Nanotechnology has revolutionized our life through its different advantages and applications . Different nanomaterials from gold  to carbon , have been used so far in biomedical applications such as diagnostics [4, 5]. The term nanobiosensor, which is the application of nanotechnology in biosensors, has developed for medical applications in various scientific papers and patents, especially electrochemical nanobiosensors [6, 7]. The electrochemical nanobiosensors are pulling together the advantages of electrochemical methods, nanotechnology and selectivity of the biological molecules [8, 9]. For that reason, they are one of the most important ways of medical diagnostics today [10, 11].
With the increasing of human knowledge about the mechanisms of development, spread, and transmission of various diseases, novel strategies are provided for prevention, diagnosis, and treatment. Although these processes have been upgraded faster and more efficient in recent years due to the advancement in science and technology, however the increasing number of cases with lethal diseases such as cancers reveals the importance and emergency of developing early detection systems that can provide rapid, precise, and low-priced diagnosis results [12, 13].
One of the most common used approaches in biomedical detection of diseases is determining the level of valuable specific biomarkers in different tissues. Biomarkers are biological molecules which can indicate particular medical or clinical conditions including the infections, emergence of a disease, mal/well-functioned biological process, etc. . Due to the intelligent nature of biomolecules, biomarkers offer higher sensitivity, selectivity, specificity, and accuracy compared to the chemical indicators. The biomarkers can be successfully used for screening, early detection, and prognosis of different diseases and malignancies. Various types of biomarkers (proteins, nucleic acids, metabolites, etc.) have been reported that in some cases are currently the common targets of commercial diagnosis kits [6, 15].
The biomarkers can be divided into two groups based on their application: diagnostic biomarkers and screening biomarkers. The evaluation of diagnosis biomarkers is frequently done by analysis of enzymes activity, exploring antigen-antibody interaction, or detecting a specific nucleic acid sequence that can be achieved via biotechnological or biochemical techniques including PCR, ELISA, FISH, IHC, etc. [10, 16-18]. An ideal diagnostic biomarker should provide high sensitivity, remarkable specificity, and agreeable accuracy in differentiating between diseases. These biomarkers should additionally have the potential for investigating the drug efficacy and estimating the result of chosen treatment, particularly in cancers [16, 18]. On the other hand, a perfect screening biomarker should be extremely specific, reduce false positive errors, be able to show the different stages of the disease, be determined easily and without the need for complex medical procedures, and also be affordable. Biomolecules that are released into the body liquids (e.g. blood, saliva, and urine) are of the beneficial targets for early screening acute and chronic illnesses, infectious diseases, genetic disorders, and cancers [19-21].
microRNAs are of the newest and most reliable biomarkers reported for the early detection, diagnosis, metastasis, prognosis, and assessment of treatment [22, 23]. In following section, these valuable tiny biomolecules are briefly discussed.
The aim of this article is to review the concept of medical diagnostics, biosensors, electrochemical biosensors and to emphasize the role of nanotechnology in nanobiosensor development and performance for application in microRNA (miRNA), as a new and reliable biomarker, detection. We have also summarized recent ideas and advancements in the field of electrochemical nanobiosensors for miRNA detection, and the important breakthroughs are explained.
microRNAs (miRNAs) as valuable biomarkers for medical diagnosis
miRNAs are a class of small, non-coding endogenous RNAs with about 22 nucleotides long that can negatively control their target gene expression at post-transcription levels. Since their discovery in 1993, the essential role of miRNAs has been frequently reported in regulating different genes of humans [24, 25]. The Figure 1 schematically shows the steps of generation and maturation of a miRNA and also, its mode of function in gene regulation.
Due to the involvement of miRNAs in regulating critical genes of vital biological pathways and mechanisms including cell cycle and apoptosis, considerable differences in their expression patterns in healthy and cancerous cells/tissues has been found so far [22, 24]. In addition, a large group of miRNAs categorized as “circulating miRNA” are released into the blood and their expression level is specifically related with the disease stage, beginning of tumorigenesis , cancer development, or metastasis [11, 26]. Circulating miRNAs are considerably stable and can be easily evaluate through blood sampling and following molecular analysis. Having all these advantageous characteristics makes these highly-conserved molecules as ideal biomarkers for being used in medical diagnosis [11, 26].
