miRNA Detection Methods Research Articles

Label-free and self-assembled fluorescent DNA nanopompom for determination of miRNA-21.

A label-free fluorescence method based on self-assembled DNA nanopompom has been developed for miRNA-21 detection. In the presence of miRNA-21, three DNA hairpin probes with split G-quadruplex assemble the DNA nanopompom. Based on the isothermal toehold-mediated DNA strand displacement reaction, the target miRNA can be catalytically recycled and trigger three DNA hairpin probes to self-assemble the DNA nanopompom and release the G-quadruplex. The formation of the G-quadruplex increases the fluorescence emission intensity of thioflavin. For thioflavin-based miRNA-21 detection, the excitation and emissionwavelengths are set to425nm and 490 nm, respectively. The limit of detection for miRNA-21 is 0.8pM according to F/F0 = 0.0031 × CmiRNA-21 + 1.0382 (R2 = 0.9978). This sensing system provides a low-cost, effective, and convenient method for miRNA detection, which holds great potential in biochemical diagnosis and clinical practice. Graphical abstract Label-free and self-assembled fluorescent DNA nanopompom for miRNA detection.

Use of Microfluidic Assays to Develop Reliable and Economic Nucleic Acid Application Technologies, Employing MicroRNAs for the Diagnostic Screening of Colon Cancer in Human Stool in Low-Resource Settings

Isolation methods that employ readily-available inexpensive supplies on the open market, which are reliable, as well as economical, such as nucleic acid amplification techniques (NAAT) based on microfluidic technology in low-resource research settings (LRRS) that meets the ASSURED guidelines are essential to develop a noninvasive diagnostic colon cancer screen in stool using micro(mi)RNA molecules. A combination of a microfluidic-based MiRNA stool test with a reliable rolling circle amplification/detection method applied to the quantification of miRNA molecules, result in an affordable sensitive and specific isothermal method for the noninvasive quantitative detection of miRNAs in LRRS. Scientists and engineers have become interested in miRNAs, and they have intensified their efforts to apply emerging simple detection tools to the important bioanalytical challenge of quantifying these small 18-26 nt long molecules. Some of the proposed approaches incorporate novel material, such as simple centrifuges and methods based on microfluidic technology, while others utilize the interesting biological properties of these molecules, such as forming branched RCA structures, allowing for the detection of these biomarker molecules at an attomolar "aM" concentration level, using low cost extraction and isothermal amplification methods in LRRS. We have been interested in studying colorectal cancer (CRC) because it is the 3rd most common malignancy worldwide, and stool can be obtained noninvasively from the patients. We have focused in this research on colon cancer (CC) because it is more common in the USA than rectal cancer (RC). The innovation of our approach lies in the exploratory use of an affordable, quantitative miRNA profiling in noninvasive stool samples in LRRS, whose extracted fragile total RNA is stabilized shortly after excretion from stool by commercially available kits, so it does not ever fragment, followed by quantitative standardized analytical tests that are neither labor intensive, nor require expensive instrumentation, in order to develop apanel of novel miRNA genes for the noninvasive diagnostic screening of early left and right sporadic colon cancers, more economically, and with higher sensitivity and specificity than any other colon cancer screening test currently available on the market. To show the clinical sensitivity and specificity of the proposed quantitative miRNA test using simple methodologies in LRRS,the miRNA results are to be correlated with FOBT, colonoscopy, and pathology data. Standardization establishes test’s performance criteria (sample selection, optimal sample running conditions, preservation and storage), in order to ensure that the assay will perform the same way in any laboratory, by any trained personnel, anywhere in low-resource laboratory settings worldwide.

Sensitive and specific microRNA detection by RNA dependent DNA ligation and rolling circle optical signal amplification

MicroRNAs (miRNAs) have been regarded as potential biomarkers in early diagnosis of cancer. Since the high sequence similarity among miRNA family members, biosensing miRNAs with single-base resolution is still a challenge, particularly when the different base is located at the terminal of miRNA. Herein, we developed two real-time fluorescence monitoring methods for miRNA detection utilizing efficient PBCV-1 DNA ligase mediated target miRNA dependent DNA ligation, followed by rolling circle signal amplification. Compared to duplex-specific nuclease (DSN) enhanced rolling circle transcription (RCT) system, nicking endonuclease (NEase) assisted rolling circle amplification (PRCA-NESA) can provide higher amplification efficiency, and achieve a limit-of-detection of 0.5 amol for miR-17 in 10 μL sample. More importantly, benefiting from the unique characteristics of PBCV-1 DNA ligase, we designed an asymmetric PRCA-NESA method, which can greatly discriminate the single-base difference at either 5′- or 3′-terminals of miRNAs. MiR-17 from various tumor cells also can be reliably detected. In conclusion, our strategy exploited the application potential of PBCV-1 DNA ligase in biosensing, and provided a new idea to highly specific miRNA detection, thereby would possess a promising potential for further application in biomedical research and early cancer diagnosis.

