miRNA Detection Methods Research Articles

Rapid and highly sensitive hairpin structure-mediated colorimetric detection of miRNA

Herein, we report a novel hairpin structure–mediated diagnostic method for the simple and rapid colorimetric detection of miRNA through the sensing of pyrophosphate. When the hairpin structure of the template DNA (h-Probe) was hybridized with the primer, the DNA primer extension mediated by nPfu special enzyme was blocked. However, this h-Probe was extended using nPfu special enzyme, upon the structural change of the template DNA, from a hairpin to a linear structure, in the presence of the target miRNA. The miRNA-hybridized template DNA sequence was cleaved by a duplex-specific nuclease (DSN), which cleaved the DNA from the RNA–DNA hybrid, thereby allowing the target miRNA to be recycled. Primer extension using nPfu special enzyme produced pyrophosphate when nucleotide triphosphate was incorporated into the DNA; this pyrophosphate was sensed in terms of a color change, from pink to colorless, when using pp Probe, a probe developed previously by our group. This novel system for the colorimetric detection of target miRNA operated with high sensitivity (LOD = 132 aM) and selectivity, with the whole detection process requiring only 30 min. Furthermore, this system could also detect miRNA fluorimetrically with similar sensitivity (LOD = 105 aM), highlighting the dual-sensing properties of pp Probe. This unique, extremely simple, and rapid system for the detection of miRNA through a highly sensitive color change would presumably be useful in applications requiring point-of-care detection.

Rapid and Sensitive Detection of miRNA Based on AC Electrokinetic Capacitive Sensing for Point-of-Care Applications.

A sensitive and efficient method for microRNAs (miRNAs) detection is strongly desired by clinicians and, in recent years, the search for such a method has drawn much attention. There has been significant interest in using miRNA as biomarkers for multiple diseases and conditions in clinical diagnostics. Presently, most miRNA detection methods suffer from drawbacks, e.g., low sensitivity, long assay time, expensive equipment, trained personnel, or unsuitability for point-of-care. New methodologies are needed to overcome these limitations to allow rapid, sensitive, low-cost, easy-to-use, and portable methods for miRNA detection at the point of care. In this work, to overcome these shortcomings, we integrated capacitive sensing and alternating current electrokinetic effects to detect specific miRNA-16b molecules, as a model, with the limit of detection reaching 1.0 femto molar (fM) levels. The specificity of the sensor was verified by testing miRNA-25, which has the same length as miRNA-16b. The sensor we developed demonstrated significant improvements in sensitivity, response time and cost over other miRNA detection methods, and has application potential at point-of-care.

Cascade Strand Displacement and Bipedal Walking Based DNA Logic System for miRNA Diagnostics.

DNA logic gated operations empower the highly efficient analysis of multiplex nucleic acid inputs, which have attracted extensive attention. However, the integration of DNA logic gates with abundant computational functions and signal amplification for biomedical diagnosis is far from being fully achieved. Herein, we develop a bipedal DNA walker based amplified electrochemical method for miRNA detection, which is then used as the basic unit for the construction of various logic circuits, enabling the analysis of multiplex miRNAs. In the bipedal walking process, target triggered strand displacement polymerization is able to produce a large number of strands for the fabrication of three-way junction-structured bipedal walkers. The following catalytic hairpin assembly ensures the walking event and the immobilization of signal probes for output. Ultrahigh sensitivity is realized due to the integration of dual signal amplification. In addition, under logic function controls by input triggered cascade strand displacement reactions, NOT, AND, OR, NAND, NOR, XOR, and XNOR logic gates are successfully established. The as-developed DNA logic system can also be extended to multi-input modes, which holds great promise in the fields of DNA computing, multiplex analysis, and clinical diagnosis.

