Sensitive Detection Of microRNA Research Articles

Sensitive fluorescent detection of exosomal microRNA based on enzymes-assisted dual-signal amplification

The analysis of microRNAs (miRNAs) in exosomes offers significant information for a rapid and non-invasive diagnosis of cancer. However, the clinical utility of miRNAs as biomarkers is often hampered by their low abundance in exosomes. Herein, we develop a dual-signal amplification biosensor for the sensitive detection of exosomal miRNA-21 (miR-21). In the presence of a cognate target, it hybridizes with a biotin-modified capture probe (Cp) to form a DNA-RNA heteroduplex that serves as a substrate for duplex-specific nuclease (DSN). With the assistance of DSN, the Cps are enzymatically hydrolyzed and numerous DNA catalysts are released, leading to the first signal amplification. After magnetic isolation, the DNA catalyst remaining in the supernatant triggers a strand displacement reaction based on the nicking-assisted reactant recycling strategy, without depleting the reactants, to implement the second signal amplification. Using this dual-signal amplification concept, our biosensor achieves a limit of detection of miR-21 of 0.34 fM, with a linear range of 0.5–100 fM. The receiver operating characteristic curve generated during clinical sample analysis indicates that the exosomal miR-21 outperforms serum carcinoembryonic antigen in discriminating between patients with gastric cancer (GC) and patients with precancerous (PC) lesions (area under the curve: 0.89 versus 0.74, n = 40). Moreover, the proposed biosensor exhibits an 83.9% accuracy in classifying patients with GC or PC lesions and healthy donors using a confusion matrix. Furthermore, patients with GC with or without metastases are discriminated using the proposed biosensor. Our technology may expand the applications of DNA-based biosensor-enabled cancer diagnostic tools.

Highly sensitive and reliable detection of microRNA for clinically disease surveillance using SERS biosensor integrated with catalytic hairpin assembly amplification technology

MicroRNAs (miRNAs) play an important regulatory role in several diseases, especially as a class of promising biomarkers for cancer diagnosis and prognosis. Here, a biosensor based on surface enhanced Raman spectroscopy (SERS) combined with catalytic hairpin assembly (CHA) amplification technology was developed for ultra-sensitive detection of miRNA-21 and miRNA-155 in breast cancer serum. By using CHA strategy, the extremely low concentration of target microRNA in human serum can be significantly amplified through the re-hybridization with thousands of hairpin probes to trigger amplification cycles. Besides, a sandwich SERS sensing chip with numerous hot spots and signal self-calibration was built through the linkage between two-dimensional Au–Si substrate and upper Ag@4-MBA@Au core-shell nanoparticles. Using this specially-designed biosensing platform, a low detection limit of 0.398 fM and 0.215 fM with a dynamic range from 1 fM to 10 nM can be achieved for the detection of miRNA-21 and miRNA-155, respectively. Additionally, the analysis of these two miRNAs in serum samples is capable of identifying the breast cancer subjects from normal ones with 100% of accuracy, as well as potentially evaluating the molecular types and prognosis for breast cancer. These results demonstrate that the proposed SERS with CHA technology would be an alternative method for highly sensitive and reliable detection of miRNA biomarkers contributing to breast cancer diagnosis and prognosis.

Label-free and sensitive microRNA detection method based on the locked nucleic acid assisted fishing amplification strategy.