There are numerous methods designed and suggested for miRNA quantification and some of them including real-time qPCR, northern blotting, microarray, and Deep sequencing of transcriptome (RNAseq) are more preferable due to their specific advantages [11, 28]. Northern blotting is the most ancient method introduced in 1977 and consists of size-based separation of RNA samples and their final hybridization with a complementary probe. The application of this technique is limited because of its low sensitivity, time-consuming procedure, and the use of dangerous radioactive labels [29, 30]. Microarray-based methods were of the interest for years due to their potential for simultaneous examinations of huge number of samples and gaining massive data. Despite of easy manual, microarray-based analysis has been known as a qualitative technique that cannot provide essential sensitivity in miRNA quantification [31, 32].
Recently, deep sequencing of transcriptome has been also used for investigating miRNA in biological samples. Deep sequencing of transcriptome benefits greatly from high-throughput technology for parallel analyses and the ability to explore expression of all annotated miRNAs [33, 34]. However, this technique has shown promising application for discovery of novel miRNAs instead of being used as a quantification handy tool for diagnosis or screening purposes. Nowadays, real-time qPCR is the most common used method for miRNA evaluation. It is a sensitive method which provides multi-analysis even in small labs . In recent years, various strategies such as using the stem-loop primers have been introduced for optimizing this method toward miRNA evaluation . However, the small size of miRNAs still limits this technique in discriminating between miRNA precursors and mature miRNA. Furthermore, real-time qPCR is generally considered as an advanced expert-dependent technique that needs expensive reagents and equipment . Based on all the advantages and shortcomings of conventional methods, the search for better strategies that can lead to rapid inexpensive miRNA determination is still essentially warranted.
Development of miRNA biosensors has become one of the most attractive field of research in last decade. This interesting approach has presented many biosensing systems that in spite of significant variation, have some key features in common . In the next parts, we will discuss the electrochemical miRNA biosensors and see how they have risen the hope for precise screening and early diagnosis of lethal diseases due to remarkable advantages over the commonly-used methods.
Electrochemical biosensors for miRNA quantification
As IUPAC defined, a biosensor is a sensor which uses biological/biochemical interaction for detection of the analyte. Biosensors generally consist of two major parts: bioregonition element also called “bioreceptor” is the one which interacts with the analyte and is made of biomolecules (protein, nucleic acids, PNA, etc.), cell receptors, enzymes, organelles, tissue, or even a whole cell . This part is assembled with a physicochemical transducer that is responsible for converting the signal resulting from bioreceptor-analyte interaction into measurable signal. The nature of transducer significantly influences the sensitivity of the biosensor and usually is the basis of biosensor classification [4, 36] (Figure 2).
Among the various types of biosensors, the electrochemical ones have been found more applications in biomedical fields due to their impressive sensitivity, high compatibility with advanced technologies, portability, and affordability . Particularly, the association of remarkable features of electrochemical methods with high-selective nature of nucleic acids has led to fabricate successful biosensors for detecting all types of nucleic acids [4, 6]. As far as the detection of specific nucleic acid sequences is really valuable in the diagnosis of bacterial/viral infections and contaminations, these electrochemical biosensing devices can be effectively helpful in biomedical, pharmaceutical, and environmental applications. In the case of miRNAs, a well-designed electrochemical analysis can promisingly make the early detection of lethal diseases possible, lead to apply more efficient treatment, and enhance the patients’ quality of life [7, 9, 38, 39].
Electrochemical biosensors can be categorized into five main groups based on the type of transducer and measurement method: Potentiometry , Amperometry , Voltammetry , Conductometry , Impedimetry , and Ion charge or field effect . The conductometry- and impedimetry-based biosensors are commonly known as label-free electrochemical biosensors and can determine the analyte according to the behavior of electroactive species on the electrode surface . However, as shown in Figure 3, the voltammetric modes are the most frequently used electrochemical techniques preferred for qualitative and quantitative analyses. In electrochemical miRNA biosensors, cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square wave voltammetry (SWV) are the most attractive techniques due to higher sensitivity [7, 46, 47].
In almost all of the miRNA biosensors, the detection procedure has been done based on the specific hybridization of miRNA target with complementary probe. The probe is generally labeled with the materials which are able to generate an electrochemical signal . In another strategy, miRNA biosensors have been fabricated using electroactive labels such as ferroceneboronic acid , methylene blue , or oracet blue  which showed higher tendency to intercalate into the double stranded nucleic acids compared to single strands. Hence, there are three main steps in fabricating an electrochemical miRNA biosensor: 1) immobilization of complementary probe on the electrode surface; 2) hybridization of miRNA target with the probe on the surface of electrode; and 3) electrochemical analysis of the modified electrode [5, 7].