Three-dimensional nanorod array for label-free surface-enhanced Raman spectroscopy analysis of microRNA pneumoconiosis biomarkers

Novel approaches are required to overcome the challenges associated with conventional microRNA (miRNA) detection methods and realize the early diagnosis of diseases. This work describes a novel label-free surface-enhanced Raman spectroscopy (SERS) method for the detection of the miRNA biomarkers for pneumoconiosis on a three-dimensional Au-coated ZnO nanorod array (Au-ZnO NRA). The Au-ZnO NRA substrate, which was fabricated via a modified seeding method combined with ion sputtering, provided a high enhancement factor and good spatial uniformity of the signal. With the Au-ZnO NRA, the SERS spectra of miRNAs were obtained in 30 s without labeling at room temperature. Density functional theory calculations were performed to understand the structural fingerprints of the miRNAs. Principal component analysis was carried out to identify the pneumoconiosis biomarkers based on their fingerprint SERS signals. Dual-logarithm linear relationships between the SERS intensity and the miRNA concentration were proposed for quantitative analysis. The label-free SERS method has limits of detection on the femtomolar level, which is much lower than the concentrations of the miRNA biomarkers for pneumoconiosis in lung fibroblasts.

Microfluidic circulating reactor system for sensitive and automated duplex-specific nuclease-mediated microRNA detection

Duplex-specific nuclease signal amplification (DSNSA) is a promising microRNA (miRNA) quantification strategy. However, existing DSNSA based miRNA detection methods suffer from costly chemical consumptions and require laborious multi-step sample pretreatment that are prone to sample loss and contamination, including total RNA extraction and enrichment. To address these problems, herein we devised a pneumatically automated microfluidic reactor device that integrates both analyte extraction/enrichment and DSNSA-mediated miRNA detection in one streamlined analysis workflow. Two flow circulation strategies were investigated to determine the effects of flow conditions on the kinetics of on-chip DSNSA reaction in a bead-packed microreactor. With the optimized workflow, we demonstrated rapid, robust on-chip detection of miR-21 with a limit-of-detection of 35 amol, while greatly reducing the consumption of DSN enzyme to 0.1 U per assay. Therefore, this microfluidic system provides a useful tool for many applications, including clinical diagnosis.

Label-free microRNA detection through analyzing the length distribution pattern of the residual fragments of probe DNA produced during exonuclease III assisted signal amplification by mass spectrometry

Biosensors based on various spectroscopic techniques discriminate the target microRNA (miRNA) from non-target ones with single nucleotide polymorphisms (SNPs) according to the differences in signal intensities which can be caused by other factors besides SNPs. As a result, they are liable to produce false positive results. Herein, we report an attempt to develop a false-positive resistance, sensitive and reliable mass spectrometric platform for miRNA detection. In the proposed platform, the qualitative and quantitative information of the target miRNA was obtained through analyzing mass spectral responses of the multiply charged ions of the residual fragments of the probe DNA produced during exonuclease III assisted signal amplification reaction using an advanced data analysis method. The proposed platform could achieve sensitive and accurate quantitative results for the target miRNA (e.g., miRNA-141) in complex medium with a detection limit of about 1 pM, and unambiguously identify non-target miRNAs with SNPs based on the length distribution patterns of residual fragments of probe DNA. The findings obtained in this study might open an avenue for the design of new miRNA detection methods based on mass spectrometry in combination with various nuclease assisted signal amplification strategies.

Highly Sensitive and Specific Mass Spectrometric Platform for miRNA Detection Based on the Multiple-Metal-Nanoparticle Tagging Strategy.