Herein, a label free and sensitive miRNA detection method with enhanced practical applicability was developed based on the locked nucleic acid (LNA) assisted repeated fishing amplification strategy. The working mechanism of the proposed method is as follows: 1) a DNA probe (i.e, L-DNA) with LNA bases is immobilized onto the surface of a gold foil. The L-DNA hybridizes with the 3' terminus of the first strands of complementary deoxyribonucleic acid (cDNA) of the target miRNA in the test samples; 2) The protruding 5' terminus of the cDNA serves as a 'fishhook' to repeatedly fish the products of a hybridization chain reaction (HCR) out from a 'reaction tube'; 3) The HCR products can be unloaded from the gold foil into a 'product tube' through temperature-controlled dehybridization; 4) The concentration of the target miRNA is determined based on the fluorescence intensity generated by the addition of SYBR-Green I (SG) into the 'product tube'. The proposed platform was applied to the detection of miRNA-122 in cell lysate samples and obtained quantitative results with accuracy comparable to the quantitative reverse transcription PCR method (qRT-PCR). It is worth pointing out that the proposed platform achieved a limit of detection value of 2.9fM for miRNA-122 by a simple but effective LNA-assisted repeated fishing amplification strategy instead of complicated enzyme-based amplification techniques. It is reasonable to expect that the proposed method provides a competitive alternative for designing practically applicable, cost-effective and label-free miRNA detection methods.

A Sample and Detection Microneedle Patch for Psoriasis MicroRNA Biomarker Analysis in Interstitial Fluid

Skin interstitial fluid (ISF) containing a great variety of molecular biomarkers derived from cells and subcutaneous blood capillaries has recently emerged as a clinically potential component for early diagnosis of a wide range of diseases; however, the minimally invasive sampling and detection of cell-free biomarkers in ISF is still a key challenge. Herein, we developed microneedles (MNs) that consist of gelatin methacryloyl (GelMA) and graphene oxide (GO) for the enrichment and sensitive detection of multiple microRNA (miRNA) biomarkers from skin ISF. The GO-GelMA MNs exhibited robust mechanical properties, fast sampling kinetics, and large swelling capacity, which enabled collecting ISF volume high to 21.34 μL in 30 min, facilitating effective miRNA analysis. It preliminarily realized the sensitive detection of three types of psoriasis-related miRNAs biomarkers either on the patch itself or in solution after release from the hydrogel by combining catalytic hairpin assembly signal amplification reaction. The automated and minimally invasive ISF miRNA detection technology of GO-GelMA MNs has great potential to monitor cell-free clinically informative biomarkers for personalized diagnosis and prognosis.

One-step isothermal amplification strategy for microRNA specific and ultrasensitive detection based on nicking-assisted entropy-driven DNA circuit triggered exponential amplification reaction

Sensitive and specific detection of microRNAs (miRNAs) is of critical significance for early diagnosis of cancers such as pancreatic cancer with atypical initial symptoms and high mortality. Despite exponential amplification reaction (EXPAR) is an attractive isothermal amplification method for detecting miRNAs, it faces the problems of the dependence difference and low specificity. To address such challenges, herein, a nicking-assisted entropy-driven DNA circuit triggered exponential amplification reaction (NAED-EXPAR) was firstly employed for ultrasensitive and specific detection of miRNA in “one-pot” manner at constant temperature. Nicking-assisted entropy-driven DNA circuit can specifically recognize the target miRNA, leading to continuous disassembly of DNA substrates via intramolecular toehold-mediated branch migration. During the reaction, the catalytic circuit can consume excess fuel DNA strands to produce a large number of primers. Then the newly formed primers can trigger EXPAR for highly efficient signal amplification. Mechanism analysis shows that the amplification efficiency of NAED-EXPAR is superior than that of single EXPAR. For miR-21, the detection limit of NAED-EXPAR can reach 100 aM, which is at least five orders of magnitude higher than the standard EXPAR that directly uses the target as primer. NAED-EXPAR shows improved specificity for identifying single nucleotide variations and enables sensitive and accurate analysis of miR-21 in human cancer cell lines. This method is expected to offer a new approach for the reliable quantification of miRNAs in complex biological matrices and provide valuable information for early cancer diagnosis.

Interference Reduction Biosensing Strategy for Highly Sensitive microRNA Detection.

