Selective Detection Of miRNA Research Articles

Flexible 3D nanofiber-based SERS biosensor for detection of miRNA-223-3p in early Laryngeal Cancer diagnosis

MicroRNAs (miRNAs) are small non-coding RNAs (18–22 nucleotides) that regulate gene expression and are associated with various diseases, including Laryngeal Cancer (LCa), which has a high mortality rate due to late diagnosis. Traditional methods for miRNA detection present several drawbacks (time-consuming steps, high cost and high false positive rate). Early-stage diagnosis and selective detection of miRNAs remain challenging.This study proposes a 3D flexible biosensor that combines nanofibers (NFs), gold nanoparticles (AuNPs), and an inverse molecular sentinel (iMS) for enzyme-free, SERS-based detection of miRNA-223-3p, evaluated as a potential LCa biomarker. The electrospun flexible nanofibers decorated with AuNPs enhance Raman signal. Selective detection of miRNA-223-3p is achieved by immobilizing an iMS-DNA probe labeled with a Raman reporter (Cyanine 3) on the AuNPs. The iMS distinctive stem-and-loop structure undergoes a conformational change upon interaction with the miRNA-223-3p, producing an “on to off” SERS signal. The proposed sensor demonstrated a linear detection range from 10 to 250 fM, with a limit of detection (LOD) of 19.50 ± 0.05 fM. The sensor selectivity was confirmed by analyzing the SERS signal behaviour in the presence of both Non-complementary miRNA and miRNA with three mismatched base pairs. This easily fabricable sensor requires no amplification and offers key advantages, including sensitivity, flexibility, and cost-effectiveness.

Microsphere-Enhanced Fluorescence-Lightened Solid-Phase Hybridization Assay: The Strategy to Highly Selective Detection of Micro Ribonucleic Acids.

The detection of low-abundance microribonucleic acid (miRNA) frequently adopted nucleic acid sequence-based amplification detection, which was found to have poor selectivity for the nonspecific amplification of template-dependent ligation in enzyme-mediated cascade reactions. Here, a highly selective detection of miRNAs was developed that combined microsphere-enhanced fluorescence (MSEF) and solid-phase base-paired hybridization. The target miRNA could be accurately and quantitatively identified through the solid-phase hybridization assay on the surface of an optical microsphere, while the detected fluorescence signal could be physically amplified by MSEF. Hereinto, the optical microsphere acted as the fluorescence amplifier and whose surface supplied the space to carry out base-paired hybridization to recognize the target miRNA via the immobilized capture DNA sequence. The detected fluorescence signal of the single-base mismatched miRNA-21 sequence was just around 12% of that of the target miRNA-21 sequence in the measurement of model miRNA-21, while the limit of detection of miRNA-21 could be 1.0 fM. The developed detection of miRNA on an optical microsphere was demonstrated to be an excellent physically amplified method to selectively and sensitively detect the target miRNA and magnificently avoid the nonspecific amplification and false-positive results, which is expected to have wide applications in pathematology, pharmacology, clinic diagnosis, and on-site screening fields as well.

Artificial nanochannels for highly selective detection of miRNA based on the HCR signal amplification

Artificial solid-state nanochannels have nanoscale spatial confinement and unique ion transport properties that have been widely used in biosensing, nanofluidic devices, and nucleic acid sequencing. However, most of the nanochannel sensors for miRNA detection are grounded on the complementary pairing of bases, lacking signal amplification, which limits its application in many fields. Herein, we proposed a signal amplification strategy for miRNA-182 detection based on introducing hybridization chain reaction (HCR) in artificial nanochannels to improve the detection sensitivity. In this method, the capture DNA (cDNA) was anchored in the nanochannel, and when the target miRNA appeared, the hairpin probes H1, H2, and initiator were introduced to trigger the HCR reaction to form long nucleic acid double strands, which significantly enhanced the negative charge and affected ion transport in nanochannel. The nanochannel sensor performed excellent selectivity to the target miRNA-182, and it can realize the detection of miRNA-182 with an ultra-low concentration of 1.65 aM, which can be further applied to the detection of miRNA-182 in actual biological samples. Moreover, the nanochannel system was proved by using the AND logic gate that cDNA, initiator, H1, H2, and miRNA-182 are indispensable for successfully triggering HCR. This work provides a simple and efficient signal amplification method for miRNA detection, which considerably expands the application of nanochannel sensors and holds great prospects in biomedical and clinical detection.