Development of a sensitive primer extension method for direct detection and quantification of miRNAs from plants.

MicroRNAs (miRNAs) are short non-coding RNA molecules that regulate target gene expression in various organisms. Functional studies are therefore required to determine their temporal and spatial expression patterns. Primer extension has been used as a sensitive and reliable approach to identify miRNAs (∼21–22 nt) in the mammalian system and can be used in other systems such as plants. However, a well-defined method is required for ease of application and reproducibility. Here, a radioactive primer extension method was developed for the quantitative detection of miRNAs found in total RNA samples from plants. As a proof of concept, miR173 and miR828 were detected by primer extension in total RNA samples isolated from Arabidopsis. The assay involved the extension reaction of the miRNA guide strand with a radiolabeled specific primer. Using a manual DNA sequencer, primers extended with reverse transcriptase were separated on a denaturing polyacrylamide gel. The gel was then dried and exposed to a PhosphorImager screen for size-dependent product identification up to a single base difference. Quantification was done based on the intensity of radioactive signals by normalizing the cDNA products to an internal control. The primer extension was proven to be efficient to detect and quantify miRNAs in plant total RNA samples without subsequent enrichment of low-molecular-weight RNA species. This method, optimized for Arabidopsis, can be applied to a wide variety of organisms for the detection and quantification of miRNAs as well as siRNAs.

Tissue microRNAs in non-small cell lung cancer detected with a new kind of liquid bead array detection system

BackgroundCommonly used miRNA detection methods cannot be applied for high-throughput analyses. However, this study was aimed to performed a liquid bead array detection system (LBAS) to detect tissue 6 miRNAs in non-small cell lung cancer (NSCLC).MethodsIn this study, evaluation of LBAS was performed to observe the precision, specificity, limitation and stability. Then, a total of 52 primary NSCLC patients who received resection operation without preoperative radiotherapy and chemotherapy between June 2013 and March 2014 were selected, and then the total RNA of the tissues were extracted. We prepared six NSCLC-related miRNAs for LBAS. After optimization and evaluation, LBAS was verified by detecting the relative expression levels of 6 microRNAs in the pathological tissues and corresponding normal tissues of 52 NSCLC patients.ResultsThe results of evaluation of LBAS showed that the Mean Fluorescence Intensity (MFI) of the reaction only added with chimeric probes and beads showed no significant change after 180 days (P > 0.05). And the intra-assay Coefficient of Variation (CV) was between 1.57 and 3.5%, while the inter-assay CV was between 4.24 and 11.27%, indicating this system was ideal for diagnostic reagents. In addition, only the beads corresponding to the additional miRNAs showed high MFIs from 8426 to 18,769, whereas the fluorescence values of the other beads were under background levels (MFIs = 20 to 55) in each reaction, indicating no cross reactivity among the miRNAs. The limit of detection of miR-21, miR-210, miR-125b, miR-155, miR-375, and miR-31 were 5.27, 1.39, 1.85, 2.01, 1.34, and 2.73 amol/μL, respectively, showing that the lowest detection limit of miRNA by this system was under pM level. Then, the relative expression levels of miR-21, miR-210, miR-125b, miR-155, miR-375, and miR-31 by using this system were significantly correlated with NSCLC (P < 0.05). And the results of AUC method indicated that specific of the LBAS system was 94.2%.ConclusionsOur findings suggest that LBAS was simple, high-throughput, and freely combined with absolute quantification. Thus, this system could be applied for tumor miRNAs detection.

A sensitive and specific method for microRNA detection and in situ imaging based on a CRISPR-Cas9 modified catalytic hairpin assembly.

We report here a method for the molecular detection of miRNAs in exosomes and imaging in living cells based on CRISPR–Cas9 and catalytic hairpin assembly. With the cascade signal amplification of catalytic hairpin assembly and high specific recognition of CRISPR–Cas9, the detection method showed a sensitivity of 23.5 fM and was applied for accurate exosomal miR-21 detection and in situ miR-21 imaging.