The multiple-metal-nanoparticle tagging strategy has generally been applied to the multiplexed detection of multiple analytes of interest such as microRNAs (miRNAs). Herein, it was used for the first time to improve both the specificity and sensitivity of a novel mass spectroscopic platform for miRNA detection. The mass spectroscopic platform was developed through the integration of the ligation reaction, hybridization chain reaction amplification, multiple-metal-nanoparticle tagging, and inductively coupled plasma mass spectrometry. The high specificity resulted from the adoption of the ligation reaction is further enhanced by the multiple-metal-nanoparticle tagging strategy. The combination of hybridization chain reaction amplification and metal nanoparticle tagging endows the proposed platform with the feature of high sensitivity. The proposed mass spectrometric platform achieved quite satisfactory quantitative results for Let-7a in real-world cell line samples with accuracy comparable to that of the real-time quantitative reverse-transcriptase polymerase chain reaction method. Its limit of detection and limit of quantification for Let-7a were experimentally determined to be about 0.5 and 10 fM, respectively. Furthermore, due to the unique way of utilizing the multiple-metal-nanoparticle tagging strategy, the proposed platform can unambiguously discriminate between the target miRNA and nontarget ones with single-nucleotide polymorphisms based on their response patterns defined by the relative mass spectral intensities among the multiple tagged metal elements and can also provide location information of the mismatched bases. Its unique advantages over conventional miRNA detection methods make the proposed platform a promising and alternative tool in the fields of clinical diagnosis and biomedical research.

Recent trends in application of nanomaterials for the development of electrochemical microRNA biosensors.

The biology of the late twentieth 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. These small RNAs (18-25 nucleotides), called microRNAs (miRNAs), are implied in several biological processes such as differentiation, metabolic homeostasis, or cellular apoptosis and proliferation. The discovery in 2008 that the presence of miRNAs in body fluids could be correlated with cancer (prostate, breast, colon, lung, etc.) or other diseases (diabetes, heart diseases, etc.) has made them new key players as biomarkers. Therefore, miRNA detection is of considerable significance in both disease diagnosis and in the study of miRNA function. Until these days, more than 1200 miRNAs have been identified. However, traditional methods developed for conventional DNA does not apply satisfactorily for miRNA, in particular due to the low expression level of these miRNA in biofluids, and because they are very short strands. Electrochemical biosensors can provide this sensitivity and also offer the advantages of mass fabrication, low-cost, and potential decentralized analysis, which has wide application for microRNAs sensing, with many promising results already reported. The present reviewsummarizessome newly developed electrochemical miRNA detection methods.

Ultrasensitive Detection of Nasopharyngeal Carcinoma-Related MiRNA through Garland Rolling Circle Amplification Integrated Catalytic Hairpin Assembly.

MiRNA is reported to be closely related to nasopharyngeal carcinoma and has the potential to be a biomarker for the early diagnosis of nasopharyngeal carcinoma. However, the detection of miRNAs remains to be improved, given their complexity and low sensitivity. Herein, we propose here a novel miRNA detection method through the integration of garland RCA and CHA. In detail, the method is composed of two important signal amplification processes. For the first signal amplification process, the target miRNA could initiate garland RCA and then generate a nicking site on the products with the assistance of Nb.BbvCI enzymes. Afterward, a CHA process is induced with a designed H probe through the two signal amplification processes; the method exhibited a much-improved sensitivity. At last, we believe that this method is a promising approach capable of being applied in screening, diagnosing, and prognosticating multiple diseases.

Construction of microRNA-21 terahertz metamaterial sensing method based on duplex-specific nuclease triggered rolling circle amplification

Objective: To construct of a terahertz metamaterial sensing method for detection of microRNA-21 (miRNA-21) based on duplex-specific nuclease triggered rolling circle amplification. Methods: First, a THz metamaterial sensing method was constructed, and verified by polyacrylamide gel electrophoresis and zeta potential; after optimizing the detection conditions of the sensor, different concentrations of miRNA-21 and other different miRNAs were detected. And this biosensor was compared with other miRNA detection methods; finally, the recovery rate of the biosensor was evaluated. Results: Under the optimal experimental conditions, through the dual signal amplification strategy of duplex-specific nuclease (DSN) cycle recognition and rolling circle amplification (RCA), the THz metamaterial sensor has a response range of 10 fmol/L to 10 nmol/L to the target miRNA-21, with a detection limit of 8.49 fmol/L. And the biosensor has good specificity with the ability to recognize the target miRNA-21 from a variety of microRNAs. And the experiment with recovery rate from 94.33% to 115.33% has been further verified in commercial human serum samples. Conclusion: The terahertz biosensor can achieve highly sensitive and specific detection of the target miRNA-21, which proves the potential for label-free diagnosis and early warning of miRNA-related diseases.