Selective miR-21 detection technology based on photocrosslinkable artificial nucleic acid-modified magnetic particles and hybridization chain reaction

Recently, microRNA (miRNA) detection in blood has attracted attention as a new early detection technology for cancer. The extraction of target miRNA is a necessary preliminary step for detection; however, currently, most extraction methods extract all RNA from the blood, which limits the detection selectivity. Therefore, a method for the selective extraction and detection of target miRNA from blood is very important. In this study, we utilized photocrosslinkable artificial nucleic acids and the hybridization chain reaction (HCR) in an attempt to improve upon the current standard method RT-qPCR, which is hampered by problems with primer design and enzymatic amplification. By introducing photocrosslinkable artificial nucleic acids to oligonucleotide probes modified with magnetic particles with a sequence complementary to that of the target miRNA and irradiating them with light, covalent bonds were formed between the target miRNA and the oligonucleotide probes. These tight covalent bonds enabled the capture of miRNA in blood, and intensive washing ensured that only the target miRNA were extracted. After extraction, two types of DNA (H1 and H2) modified with fluorescent dyes were added and the fluorescence signals were amplified by the HCR in the presence of the target miRNA bound to the photocrosslinkable artificial nucleic acids, allowing for isothermal and enzyme-free miRNA detection. The novel method is suitable for selective miRNA detection in real blood samples. Because the reaction proceeds isothermally and no specialized equipment is used for washing, this detection technology is simple and selective and suitable for application to point-of-care technology using microfluidic devices.

Three-Component Covalent Organic Framework Nanosheets for the Detection of MicroRNAs

The development of new techniques for the detection of microRNAs (miRNAs) is highly desirable. Herein, a new crystalline three-component covalent organic framework (COF) termed EB-TAPB-TFP COF was synthesized under solvothermal conditions utilizing 1,3,5-triformylphloroglucinol, 1,3,5-tris(4-aminophenyl)benzene and ethidium bromide as monomers. Interestingly, EB-TAPB-TFP COF can be self-exfoliated into two-dimensional nanosheets (NSs) in an aqueous medium. The obtained EB-TAPB-TFP NSs exhibited a remarkable fluorescence intensity enhancement in the presence of a DNA-miRNA heteroduplex when compared to the presence of single-stranded DNA and other phosphate-based small molecules, making it promising in the detection of miRNA without tagging any fluorescent marker. Moreover, the EB-TAPB-TFP NSs can also be used as sensing material for the detection of a DNA-miRNA heteroduplex using the quartz crystal microbalance technique, which is in good agreement with the fluorescence sensing result. The exploration of COF-based sensors in this work demonstrates a new pathway for the selective detection of miRNAs.

Highly sensitive detection and intracellular imaging of MicroRNAs based on target-triggered cascade catalytic hairpin assembly

MicroRNAs (miRNAs) have been identified as important biomarkers with great significance for diagnosis and treatment of various diseases. However, their unique properties, such as small size, high sequence homology, and low abundance, make quantitative analysis of miRNAs extremely challenging. Herein, we reported a cascade catalytic hairpin assembly (CCHA) for sensitive and selective detection of miRNA with three kinds of hairpin probes (HP1, HP2, and HP3). In the presence of target miRNA, a series of toehold-mediated intermolecular DNA strand displacement and hybridization was activated among HP1, HP2, and HP3 to assembly numbers of DNA nanoobjects. During this period, the fluorescence response was greatly intensified to indicate the presence and expression level of interested target miRNA. We have demonstrated that the proposed method exhibits a high assay sensitivity to detect low concentration target and an excellent sequence specificity to distinguish even a single-nucleotide difference in vitro. Moreover, we also demonstrated that our design enables the intracellular imaging of miRNA in live cancer and normal cells. These results showing the promising potential of our CCHA for powerful biosensing, clinic diagnosis, or prognosis.