Diagnostic and prognostic value of microRNAs for Alzheimer's disease: a comprehensive meta-analysis.

Background and aimsRecent research has shown that microRNAs (miRNAs), a class of sequences regulating gene expression without undergoing translational processes, have been accepted as novel biomarkers of diseases. In the present meta-analysis, our main objective was to evaluate the diagnostic value of miRNAs expressed in different body fluids for Alzheimer’s disease (AD), more exactly to analyze the discriminative value of miRNAs between AD and control subjects.MethodsMedline and EMBASE were searched for articles written in English, and because the result reporting modalities were extremely different in the studies included in the analysis, the current article comprises 2 meta-analyses, each of them using different statistical indicators. The first meta-analysis reviewed 10 studies, which were required to provide sufficient information to allow the calculation of AUC or Cohen’s d for size effect. We proposed a second meta-analysis, starting from the drawbacks identified in this first approach, which used different statistical indicators (fold change) provided by other studies (8 studies).ResultsThe present study offers an encouraging role of miRNA families in diagnosing AD. The heterogeneity of miRNA expression between the hippocampus, CSF and peripheral blood, the small sample size of each research study, as well as the different methods for miRNA detection remain the main obstacles in interpreting these results.ConclusionsThere is a need (in a future perspective) to establish the right miRNA combinations as potent diagnostic biomarkers for AD.

Sensitive detection of microRNA using QCM biosensors: sandwich hybridization and signal amplification by TiO2 nanoparticles.

MicroRNA-21 (miR-21) is known to act as an important biomarker for cancer, in that its up-regulation is closely related to several types of malignant tumor. Sensitive and accurate detection of miR-21 using a biosensor is highly challenging. In this study, sensitive and selective detection technology for miR-21 molecules using a quartz crystal microbalance (QCM) biosensor was developed. Sandwich hybridization between miR-21 and specially designed probes and a subsequent TiO2 photocatalytic silver enhancement reaction were the driving forces for sensitive detection with high selectivity for miR-21. Using this strategic approach under optimal conditions, the novel QCM biosensor can detect miR-21 with a LOD of 0.87 pM over the entire linear range from 0.1 pM to 10 μM, with a correlation coefficient of 0.988. In addition, the developed QCM biosensor was very effective in the quantification of miR-21 in serum samples, so the proposed miRNA detection method offers great potential for the diagnosis of early disease, such as cancer and vascular diseases, and could be an excellent alternative for biological research and clinical diagnosis.

Bright carbon nanodots for miRNA diagnostics coupled with concatenated hybridization chain reaction.

We developed a bright carbon nanodot-based miRNA detection method with signal amplification by concatenated hybridization chain reaction (CHCR). In the presence of target miRNA, CHCR was triggered and a frond-like DNA product was formed, which recovered remarkable fluorescence. The location and level of the target miRNA were then indicated.

Fluorescence Signal Amplification Strategies Based on DNA Nanotechnology for miRNA Detection

In this review, the most recent progresses in the field of fluorescence signal amplification strategies based on DNA nanotechnology for miRNA are summarized. The types of signal amplification are given and the principles of amplification strategies are explained, including rolling circle amplification(RCA), catalytic hairpin assembly (CHA), hybridization chain reaction(HCR) and DNA walker. Subsequently, the application of these signal amplification methods in biosensing and bioimaging are covered and described. Finally, the challenges and the outlook of fluorescence signal amplification methods for miRNA detection are briefly commented.

Target-dependent dual strand extension recycling amplifications for non-label and ultrasensitive sensing of serum microRNA

MicroRNAs (miRNAs) are important biomarkers because their abnormal expressions or mutations can usually indicate the development of cancers or other diseases. However, their high family sequence homology and low abundance pose a major challenge for the analysis of miRNAs. In the present work, we developed a dual strand extension recycling signal amplification strategy for non-label detection of miRNA-155 with high sensitivity on the basis of DNA polymerase and the G-quadruplex/thioflavin T (ThT) complexes. The specific miRNA-155 target sequences could initiate two strand extension-based independent recycling cycles under the function of the DNA polymerase, resulting in the production of many active G-quadruplex segments. The dye of ThT further bound the G-quadruplexes to induce greatly enhanced fluorescence for ultra-sensitively detecting miRNA-155 sequences at the low concentration of 35 fM in the dynamic range from 0.1 pM to 100 pM. The proposed detection strategy used the completely unmodified and synthetic DNA as probes and could be performed in homogeneous solutions. This method with a high selectivity could also be used for diluted serum samples. The demonstration of our new method for miRNA detection thus offers it with high potentials for miRNA biomarker analyses with the purpose of clinical diagnostics and biomedical applications.

