Highly sensitive electrochemical biosensor based on naturally reduced rGO/Au nanocomposite for the detection of miRNA-122 biomarker

Abstract

Herein, we have developed a novel high-resolution electrochemical biosensor for the detection of the microRNA-122 (miRNA-122) using gold nanoparticles dotted reduced graphene oxide (rGO/Au) nanocomposite. The natural soapnut solution was used as a reducing agent for the synthesis of the nanocomposite. The naturally reduced rGO/Au nanocomposite was confirmed through various characterization techniques. When rGO/Au nanocomposite was coated on to the gold electrode and checked the electrochemical performance, the nanocomposite shows superior analytical performance. The probe DNA was anchored onto the binding sites of rGO/Au nanocomposite through thiol linker and recognized the target miRNA-122. The developed rGO/Au based electrochemical biosensor demonstrated a linear response for various target miRNA-122 concentrations with a range from 10 μM to 10 pM and, with a detection limit of 1.73 pM. The developed biosensor also shows good stability and reproducibility, could be used for the detection of miRNA-122 and, also can be used for the basic research and clinical studies. Besides, the demonstrated rGO/Au nanocomposite-based sensing strategy could be used to detect various miRNA and protein biomarkers. Furthermore, the green synthesis approach could also be useful for the synthesis of various nanomaterials and nanocomposites for various biomedical applications.

Introduction

MicroRNA-122 (miRNA-122) is a specific type of RNA, highly expressed in the human liver and accounts for up to 70% of total hepatic microRNA presence [1]. The alterations of miRNA-122 expression level changes can cause liver diseases including hepatocellular carcinoma (HCC), responsible for 841,000 new diagnosed cancer cases and became the fourth leading cause of cancer deaths, 742,000 deaths worldwide in 2018 alone [2], [3], [4]. Increasing studies prove that the miRNA-122 present in blood serum can be used as a biomarker to detect liver diseases including hepatitis C and HCC [2], [5], [6]. Therefore, there is an urgent requirement for the early detection of miRNA-122 to control liver-based diseases. The most standard methods used to detect miRNA including surface plasmon resonance imaging, surface-enhanced Raman spectroscopy, polymerase chain reaction, and fluorescence have shown major limitations including time-consumption steps, large sample requirement, slow detection, and less sensitivity [7], [8], [9], [10], [11], [12], [13]. Hence, the development of a simple and reliable method for the detection of miRNA-122 for early control of acute infections is required.

Although several reported methods are available for various miRNA target-based detection, still there are few difficulties involved such as many steps to recognize the target miRNA and required an indicator for the sensing signal [14]. However, Electrochemical based early detection of disease has emerged as an advanced reliable technology due to less expensive, miniaturized setup, rapid detection with high sensitivity and specificity [15], [16], [17], [18], [19], [20]. Hence, the electrochemical technique with the combination of nanomaterials as a probe immobilization surface could be used for the development of less complicated, label-free, and highly sensitive miRNA biosensors to detect miRNA-122.

Graphene is an interesting 2D carbon material with superior properties like high mechanical strength and electrical conductivity, super-fast electron transfer, and high surface to volume ratio, which have been used in various applications including high-performance detection devices [21], [22]. When the graphene combined with nanomaterials such as gold (Au), platinum, and palladium nanoparticles/nanorods, the resulted nanocomposite emerged as a new class of material, can multiply the sensing properties into many folds due to the synergistic effect, and thus, has been used as an electrode material for high-performance electrochemical biosensors [23], [24], [25], [26], [27]. Particularly, Au nanoparticle-based reduced graphene oxide (rGO/Au) nanocomposite has shown more interest due to the excellent charge carrier characteristics of rGO with high biocompatibility and ease of biomolecule immobilization capability of Au nanoparticles, leads the nanocomposite suitable for the construction of electrochemical biosensors. Here, the receptor molecules can be easily immobilized by covalently on Au nanoparticles through thiol (–SH) linker by enabling the bond between sulfur and Au, and eventually can succeed to capture the target molecules [27]. As we understand from the synthesis of nanocomposite materials like graphene/gold is a challenging assignment, as many of the reducing agents involve toxic chemicals like NaBH₄, Hydrazine, etc. [28], [29], [30]. However, there are few green reagents reported for the synthesis of nanomaterials and reduced graphene (rGO) without any use of toxic chemicals [31], [32], [33]. In this context, the natural soapnut (Sapindus mukorossi) solution used for the synthesis of nanomaterials [34], [35], [36], could be a good reducing agent for the reduction of graphene oxide (GO) and gold precursors simultaneously.

In this work, we fabricated an effective, facile, and sensitive electrochemical RNA biosensor for miRNA-122 detection based on Au loaded rGO synthesized by a simple hydrothermal reflux method with natural soapnut solution as a reducing agent. Various characterization techniques have been used to confirm the structure and formation of the synthesized rGO/Au nanocomposite. The cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) was used to confirm the immobilization of probe DNA and hybridization of target miRNA, and also estimated the optimum time for the immobilization and hybridization. To estimate the detection limit of the sensor, a series of target miRNA-122 concentration in the range from 10 pM to 10 μM was tested. The overall green synthesized rGO/Au nanocomposite-based method could be advantageous and reliable for the development of various biosensors.