MicroRNAs are potential biomarkers for human cancers and other diseases due to their roles as post-transcriptional regulators for gene expression. However, the detection of miRNAs by conventional methods such as RT-qPCR, in situ hybridization, northern blot-based platforms, and next-generation sequencing is complicated by short length, low abundance, high sequence homology, and susceptibility to degradation of miRNAs. In this study, we developed a nicking endonuclease-mediated interference reduction rolling circle amplification (NEM-IR-RCA) strategy for the ultrasensitive and highly specific detection of miRNA-21. This method exploits the advantages of the optical properties of long-lived iridium(III) probes, in conjunction with time-resolved emission spectroscopy (TRES) and exponential rolling circle amplification (E-RCA). Under the NEM-IR-RCA-based signal enhancement processes, the limit of detection of miRNA-21 was down to 0.0095 fM with a linear range from 0.05 to 100 fM, which is comparable with the conventional RT-qPCR. Unlike RT-qPCR, the strategy was performed at a lower and constant temperature without heating/cooling cycles and reverse transcription. The strategy could clearly discriminate between matched and mismatched targets, demonstrating high specificity. Moreover, the potential application of this method was demonstrated in cancer cells and mouse serum samples, showing good agreement with RT-qPCR results. Apart from miRNA-21 detection, this platform could be also adapted for detecting other miRNAs, such as let-7a and miRNA-22, indicating its excellent potential for biomedical research and clinical diagnostics.

A dual-amplification mode and Cu-based metal-organic frameworks mediated electrochemical biosensor for sensitive detection of microRNA

In this work, we developed a novel label-free and highly sensitive electrochemical (EC) biosensor for detection of microRNA (miRNA), which was based on the target-triggered and the Cu-based metal-organic frameworks (Cu-MOFs) mediated CHA-HCR dual-amplification process. Initially, the target miRNA triggered the catalytic hairpin assembly (CHA) process of hairpin DNA 1 (H1) and hairpin DNA 2 (H2) to produce massive double-stranded DNA (H1/H2) which could hybridize with the single-stranded DNA 1 (P1) to form capture probe (P1/H1/H2) on electrode surface, realizing the first amplification of input signals. Subsequently, hybridization chain reaction (HCR) between signal probe (H3-AuNPs/Cu-MOFs) and hairpin DNA 4 (H4) was activated by above capture probe (P1/H1/H2), leading to the second amplification of input signals. After the HCR process, numerous Cu-MOFs were immobilized on the electrode surface, which brought out the enhancement of electrochemical signals generating by Cu-MOFs. Herein, Cu-MOFs not only offered the lager surface area to decorate with gold nanoparticles (AuNPs) and hairpin DNA 3 (H3), but also served as the signal probe (H3-AuNPs/Cu-MOFs) to produce electrochemical signals by hybridizing with the capture probe on electrode surface. Therefore, the ingenious design of CHA-HCR-Cu-MOFs scheme enables the sensitive analysis of microRNA-21 (miR-21) with a broad linear range from 0.1 fM to 100 pM and a lower LOD of 0.02 fM. In addition, the outstanding specificity of this sensing strategy allows it successfully to be applied for determining miR-21 in real biological samples.

Rapid and Sensitive Detection of Non-Small Cell Lung Cancer-Related Micrornas by a Pump-Free Sers Microfluidic Chip

Rapid and Sensitive Detection of Non-Small Cell Lung Cancer-Related Micrornas by a Pump-Free Sers Microfluidic Chip

Highly Sensitive Detection and Intracellular Imaging of Micrornas Based on Target-Triggered Cascade Catalytic Hairpin Assembly

Highly Sensitive Detection and Intracellular Imaging of Micrornas Based on Target-Triggered Cascade Catalytic Hairpin Assembly

Superior performance of a graphdiyne self-powered biosensor with exonuclease III-assisted signal amplification for sensitive detection of microRNAs.