MicroRNA expression signatures of atrial fibrillation: The critical systematic review and bioinformatics analysis.

Association between microRNA (miRNA) expression signatures and atrial fibrillation has been evaluated with inconsistent findings in different studies. This study aims to identify miRNAs that actually play vital role in pathophysiological process of atrial fibrillation and explore miRNA-targeted genes and the involved pathways. Relevant studies were retrieved from the electronic databases of Embase, Medline, and Cochrane Library to determine the miRNA expression profiles between atrial fibrillation subjects and non-atrial fibrillation controls. Robustness of results was assessed using sensitivity analysis. Subgroup analyses were performed based on species, miRNA detection method, sample source, and ethnicity. Quality assessment of studies was independently conducted according to QUADAS-2. Bioinformatics analysis was applied to explore the potential genes and pathways associated with atrial fibrillation, which were targeted by differentially expressed miRNAs. Form of pooled results was shown as log10 odds ratios (logORs) with 95% confidence intervals (CI), and random-effects model was used. In total, 40 articles involving 283 differentially expressed miRNAs were reported. And 51 significantly dysregulated miRNAs were identified in consistent direction, with 22 upregulated and 29 downregulated. Among above-mentioned miRNAs, miR-223-3p (logOR 6.473; P &lt; 0.001) was the most upregulated, while miR-1-5p (logOR 7.290; P &lt; 0.001) was the most downregulated. Subgroup analysis confirmed 53 significantly dysregulated miRNAs (21 upregulated and 32 downregulated) in cardiac tissue, with miRNA-1-5p and miRNA-223-3p being the most upregulated and downregulated miRNAs, respectively. Additionally, miR-328 and miR-1-5p were highly blood-specific, and miR-133 was animal-specific. In the detection method sub-groups, miRNA-29b and miRNA-223-3p were differentially expressed consistently. Four miRNAs, including miRNA-223-3p, miRNA-21, miRNA-328, and miRNA-1-5p, were consistently dysregulated in both Asian and non-Asian. Results of sensitivity analysis showed that 47 out of 51 (92.16%) miRNAs were dysregulated consistently. Totally, 51 consistently dysregulated miRNAs associated with atrial fibrillation were confirmed in this study. Five important miRNAs, including miR-29b, miR-328, miR-1-5p, miR-21, and miR-223-3p may act as potential biomarkers for atrial fibrillation. Impact statement Atrial fibrillation (AF) is considered as the most common arrhythmia, and it subsequently causes serious complications including thrombosis and heart failure that increase the social burden. The definite mechanisms underlying AF pathogenesis remain complicated and unclear. Many studies attempted to discover the transcriptomic changes using microarray technologies, and the present studies for this hot topic have assessed individual miRNAs profiles for AF. However, results of different articles are controversial and not each reported miRNA is actually associated with the pathogenesis of AF. The present systematic review and meta-analysis identified that 51 consistently dysregulated miRNAs were associated with AF. Of these miRNAs, five miRNAs (miRNA-1-5p, miRNA-328, miRNA-29b, miRNA-21, and miRNA-223-3p) may act as novel biomarkers for AF. The findings could offer a better description of the biological characteristics of miRNAs, meanwhile might serve as new target for the intervention and monitoring AF in future studies.

Exponential amplification reaction and triplex DNA mediated aggregation of gold nanoparticles for sensitive colorimetric detection of microRNA

Abnormal expression of specific microRNAs (miRNAs) is associated with the occurrence, development and prognosis of many diseases. In this study, a miRNA detection method based on exponential amplification reaction (EXPAR) and triplex DNA mediated aggregation of gold nanoparticles (AuNPs) was established. Specifically, one class of AuNPs is conjugated with an EXPAR probe, on which there is a complementary sequence of the target miRNA. The EXPAR reaction is triggered and duplex DNA is formed on the surface of AuNPs when the target miRNA exists. Then, single DNA probe on another class of AuNPs interacts with the duplex DNA to form triplex DNA, leading to the aggregation of the two classes of AuNPs, which could be quantified by UV–vis. The proposed method is highly selective and can afford a detection limit of 0.23 fM. Notably, all the ingredients needed for the analysis can pre-add to a tube and only 30 min is needed for the whole detection process. The method is simple, fast and with considerable selectivity and accuracy, so a great potential for this method is expected to meet the need of point-of-care testing of miRNA.