Hemoglobin assisted carbon nanofiber preparation for selective detection of miRNA molecules

A selective miRNA detection is an important factor for the early-stage diagnosis of the diseases and determination of an appropriate treatment method. In this regard, hemoglobin assisted carbon nanofibers (CNFs) were prepared via electrospinning of the precursor polyacrylonitrile/hemoglobin (PAN/Hb) hybrid nanofibers and the following heat treatment process. Addition of low ratio Hb in the precursor PAN nanofibers caused a catalytic effect on the reaction taken place during the stabilization process that helps the formation of more graphitic structure during the carbonization process. But, increasing Hb ratio in the PAN/Hb nanofibers caused an inhibiting effect on the related reactions. Guanine oxidation signals of miRNA molecules were determined via differential pulse voltammetry (DPV) measurement. In this regard, the attachment of anti-miRNA molecules on the CNFs immobilized screen-printed electrodes (SPEs) and a following hybridization of the attached anti-miRNA with miRNA molecules were carried out. Three different miRNA molecules including the target (miRNA), single-base mismatched (SM.miRNA), and non-complementary (NC.miRNA) were hybridized with the previously attached anti-miRNA molecules on the Hb-CNFs immobilized SPEs. The enhancement of the guanine oxidation signal level was observed by using Hb-CNFs instead of using CNFs. That could be attributed to the increase of the graphitic level with low Hb addition to the precursor PAN/Hb nanofibers that causes a catalytic effect on carbonization process. The prepared biosensory system could be used for the selective detection of miRNA molecules.

Lawsone assisted preparation of carbon nanofibers for the selective detection of miRNA molecules

AbstractBACKGROUNDmicroRNA (miRNA) molecules are considered as biomarkers and have promising future for early‐stage cancer diagnosis. Lawsone (Law) enriched carbon nanofibers (CNFs) were prepared via electrospinning process for the selective miRNA detection. Anti‐miRNA molecules were attached on the CNFs immobilized screen printed electrodes (SPE) and used for the detection of miRNA molecules by observing guanine oxidation signal via differential pulse voltammetry (DPV) method. Three different miRNA molecules including the target (miRNA), single‐base mismatched (SM.miRNA) and non‐complementary (NC.miRNA) were hybridized with the previously attached anti‐miRNA molecules (with guanine [PolyT(G)] and without guanine [PolyT(I)]) on the CNFs immobilized SPE surfaces.RESULTSGuanine oxidation signal decreased after the hybridization of miRNA with anti‐miRNA molecules on CNF immobilized SPE surfaces. Guanine oxidation signal was not detected on the PolyT(I) attached samples, but the existence of guanine oxidation signal was clear detected after the addition of miRNA molecules on the previously attached PolyT(I). Because no hybridization of anti‐miRNA molecules with the non‐complementary miRNA (NC.miRNA) molecules occurred, the guanine oxidation signal was not significantly decreased after the interaction of NC.miRNA with the attached PolyT(G). Since there was no hybridization between the NC.miRNA molecules with the attached PolyT(I) on the SPEs, weak guanine peaks were detected at PolyT(I)‐NC.miRNA samples as a result of the existence of residual NC.miRNA molecules on SPE after washing.CONCLUSIONSThe measurement results confirm the enhanced selectivity of the miRNA molecules by using CNFs with SPEs and the developed biosensory system could be used to detect the specific RNA molecules. © 2021 Society of Chemical Industry (SCI).

Dual-Signal Amplification Strategy for Sensitive MicroRNA Detection Based on Rolling Circle Amplification and Enzymatic Repairing Amplification.