Magnetic nanoparticle-based amplification of microRNA detection in body fluids for early disease diagnosis.

Circulating biomarkers such as microRNAs (miRNAs), short noncoding RNA strands, represent prognostic and diagnostic indicators for a variety of physiological disorders making their detection and quantification an attractive approach for minimally invasive early disease diagnosis. However, highly sensitive and selective detection methods are required given the generally low abundance of miRNAs in body fluids together with the presence of large amounts of other potentially interfering biomolecules. Although a variety of miRNA isolation and detection methods have been established in clinics, they usually require trained personnel and often constitute labor-, time- and cost-intensive approaches. During the last years, nanoparticle-based biosensors have received increasing attention due to their superior detection efficiency even in very low concentration regimes. This is based on their unique physicochemical properties in combination with their high surface area that allows for the immobilization of multiple recognition sites resulting in fast and effective recognition of analytes. Among various materials, magnetic nanoparticles have been identified as useful tools for the separation, concentration, and detection of miRNAs. Here, we review state-of-the-art technology with regard to magnetic particle-based miRNA detection from body fluids, critically discussing challenges and future perspective of such biosensors while comparing their handling, sensitivity as well as selectivity against the established miRNA isolation and detection methods.

Ultrasensitive homogeneous detection of microRNAs in a single cell with specifically designed exponential amplification.

We firstly developed an ultrasensitive method based on specifically designed exponential amplification for miRNA detection with simple operation in homogeneous solutions. The proposed assay can detect miRNAs at a concentration as low as 1 aM and can be successfully applied for routine miRNA detection in a single cell.

A versatile magnetic bead-based flow cytometric assay for the detection of thyroid cancer related hsa-miR-221-3p in blood and tissues.

In vitro detection of low abundance biomolecules including microRNAs (miRNAs) is essential to biological research and early clinical diagnosis. In this work, a versatile magnetic bead (MB)-based flow cytometric assay was developed for the detection of hsa-miR-221-3p, which is strongly associated with papillary thyroid carcinoma (PTC). In the presence of hsa-miR-221-3p, the complementary DNA probe attached to the surface of MBs is hybridized with the target to form DNA/RNA heteroduplexes. After the recognition of the DNA/RNA heteroduplexes by PicoGreen, the fluorescence signals of each MB were readily detected using a flow cytometer. This assay can selectively detect hsa-miR-221-3p with a detection limit of 2.1 pM. The practicality of the assay is demonstrated by the discrimination of thyroid cancer tissues from normal tissues, and a satisfactory result is obtained. Moreover, this assay can be rapidly carried out in one step at room temperature, providing a generic method for the sensitive detection of miRNAs in molecular diagnosis.

NEase-based amplification for detection of miRNA, multiple miRNAs and circRNA

MiRNAs are particularly desirable biomarkers for the diagnosis of many cancers, but the methods for simultaneous detection of multiple miRNAs are rarely published. In addition, circRNAs are a novel class of non-coding RNAs and it has been accepted that circRNAs participate in regulating various biological processes. Herein, we design a method using NEase which has highly selectivity and activity to amplify fluorescence signal, so single miRNA, circRNA and multiple miRNAs could be simply quantified through our proposed assay. Furthermore, this method also can detect miRNA level in different cell lines.