Simultaneous multiplexed detection of exosomal microRNAs and surface proteins for prostate cancer diagnosis

Since the tumor is extremely heterogeneous, a single biomarker cannot reflect the exact symptoms of the disease or its stage. Exosomes are biomarker reservoirs that provide disease information with a high accuracy, especially when specific markers, including microRNAs (miRNAs) and proteins, are combined. However, currently available exosomal miRNA and protein detection methods are time consuming, expensive, and laborious. Meanwhile, simultaneous detection of an exosomal miRNA and protein in a single reaction is even more challenging. Thus, development of an efficient method for detecting multiple miRNAs and proteins in a single exosomal reaction is highly needed. Herein, to increase the value of using exosomes over other circulating biomarkers for prostate cancer (PCa) liquid biopsy, a method for simultaneous multiplexed in situ detection of exosomal miRNAs and proteins was developed. Exosomal miRNAs and surface proteins were simultaneously detected in captured exosomes with a high specificity, using nano-sized molecular beacons and fluorescent dye-conjugated antibodies. The method allowed the quantitative analysis of various disease-specific miRNAs and surface proteins in PCa cell-derived exosomes in a single exosomal reaction. Overall, simultaneous multiplexed in situ detection of exosomal miRNAs and surface proteins can be developed as a simple, cost-effective, non-invasive liquid biopsy method for diagnosing PCa.

Sensitivity Enhancement of MicroRNA Detection Using a Power-free Microfluidic Chip.

We present a microRNA (miRNA) detection method that achieves enhanced sensitivity by means of a power-free microfluidic chip without the requirement of an external power source. The miRNA detection is completed by sandwich hybridization between probe DNAs and target miRNA with small sample volume (0.5 μL) within 20 min. Fluorescence signals after hybridization were amplified by laminar flow-assisted dendritic amplification (LFDA) using fluorescein isothiocyanate (FITC)-labeled streptavidin (F-SA) and biotinylated anti-streptavidin (B-anti-SA) as amplification reagents. To enhance the sensitivity of on-chip miRNA detection, the hybridization buffer solution was newly optimized with three main components-sodium dodecyl sulfate (SDS), formamide and dextran sulfate-that are known to strongly influence hybridization. An on-chip miRNA detection test in the newly optimized hybridization buffer (0.2% SDS, 5% formamide and 1% dextran sulfate) revealed dramatic increases in both the LFDA signal in the sample channel and the signal-to-background ratio (S/B ratio). Moreover, the LFDA signals in a blank reference channel remained low due to the suppression of non-specific bindings and hybridizations. By changing the hybridization buffer, we obtained an improved limit of detection (LOD) that was 0.045 pM (miRNA-196a) and 0.45 pM (miRNA-331), which are around 30- and 10-fold better than that of when control hybridization buffer was used. The improved performance of our miRNA detection system with short running time and high sensitivity could contribute to future research, including point-of-care diagnostic systems.

Chameleon silver nanoclusters for ratiometric sensing of miRNA

Chameleon silver nanoclusters (AgNCs) have attracted substantial attention over traditional AgNCs, which provide new possibilities for the construction of ratiometric fluorescent biosensors. Herein, a novel method for sensitive and selective detection of miRNA is developed coupling chameleon AgNCs with hybridization chain reaction. Generally, the signal DNA probe is designed to contain two pieces of DNA templates for the synthesis of AgNCs, which are in close proximity due to the hairpin structure. A series of hybridization reactions can be triggered by target miRNA, which open the hairpin of signal probe and separate the two pieces of segmental AgNCs. Controllable color switching from red to yellow is thus achieved. Note that signal generation and amplification are integrated into one step, which is a great advance compared with previous amplified systems. The proposed strategy provides a sensitive fluorescent detection of miRNA down to 2.8 pM with high selectivity. Therefore, this work not only brings novel insights into fluorescence regulation of DNA-templated AgNCs, but also holds great potential for the application of miRNA diagnostics.

Biological Significance of microRNA Biomarkers in ALS-Innocent Bystanders or Disease Culprits?

