1 do not lead to any significant current change. A very low detection limit of ca. 8 fM is achieved with this sensor.

Introduction

The biology of the late 20th century was marked by the discovery in 1993 of a new class of small non-coding ribonucleic acids (RNAs) which play major roles in regulating the translation and degradation of messenger RNAs (Lee et al., 1993, Wightman et al., 1993). These small RNAs (18–25 nucleotides), called microRNAs (miRNAs), are implied in several biological processes such as differentiation, metabolic homeostasis, cellular apoptosis and proliferation (Iorio and Croce, 2009, Brase et al., 2010).

The discovery in 2008 that the presence of miRNAs in body fluid is in correlation with cancer (prostate, breast, colon, lung, etc.) or other diseases (diabetes, heart diseases, etc.) has made them new key players as biomarkers (Lawrie et al., 2008, Catuogno et al., 2011, Chen et al., 2008). Actually, more than 1200 miRNAs have been identified (Liu et al., 2012), among which miR-141 is detected at elevated level in blood of patients having metastatic prostate cancer (Mitchell et al., 2008).

Current standard methods for identification and quantification of miRNAs are based on traditional molecular biology techniques (Northern blot, microarray, qRT-PCR). These approaches although very sensitive and reliable are often expensive, time consuming, and need highly trained technicians (Hunt et al., 2009, Planell-Saguer and Rodicio, 2011). That is why a real challenge is to develop devices able to detect and quantify easily and simultaneously different miRNA sequences at sub-picomolar levels (Wang et al., 2012). Ideally, these new bioanalytical tools should be easy to manufacture, need low power, and allow reagentless and label-free detection. Few work deal with such strategy, and particularly very few when electrochemical transduction is involved. Electrochemical biosensors offer the advantages of mass fabrication, low cost and potential decentralized analysis (Paleček and Bartošík, 2012).

Lusi et al. (2009) reported amperometric detection based on oxidation of RNA nucleobases. This system allows detection at sub-picomolar level (0.1 pM), but the current depends on the number of guanine and needs high oxidation potentials, which may generate side-oxidations. Using enzyme-labeled detection probes, Kilic et al. (2012) reported detection for miR-21 with a detection limit of 1 μM. Gao and Peng (2011) achieved a detection limit of 10 fM. Allosteric molecular beacons able to bind HRP enzyme were used by Cai et al. (2003), with a detection limit of 44 amol in a volume of 4 μL, i.e. 11 pM. Yin et al. (2012) have shown a detection limit of 60 fM for miR-21 with gold NPs bearing HRP. Using a polymerase-labeled DNA probe and impedance measurements, Shen et al. (2013) reported a LOD of 2 fM for a S/N of 3. Very high sensitivity (0.1 fM) was obtained using peptide nucleic acid (PNA) probes (Zhang et al., 2009). Qavi et al. (2010) proposed an excellent review on miRNA analysis.

Conducting polymers constitute a powerful platform to immobilize short DNA or RNA sequences while maintaining their stability, accessibility and activity (Gerard et al., 2002, Cosnier, 2003, Cosnier, 1999). Unfortunately, label-free electrochemical biosensors based on polymer-modified electrodes are known to suffer from lack of sensitivity (Cosnier and Holzinger, 2011). To enhance sensitivity, carbon nanotubes (CNTs) were frequently reported (Wohlstadter et al., 2003, Wang, 2005) to increase the electroactive area and decrease the electrical resistance of the working electrodes, leading to 3D conductive materials (Peigney et al., 2001, Kulesza et al., 2006, Acevedo et al., 2008). Qi et al. (2007) fabricated an electrochemical DNA biosensor based on electropolymerised polypyrrole and carbon nanotubes, using ethidium bromide as redox indicator with high sensitivity, ca. 85 pM. Very few works were related to label-free and reagentless biosensors.

Okuno et al. (2007) described a label-free and reagentless immunosensor for prostate-specific antigen based on single-walled CNT-modified microelectrodes with low detection limit (0.25 ng mL⁻¹). The current being derived from oxidation of amino acid residues (tyrosine and tryptophan), it is then dependent on the presence of these residues in the target sequence. Zhang et al. (2011) described a strategy for label-free and reagentless electrochemical DNA sensing based on SWNTs and an immobilized redox probe. This system allows very specific detection of DNA but the limit of detection is only 0.1 µM. 3D structures obtained using CNTs may induce high capacitance which may introduce distortion on cyclic voltammograms (Peng et al., 2007). In order to minimize this effect, pulsed methods such as differential pulse voltammetry (DPV) or square wave voltammetry (SWV) are currently used. Impedance methods combining polymer and carbon nanotubes have also been widely used in reagentless formats (Xu et al., 2004, Xu et al., 2006, Cai et al., 2003). In this paper, we describe a label-free and reagentless miRNA sensor based on an interpenetrated network of carbon nanotubes and electroactive polymer. The nanostructured polymer film presents very well-defined electroactivity in neutral aqueous medium from the quinone group embedded in the polymer backbone. When the miRNA-141 target is added (miR-141, a prostate biomarker) a “signal-on” response, i.e. a current increase, is observed while no current change occurs with non-complementary miRNAs such as miR-103 (a colorectal cancer biomarker; Chen et al., 2012) or miR-29b-1 (a lung cancer biomarker; Fabbri et al., 2007). The biosensor presents a very low detection limit of ca. 8 fM.