MicroRNAs (miRNAs) play crucial regulatory roles as post-transcriptional regulators for gene expression and serve as promising biomarkers for diagnosis and prognosis of diseases. Herein, a dual-signal amplification method has been developed for sensitive and selective detection of miRNA based on rolling circle amplification (RCA) and enzymatic repairing amplification (ERA) with low nonspecific background. This strategy designs a padlock probe that can be cyclized in the presence of target miRNA to initiate the RCA reaction, after which the TaqMan probes that are complementary to the RCA products can be cyclically cleaved to produce obvious fluorescence signals with the help of endonuclease IV (Endo IV). Attributed to the dual-signal amplification procedure and the high fidelity of Endo IV, the RCA–ERA method allows quantitative detection of miR-21 in a dynamic range from 2 pM to 5 nM with a low background signal. Moreover, it has the ability to discriminate single-base difference between miRNAs and shows good performance for miRNA detection in complex biological samples. The results demonstrate that the RCA–ERA assay holds a great promise for miRNA-based diagnostics.

A light-up “G-quadruplex nanostring” for label-free and selective detection of miRNA via duplex-specific nuclease mediated tandem rolling circle amplification

MicroRNA (miRNA) has emerged as a promising genetic marker for cancer diagnosis and therapy because its expression level is closely related to the progression of malignant diseases. Herein, a label-free and selective fluorescence platform was proposed for miRNA based on light-up “G-quadruplex nanostring” via duplex-specific nuclease (DSN) mediated tandem rolling circle amplification (RCA). First, a long DNA generated from upstream RCA was designed with the antisense sequences for miR-21 and downstream RCA primer. Upon recognizing miR-21, the resulting DNA–RNA permitted DSN digestion and triggered downstream two-way RCA, and generation of abundant “G-quadruplex nanostring” binding with ZnPPIX for label-free fluorescent responses. In our strategy, the strong preference of DSN for perfectly matched DNA/RNA ensures its excellent selectivity. The developed method generated wide linear response with LOD of 1.019 fM. Additionally, the miR-21 levels in cell extracts have been evaluated, revealing the utility of this tool for biomedical research and clinical diagnosis.

DNA Walking and Rolling Nanomachine for Electrochemical Detection of miRNA.

miRNAs, a class of endogenous noncoding RNAs, are involved in many crucial biological processes, which have emerged as a new set of biomarkers for disease theranostics. Exploring efficient signal amplification strategy is highly desired to pursue a highly sensitive miRNA biosensing platform. DNA nanotechnology shows great promise in the fabrication of amplified miRNA biosensors. In this work, a novel DNA walking and rolling nanomachine is developed for highly sensitive and selective detection of miRNA. Particularly, this approach programs two forms of dynamic DNA nanomachines powered by corresponding enzymes, which are well integrated. It is able to achieve a limit of detection as low as 39 × 10-18 m, along with excellent anti-interfering performance and clinical applications. In addition, by designing pH-controlled detachable intermolecular DNA triplex, the main sensing elements can be conveniently reset, which fulfills the requirements of point-of-care profiling of miRNA. The high consistency between the proposed approach and quantitative real-time polymerase chain reaction validates the robustness and reliability. Therefore, it is anticipated that the DNA walking and rolling nanomachine has attractive application prospects in miRNA assay for biological researches and clinical diagnosis.

A Label-Free Fluorescent Sensor Based on the Formation of Poly(thymine)-Templated Copper Nanoparticles for the Sensitive and Selective Detection of MicroRNA from Cancer Cells

In this work, a simple and label-free fluorescence “off” to “on” platform was designed for the sensitive and selective detection of microRNA (miRNA) in cancer cells. This method utilized a padlock DNA-based rolling circle amplification (P-RCA) to synthesize fluorescent poly(thymine) (PolyT) which acted as a template for the synthesis of copper nanoparticles (CuNPs) within 10 minutes under mild conditions. While the repeated PolyT sequence was used as the template for CuNP synthesis, other non-PolyT parts (single strand-DNAs without the capacity to act as the template for CuNP formation) served as “smart glues” or rigid linkers to build complex nanostructures. Under the excitation wavelength of 340 nm, the synthesized CuNPs emitted strong red fluorescence effectively at 620 nm. To demonstrate the use of this method as a universal biosensor platform, lethal-7a (let-7a) miRNA was chosen as the standard target. This sensor could achieve highly sensitive and selective detection of miRNA in the presence of other homologous analogues for the combination of P-RCA with the fluorescent copper nanoparticle. Overall, this novel label-free method holds great potential in the sensitive detection of miRNA with high specificity in real samples.