Although the design of biosensor structure including the probe immobilization methodology, the biosensing platform, and the modification of electrode surface play the crucial role in fabricating an electrochemical biosensor and is usually presented as the identifying features of a device. however, the target is also considered as an important part that should be valuable to be screened or measured. Figure 4 shows the frequent miRNAs chosen as the target of reported miRNA biosensors. As it can be seen, miR-21 is the most common miRNA among the others (32%) that can be explained due to established contribution of this miRNA in numerous types of malignancies .
Moreover, the electrochemical miRNA biosensors have been presented as the more sensitive devices with promising medical application, but, enhancing the sensitivity, increasing the selectivity, expanding the linear range, lowering the detection limit, decreasing the device size, and facilitating the portability have been always the desirable criteria to meet [52, 53]. Therefore, various approaches have been presented in order to reach an ideal miRNA biosensor. Using different enzymes involved in oxidoreduction reactions has been reported in lots of electrochemical miRNA biosensors while the enzymes are responsible for generating the final electrochemical signal [54, 55]. In these biosensors, the substrate of the enzyme is added to the reaction zone as the label of the probe. It worth noting that such enzyme-based biosensors need strict optimized condition in order to keep the enzyme stable and active during biosensor fabrication and electrochemical measurement . Another idea is employing conductive polymers for electrode modification. These polymers and their derivatives can considerably expedite the electron transfer on the electrode that lead to improvement of electrochemical behavior of electrode and amplification of detection signal . Furthermore, during the recent decade, the engagement of nanomaterial in biosensor technology has opened interesting new doors for miniaturizing the electrochemical biosensors while improving their analytical capabilities . The great impact of nanotechnology in electrochemically miRNA biosensors construction will be discussed in the next section.
Electrochemical nanobiosensors for miRNA quantification
In recent years, the advancements of science and technology at the nanoscale have entered extensively into the biosensor technology, especially electrochemical ones . Nanotechnology is defined as the potentials for producing, fabricating, and manipulating materials at molecular/atomic levels in order to control their properties and applications . Since nanomaterials (with a size range of 1-100 nm) generally show novel and different optical, electrical, electrochemical, and magnetic properties compared to bulk forms, using them in design and construction of biosensing systems not only reduces the device size, but also adds extra useful characteristics to biosensors leading to effective improvements in accuracy and reproducibility of bioanalysis result [58, 59]. Remarkable surface to volume ratio and a size range similar to the biomolecules are responsible for efficient non-destructive reactions between nanomaterials and biomarkers [7, 53, 60].
A wide range of nanoparticles, nanorods, nanowires, nanoclusters, nanocomposites, etc. have been used in electrochemical biosensor fabrication for miRNA determination (Figure 5). These nanomaterials have mainly participated in electrochemical signal amplification, bioseparation, and biomolecule immobilization platform [52, 53].
Most of the recent advances in the field of electrochemical nanobiosensors for miRNA quantification have been summarized in the Table 1. The Table does contain specifications of nanobiosensors such as detected miRNA, Electrode and Electrochemical method, Mechanism, applied nanomaterials, Dynamic range and Limit of Detection (LOD).
One of the first studies on electrochemical miRNA nanobiosensors was published in 2006 where the authors reported the use of electrocatalytic OsO2 nanoparticles for signal amplification . The detection procedure was begun with miRNA peroxidation for generating dialdehydes at the 3′ end. After the hybridization of oxidized miRNA with previously-immobilized probe on the electrode surface, the isoniazid-modified OsO2 nanoparticles were added. The final determination was done via oxidization of hydrazine. The detection limit of this biosensor was evaluated to be 80 fM . In another study by Jolly et al., a highly sensitive dual mode electrochemical platform has been developed for miRNA detection . As the Figure 6A demonstrates, the gold electrode was first modified with a thiolated peptide nucleic acids probe sequence and 6-mercapto-1-hexanol (MCH). After hybridization with the target miR-145, positively-charged gold nanoparticles have been added and by using thiolated ferrocene, the square wave voltammograms have been recorded. A wide dynamic range from 1 fM to 100 nM along with a limit of detection of 0.37 fM have been reported for this biosensing platform . In another study by Dong et al., a miRNA biosensor was fabricated using oligonucleotide encapsulated silver nanoclusters as electrochemical probe. This nanoprobe played a dual role in specific detection of miRNA sequence and effective electrochemical signal amplification. The silver nanoclusters mimicked enzymatic behavior for reducing H2O2 and generating the final detection signal. This miRNA nanobiosensor showed a linear range of 100 fM-10 nM and reached a detection limit of 67 fM .