MicroRNA (miRNA) biosensors are analytical devices that involve interactions between the target miRNA strands and recognition element on a detection platform to produce signals that can be measured to indicate levels or the presence of the target miRNA. Research into miRNA biosensors shows shorter readout times, increased sensitivity and specificity of miRNA detection and lower fabrication costs than conventional miRNA detection methods.

miRNAs are a category of small, non-coding RNAs in the range of 18-25 base pairs in length. miRNAs regulate cellular processes such as gene regulation post-transcriptionally, and are abundant in body fluids such as saliva, urine and circulatory fluids such as blood. Also, miRNAs are found in animals and plants and have regulatory functions that affect cellular mechanisms. miRNAs are highly associated with diseases such as cancers and cardiovascular diseases. In cancer, miRNAs have oncogenic or tumor suppressor roles and are promising biomarkers for disease diagnosis and prognosis. Many techniques exist in clinical and research settings for analyzing miRNA biomarkers. However, inherent limitations with current methods, such as high cost, time and personnel training requirements, and low detection sensitivity and specificity, create the need for improved miRNA detection methods.

Background miRNAs are associated with physiological and pathological processes; hence, measuring them in fields like human health, agriculture, and environmental testing is in demand. Here are some key aspects of the necessity of detection of miRNAs: Potential biomarkers: miRNAs have specific expression in diseases such as cancer, cardiovascular diseases, and autoimmune diseases, which can be beneficial for early detection, prognosis and monitoring for response to treatments. Furthermore, because miRNAs are in body fluids like urine, saliva, and blood, detecting miRNAs is less invasive than methods such as biopsies. This is more comfortable for patients and can facilitate more frequent monitoring of their disease. Molecular mechanisms: As miRNAs have regulatory roles in gene expression and signaling pathways, studying them can give the etiology of diseases and targeting them can provide therapeutic options. Personalized medicine: Because the specific expression of miRNAs offers a promising avenue for enhancing personalized medicine, they provide a deeper understanding of individual disease risk, treatment response, and prognosis, which help clinicians make better informed clinical decisions.

History of miRNA detection technology

Early and current detection methods The first miRNA (lin-4) was detected by Victor Ambros in Caenorhabditis elegans in 1993. The first detection method was Northern blotting (1977), which had low sensitivity. Following that was Reverse Transcription Polymerase Chain Reaction (RT-PCR) (1990), which had high detection sensitivity. Northern Blotting: Northern blotting involves hybridizing miRNA probes (short nucleic acid sequences) with miRNAs, followed by their separation on a gel and transfer to a membrane. The probes are labeled with radioactive isotopes (raising safety and environmental concerns), enzymes, or fluorescent markers. The quantity of RNA present is inferred from the probe signal's intensity. While Northern Blotting is highly specific and helpful for validating high-throughput methods like RNA-seq, it requires a large sample volume, is time-consuming, and lacks precision in quantification analysis. Real-Time Reverse Transcription–Polymerase Chain Reaction (Real-time RT-PCR): This method starts with converting miRNA into cDNA using reverse transcriptase enzymes. The cDNA is then amplified using sequence-specific primers, a process monitored by fluorescent dyes or probes. Real-time RT-PCR is noted for its sensitivity and specificity. However, it faces challenges such as the need for standardization, technical complexities (e.g., primer design, sample preparation), time-intensive processes, and high costs.

High-throughput Methods: Microarrays (1990): Microarrays enable the detection of thousands of miRNAs in a single experiment. They consist of a solid surface to which complementary miRNA sequences are attached. Introducing miRNAs allows them to bind to these probes, with the amount of miRNA measured by the fluorescence intensity. Microarrays are cost-effective compared to real-time RT-PCR and NGS but have limitations in detecting low quantities of miRNAs and distinguishing between miRNAs with similar sequences. Next Generation Sequencing (NGS) (2005): NGS begins with RNA extraction and reverse transcription into cDNA, followed by adaptor ligation and amplification. The cDNA is then sequenced on an NGS platform, producing millions of short reads. Expert bioinformaticians and sophisticated tools must align and analyze the data and map reads to reference miRNA sequences for miRNA discovery and identification. NGS offers high sensitivity and specificity for detecting low-quantity miRNAs and identifying miRNAs differing by a single nucleotide.

Principles of microRNA biosensors Three essential elements make up miRNA biosensors: Biological recognition element: they can detect specific target molecules and have different types, including antibodies, antigens, DNA/RNA, aptamers, enzymes, and MIPs (molecularly imprinted polymers). Transducer: following recognition, the transducer is an element required to convert changes in the recognition element to a measurable signal. Based on the type of signal they produce, they are categorized into electrochemical, optical, and mechanical transducers.  Signal processor: computational elements that amplify and process the signals produced from transducers and can be demonstrated by numerical values and digital readouts.