MicroRNAs (miRNAs) represent potential biomarkers for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). However, whether expression changes of individual miRNAs are simply an indication of cellular dysfunction and degeneration, or actually promote functional changes in target gene expression relevant to disease pathogenesis, is unclear. Here we used bioinformatics to test the hypothesis that ALS-associated miRNAs exert their effects through targeting genes implicated in disease etiology. We documented deregulated miRNAs identified in studies of ALS patients, noting variations in participants, tissue samples, miRNA detection or quantification methods used, and functional or bioinformatic assessments (if performed). Despite lack of experimental standardization, overlap of many deregulated miRNAs between studies was noted; however, direction of reported expression changes did not always concur. The use of in silico predictions of target genes for the most commonly deregulated miRNAs, cross-referenced to a selection of previously identified ALS genes, did not support our hypothesis. Specifically, although deregulated miRNAs were predicted to commonly target ALS genes, random miRNAs gave similar predictions. To further investigate biological patterns in the deregulated miRNAs, we grouped them by tissue source in which they were identified, indicating that for a core of frequently detected miRNAs, blood/plasma/serum may be useful for future profiling experiments. We conclude that in silico predictions of gene targets of deregulated ALS miRNAs, at least using currently available algorithms, are unlikely to be sufficient in informing disease pathomechanisms. We advocate experimental functional testing of candidate miRNAs and their predicted targets, propose miRNAs to prioritise, and suggest a concerted move towards protocol standardization for biomarker identification.

Evaluation of miRNA detection methods for the analytical characteristic necessary for clinical utilization.

miRNAs are promising biomarkers but methods for their measurement are not clear. We therefore examined three miRNA detection technologies and considered the analytical characteristics essential for clinical utilization. TaqMan assays, SplintR-qPCR and miREIA were compared for their absolute quantification bias, conformity and robustness. Absolute concentrations of miR-142-5p, miR-23a-3p and miR-93-5p were measured with all three methods using 30 samples. Robustness was evaluated by measurement of miR-21-5p in five uniform experiments. Correlations were miRNA-specific, but we observed a different absolute concentration range in RT-qPCR (fmol/μl) and methods evading the RT process (amol/μl). Consistently, RT-less methods reported better robustness (CV 8-19%) than RT-qPCR (CV 39-50%). The calibration curve in TaqMan Advanced assay was influenced by dilution media. Methods avoiding RT seem to be a promising future alternative for miRNA measurement.

Fluorescent sensor for detection of miR-141 based on target-induced fluorescence enhancement and PicoGreen

Studies have shown that microRNAs affect the development of tumors. In many cases, they can be applied as biomarkers for the diagnosis of cancer; therefore, simple and sensitive analytical methods for detection of miRNAs are necessary. In this study, miR-141, which is used to diagnose several types of cancer, was detected in water and serum samples using a biosensor designed based on streptavidin-coated magnetic beads (SMBs), complementary sequences of miR-141 and PicoGreen as the fluorescent dye. The method is relatively fast and simple. Briefly, in the presence of miR-141, the complementary sequence forms a DNA:RNA double–strand on the surface of SMBs with intercalated PicoGreen. Upon attachment of the PicoGreen, the fluorescence intensity increased significantly (1000-fold). In the absence of a target, only single-stranded DNA (complementary strand of miR-141) existed on the surface of the SMBs. The fluorescence of the PicoGreen was low. The results revealed that the detection limits of the biosensor for miR-141 were 70 and 113.8 nmol L−1 in deionized water and serum samples, respectively.

Investigation of Electrical Stability and Sensitivity of High Field Modulated Extended Gate FET Biosensors for miRNA Detection

miRNAs have been proven to be highly potential biomarkers for a variety of cancers and cardiovascular diseases. Typically miRNA detection method uses polymerase chain reaction (PCR) to amplify the miRNA concentration. This method has the disadvantage of being time-consuming and unable to directly detect the original concentration. Therefore, this study intends to design a FET biosensor to detect miRNA. In order to design a stable and highly sensitive miRNA FET biosensor, this study used different metal deposition techniques to form sensing electrodes and observed sensor response in different temperatures and aqueous solutions to evaluate the fluctuation of the electrical signal, and found that the ions in the solution will follow the mechanism of Brownian motion. While the Brownian motion reduces, the stability of the electrical signal will be improved. Then we used different concentrations of ssDNA probe to functionalize the sensor chip to detect target miRNA concentration, to compare the dynamic range and sensitivity. The optimization can help us to make a miRNA FET biosensor with high sensitivity, high stability and large detection range. Finally, the target detection experiments with the mismatch sequence demonstrate the high selectivity of the sensor. Comparison of ELISA based fluorescent signals with FET signals shows that FET biosensors have better detection limits. These data proved that the miRNA FET biosensor can directly measure the original concentration of miRNA without amplifying the concentration by PCR, which can effectively solve the problems faced by miRNA detection in the present time. Figure 1