Ultrasensitive Detection of MicroRNA via a Au@Ag Nanosnowman.

MicroRNAs (miRNAs) play key roles in many serious diseases, such as cancer. As a consequence, miRNAs are of great interest as biomarkers in clinical diagnostics. Simple, fast, selective, and sensitive detection of miRNAs, however, is challenged by their short length, homogeneous sequence, susceptibility to degradation, and low abundance in human serum. Here, we present a new strategy for highly sensitive and selective detection of miRNA based on the formation of a plasmonic Au@Ag nanosnowman. When triggered by miRNA-21, bimetallic nanoparticles with an asymmetric Au@Ag head-body structure were formed with significant red shift of the localized surface plasmon resonance (LSPR) scattering wavelength and clear color change from green to red. When combined with exonuclease III (Exo III)-assisted target recycling and hybridization chain reaction (HCR) amplification strategy, the proposed bioassay showed excellent selectivity toward miRNA-21 with a proportional band from 1 fM to 100 pM and ultrahigh sensitivity with a limit of detection of 0.60 fM under dark-field microscopy. The proposed strategy is universal, which shows good application prospects in clinical analysis.

A new QCM signal enhancement strategy based on streptavidin@metal-organic framework complex for miRNA detection

Sensitive and selective detection of miRNA is of great significance for the early diagnosis of human diseases, especially for cancers. Quartz crystal microbalance (QCM) is an effective tool for detecting biological molecules; however, the application of QCM for miRNA detection is still very limited. One of the great needs for QCM detection is to further improve the QCM signal. Herein, for the first time, we promote a new signal enhancement strategy for the detection of miRNA by QCM. First, a hairpin biotin-modified DNA was used as a probe DNA, which exposes the biotin site when interacting with target miRNA. Then, a streptavidin@metal-organic framework (SA@MOF) complex formed by electrostatic attractions between SA and a MOF was introduced into the QCM detection system. The SA@MOF complexes serve as both a signal amplifier and a specific recognition element via specific biotin-SA interactions. The strategy was applied to the detection of a colorectal cancer marker, miR-221, by using a stable Zr(IV)-MOF, UiO-66-NH2. The detection linear range was 10 fM-1 nM, the detection limit was 6.9 fM, and the relative standard deviation (RSD) (n = 5) was lower than 10% in both simulated conditions and the real serum environment. Furthermore, the detection limit reached 0.79 aM when coupled with the isothermal exponential amplification reaction (EXPAR).

Progress in miRNA Detection Using Graphene Material-Based Biosensors.

MicroRNAs (miRNAs) are short, endogenous, noncoding RNAs that play critical roles in physiologic and pathologic processes and are vital biomarkers for several disease diagnostics and therapeutics. Therefore, rapid, low-cost, sensitive, and selective detection of miRNAs is of paramount importance and has aroused increasing attention in the field of medical research. Among the various reported miRNA sensors, devices based on graphene and its derivatives, which form functional supramolecular nanoassemblies of π-conjugated molecules, have been revealed to have great potential due to their extraordinary electrical, chemical, optical, mechanical, and structural properties. This Review critically and comprehensively summarizes the recent progress in miRNA detection based on graphene and its derivative materials, with an emphasis on i) the underlying working principles of these types of sensors, and the unique roles and advantages of graphene materials; ii) state-of-the-art protocols recently developed for high-performance miRNA sensing, including representative examples; and iii) perspectives and current challenges for graphene sensors. This Review intends to provide readers with a deep understanding of the design and future of miRNA detection devices.

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.