A pico-HPLC-LIF system for the amplification-free determination of multiple miRNAs in cells

MicroRNAs are a class of important biomarkers, and the simultaneous detection of multiple miRNAs can provide valuable information about many diseases and biological processes. Amplification-free determination has been developed for the analysis of multiple miRNAs because of its characteristic low cost and high fidelity. Herein, a method for the amplification-free analysis and simultaneous detection of multiple miRNAs based on a so-called pico-HPLC-LIF system is described. In this process, a bare open capillary with an inner diameter of 680nm is used as a separation column for a sample volume of several hundreds of femtoliters (300fL), followed by separation and detection. The technique has a zeptomolar limit of detection. The method was applied to detect cellular miRNA from adenocarcinomic human alveolar basal epithelial (A549) cell extracts, and the simultaneous detection of the mir-182, miR-155, and let-7a was achieved. The results showed that the expression of mir-182 and miR-155 was up-regulated and that of let-7a was down-regulated in A549 cells. This method for multiple miRNAs detection is expected to have broad applications in miRNA-based disease diagnosis, prognosis, treatment, and monitoring.

A label-free mass spectrometry detection of microRNA by signal switching from high-molecular-weight polynucleotides to highly sensitive small molecules

MicroRNAs (miRNAs) are associated with various cellular processes and have been recognized as potential biomarkers for many human diseases. The sensitive and accurate determination of miRNA expression levels in biological specimens is highly significant for understanding their biological functions and clinical diagnosis. Mass spectrometry (MS) has shown its potential to study bioactive molecules, however, direct MS analysis of miRNAs is often hampered by limited sensitivity. For sensitive detection of miRNAs, indirect methods are generally employed through the use of DNA probes labeled with peptides or metal elements. In this work, we proposed a novel MS-based label-free strategy for miRNA quantification. A dual-amplification system was developed by using a padlock probe containing the poly(thymine) sequence in combination with rolling circle amplification (RCA). The specific recognition of target miRNA by the padlock probes produced long single-stranded DNAs containing poly (adenine) segments, which guaranteed the specificity of detection and realized primary amplification. Then the RCA products were extracted and treated with acid to release a large number of free adenines as reporter molecules for secondary signal amplification. Overall, the quantification of target miRNA was carried out by signal switching from high-molecular-weight RCA products to highly sensitive small molecule of adenine. The developed method achieved a linear detection range from 200 amol to 100 fmol for miRNA-21 with a limit of detection of 50 amol, and successfully applied to detect endogenous miRNA-21 levels from lung cancer cells. Overall, the present study provides a sensitive, specific MS-based method for miRNA detection and holds great potential for further application of MS technology to detect other biomarkers in biomedical research and early clinical diagnosis.

SERS Platform Based on Bimetallic Au-Ag Nanowires-Decorated Filter Paper for Rapid Detection of miR-196ain Lung Cancer Patients Serum

Detecting microRNA (miRNA) biomarkers expression is of great significance for the diagnosis and treatment of lung cancer. Surface-enhanced Raman scattering (SERS) has achieved microRNA sensing for the diagnosis of primary liver cancers. In this work, we developed a SERS technology for the rapid detection of lung cancers-related miRNA (miR-196a) using bimetallic Au-Ag nanowire (AgNW@AuNPs) substrates coupled with the target hairpin DNA. The finite-difference time-domain simulation proved that a large number of “hot spots” were generated between the AgNW and AuNPs, which resulted in a huge enhancement of the signal of Raman reporters. Filter paper treated by hexadecenyl succinic anhydride hydrophobic and modified with AgNWs@AuNPs was used as capturing substrate. The detection limits of miR-196a in PBS and serum were as low as 96.58 aM and 130 aM, respectively. Studies on nonspecific sequence and single-base mismatch of miRNA demonstrated that SERS-based platform was highly selective, excellent uniform, and reproducible. Finally, the platform was used to show that the miR-196a expression in the serum of lung cancer patients was much higher than that in healthy people. The detection results indicated that the SERS platform had potential applications in cancer diagnosis and might be a viable alternative to the conventional miRNA detection method, the real-time polymerase chain reaction (RT-PCR) technology.

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.

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.