Specificity in miRNA detection The term "specificity" in the context of miRNA biosensors refers to the ability of the biosensor to identify a particular miRNA within a sample that contains various components and miRNAs with similar sequences. The challenge in achieving this specificity derives from the small size of miRNAs, which may differ from each other by only one nucleotide. Consequently, designing biosensors capable of precisely recognizing the target miRNA is essential.

Sensitivity in miRNA detection Sensitivity in miRNA biosensors refers to their ability to detect target miRNAs in low concentrations within samples. Since miRNAs are typically found in small amounts, biosensors are engineered to identify concentrations as low as femtomolar (10^-15) or attomolar (10^-18) levels. Achieving such high sensitivity involves enhancements to recognition elements, amplification, and signal processing techniques. The LoD (limit of detection) is used to determine the concrete value of sensitivity in biosensors, which indicates the lowest concentration of miRNA that can be separated from the background (zero) signal with a specified level of confidence.

The dynamic range in miRNA biosensors refers to the concentrations over which the biosensor can accurately detect the target miRNAs, extending from the lowest detectable LoD to the maximum concentration that can be measured without necessitating sample dilution.

Types of microRNA biosensors

Electrochemical biosensors

Electrochemical biosensors present significant advantages to miRNA detection over conventional miRNA analysis methods. Using simple electronics reduces production costs and increases ease of use in portable system configurations. This allows for a broader scope of use, including environmental, clinical and food analysis applications.

miRNA electrochemical biosensor detection relies on measuring the changes in the electrode-property or electroactive compound redox signal in the transduction of electrochemically active reporter species and hybridization between the target miRNA and complementary probe. Various materials can be made into the transduction element, including silver, gold, graphite or nanoparticle variations of such materials. Detection of electrochemical property changes allows for real-time analysis and kinetics data, an advantage biosensor methods such as optical biosensors lack. Light pollution is not a limitation of electrochemical miRNA biosensors. However, amplification techniques such as rolling circle amplification (RCA) may be required when miRNA concentrations are insufficient to produce an electrical signal.

1. Voltammetric and amperometric electrochemical biosensors Electrochemical miRNA biosensors can be designed to infer voltammetric or amperometric measurements. Upon hybridization of the miRNA target with its complementary probe sequence, voltammetric miRNA biosensors detect the change in current based on a controlled increase or decrease in electric potential on the detection platform. Amperometric-based biosensors detect the change in electric current at a fixed positive electric potential. Recent developments in voltammetric and amperometric miRNA biosensors can be classified as label-based or label-free biosensors, indicating whether or not electroactive labels on the miRNA target are used as the naming suggests. Voltammetric and amperometric label-free (direct detection) miRNA biosensors

First published in 2009, label-free (direct detection) electrochemical miRNA biosensors function without labelling the target miRNA with electrocatalytic nanoparticle tags or hybridization indicators. Label-free miRNA biosensors were initially based on DNA detection through guanine electrooxidation measurements, with the lower detection limit being 5 nM of miRNA. Since then, electrode materials have been developed to increase the sensitivity of detection down to less than 1 pM, such as with graphene and ionic-liquid modified electrodes. For example, Wu et al. (2013) increased the conductivity of the electrode surface of an amperometric biosensor with a multilayer consisting of Nafion, thionine and palladium nanoparticles, which immobilized the target miRNA on the electrode surface for a lower limit of detection of 1.87 pM. Label-free miRNA biosensors detect signals before and after the hybridization of electroactive nucleic acid bases. For instance, doxorubicin-loaded gold nanoparticles (AuNps) have been integrated with a double-loop hairpin probe that hybridizes with the target miRNA to form heteroduplexes, in which duplex specific nucleases hydrolyze DNA in the heteroduplex structures to release target miRNA strands for amplification in a signal amplification system. The limit of detection in such a system is 0.17pM. Voltammetric and amperometric label-based (indirect detection) miRNA biosensors

Label-based (indirect detection) electrochemical miRNA biosensors require electrocatalytic or redox active molecule or nanoparticle labelling of the miRNA target or complementary capture probes for detection. Generally, label-based approaches offer significantly greater sensitivity of miRNA detection than label-free methods, with sensitivity reaching the fM-aM range.

An example is AuNp-superlattice-based miRNA biosensors utilizing the small molecule cationic dye toluidine blue to detect miRNA-21. Toluidine blue acts as a miRNA intercalative label through electrostatic interaction with the negatively charged backbone phosphate groups. On the biosensor, toluidine blue is a redox indicator to measure the oxidation peak current of toluidine blue and indicated hybridization of miRNA. The LoD levels reached 78 aM.

2. Amplification (enzyme)-based electrochemical miRNA biosensors