Electrical Characterization of Electrolyte/β- Ga2O3 Junction for Biosensing Applications

Electrolyte/semiconductor junctions have attracted considerable attention from different disciplines such as physical chemistry and electrochemistry in recent years. Here, we investigated a single-crystal gallium oxide (β-Ga2O3)-based electrolyte/semiconductor junction behaving similar to a Schottky type diode, determined its electrical properties and analyzed the junction characteristics under certain conditions (e.g., ionic strength, temperature, etc.). The junction was formed by adding an electrolyte layer with a specific ionic concentration (150 × 10−3 M) on top an n-type β-Ga2O3 substrate and a metal contact to assist I-V measurement. The junction diode exhibited rectifying behavior and good electrical characteristics such as an ideality factor of 1.8, a threshold potential (Vbi) of 0.74 eV and a Schottky barrier height (ØB) of 1.05 eV. The threshold voltage of the diode can be tuned by changing the ionic strength in the junction layer and the conductivity can be varied by changing the measurement temperature. The sensitivity of the conductivity of the diode to changes in the electrolyte concentration means that it can be used in various applications. As an application of the semiconductor/electrolyte junction, the selective detection of miRNA (let-7a, let-7f) molecules was demonstrated.

G-triplex based molecular beacon with duplex-specific nuclease amplification for the specific detection of microRNA.

High sequence homology among miRNA members challenges miRNA analysis. In this paper, we developed a simple and highly selective method based on a novel G-triplex molecular beacon (MBG3) and duplex-specific nuclease signal amplification (DSNSA) for miRNA detection. Herein, this MBG3 is a label-free molecular beacon with a special G-triplex probe. The excellent controllablity of the G-triplex probe allowed our MBG3 to have a short stem, which protected it from DSN digestion. Therefore, DSN cleavage induced false positive signals in most DSN signal amplification strategies have been minimised efficiently without special modifications. Importantly, the improved recognition ability of MBG3, together with the sensitive substrate selectivity of DSN, makes our novel method suitable for miRNA detection with high selectivity. The signal response of similar miRNA sequences with one-base difference has been reduced from 37% to 8% compared to the traditional linear ssDNA probe-DSN-based method.

Direct Sensing of Single Native RNA with a Single-Biomolecule Interface of Aerolysin Nanopore.

RNA sensing is of vital significance to advance our comprehension of gene expression and to further benefit medical diagnostics. Taking advantage of the excellent sensing capability of the aerolysin nanopore as a single-biomolecule interface, we for the first time achieved the direct characterization of single native RNA of Poly(A)4 and Poly(U)4. Poly(A)4 induces ∼10% larger blockade current amplitude than Poly(U)4. The statistical duration of Poly(A)4 is 18.83 ± 1.08 ms, which is 100 times longer than that of Poly(U)4. Our results demonstrated that the capture of RNA homopolymers is restricted by the biased diffusion. The translocation of RNA needs to overcome a lower free-energy barrier than that of DNA. Moreover, the strong RNA-aerolysin interaction is attributed to the hydroxyl in pentose, which prolongs the translocation time. This study opens an avenue for aerolysin nanopores to directly achieve RNA sensing, including discrimination of RNA epigenetic modification and selective detection of miRNA.

Cover Picture: MicroRNA‐Directed Intracellular Self‐Assembly of Chiral Nanorod Dimers (Angew. Chem. Int. Ed. 33/2018)

The chiral self-assembly of plasmonic nanorod dimers in living cells, driven by complementary binding to a target miRNA sequence, and leading to specific plasmonic circular dichroism, surface-enhanced Raman scattering (SERS), and fluorescence signals is described in the Communication by H. Kuang, L. M. Liz-Marzán et al. on page 10544 ff. The method allows in situ imaging as well as highly sensitive and selective miRNA detection. It can also be extended to the study of biomolecular interactions, trafficking, and kinetics inside single living cells. The chiral self-assembly of plasmonic nanorod dimers in living cells, driven by complementary binding to a target miRNA sequence, and leading to specific plasmonic circular dichroism, surface-enhanced Raman scattering (SERS), and fluorescence signals is described in the Communication by H. Kuang, L. M. Liz-Marzán et al. on page 10544 ff. The method allows in situ imaging as well as highly sensitive and selective miRNA detection. It can also be extended to the study of biomolecular interactions, trafficking, and kinetics inside single living cells. Molecular Knots Kinetic Resolution DNA Nanotechnology