Graphdiyne (GDY) is an sp and sp2 co-hydrocarbon allotrope whose particular structure endows it with many fascinating properties, including abundant chemical bonds, high conjugation, natural pores, high carrier mobility, high conductivity and stability, etc. In this work, two-dimensional graphdiyne is prepared as an electrode substrate material coupling with an exonuclease III-assisted amplification strategy to construct a superior-performance self-powered biosensor based on enzymatic biofuel cells for highly sensitive detection of the tumour marker miRNA-21. Glucose oxidase (GOD) is first immobilized on the GDY/AuNP composite to prepare a bioconjugate. GDY/AuNP modified carbon cloth is used as an enzyme biofuel cell electrode, which is then modified with bilirubin oxidase as a biocathode. The bioconjugate binds to GOD through specific binding to the bioanode. When miRNA-21 is present, specific recognition by exonuclease III in the system results in cleavage of the capture probe, and miRNA-21 is recovered and involved in the cycle. The target miRNA-21 then causes corresponding changes in the open-circuit voltage of the self-powered system. Based on this, a sensitive detection method was constructed, within the scope from 0.1 fM to 0.1 nM with a shallow detection limit of 55.2 aM (S/N = 3). The new approach triumphantly has been used to detect miRNA-21 in serum, which provides a compelling new way for early diagnosis of related cancers.

A Fluorogenic Biosensor Based on Magnetic-Assisted Hybridization Chain Reaction and DNA-Templated Silver Nanoclusters for Sensitive MicroRNA Detection

A Fluorogenic Biosensor Based on Magnetic-Assisted Hybridization Chain Reaction and DNA-Templated Silver Nanoclusters for Sensitive MicroRNA Detection

Au-quantum dot nanocluster electrochemiluminescence coupled with cycling-amplification for sensitive microRNA detection

A novel polyamidoamine (PAMAM) dendrimer-Au nanocluster composite was synthesized, and used to fabricate a new amplified electrochemiluminescence (ECL) signal probe for sensitive detection of microRNA by multiple strand displacement amplification (SDA) strategy. The as prepared PAMAM-Au nanocluster with many amino groups could assemble a large number of quantum dots (QDs) to greatly amplify ECL of the probe. In addition, a new sliver nanocluster (NC) with excellent conductivity and many reactive carboxyl groups was prepared, and used to immobilize a large amount of capture (c1) DNA molecules on the electrode. Moreover, by using bifunctional DNA strand displacement reaction-mediated multiple cycling-amplification technique, a small number of target miRNA could induce to generate abundant DNA (t1) fragments, which was used as a linker to hybridize with c1 DNA on the electrode, and then conjugate many amplified QDs probe. Thus an amplified ECL analytical method for detecting target miRNA was designed, and highly sensitive detection of miRNA was achieved. This newly established strategy paves a new way for homogeneous microRNA detection, which hold great potential for application in early clinical diagnosis.

Ultrasensitive Photoelectrochemical Biosensor Based on Novel Z-Scheme Heterojunctions of Zn-Defective CdS/ZnS for MicroRNA Assay.

The sensitive and accurate detection of microRNA (miRNA) has meaningful values for clinical diagnosis application as an early stage of tumor markers. Herein, a novel photoelectrochemical (PEC) biosensor was developed for the ultrasensitive and highly selective detection of microRNA-122 (miRNA-122) based on a direct Z-scheme heterojunction of Zn vacancy-mediated CdS/ZnS (CSZS-VZn). Impressively, the prepared Z-scheme heterojunction nanocomposite with defect level properties could make the photogenerated charges stay at the Zn vacancy defect levels and combine photogenerated holes in the valence bands of CdS, thus significantly achieving a better charge carrier separation efficiency and broadening the absorption of visible light and demonstrating 5-8 times enhancement of PEC response compared to single-component materials. Simultaneously, an exonuclease III (Exo-III)-assisted signal amplification strategy and a strand displacement reaction were combined to improve the conversion efficiency of the target and further increase the detection sensitivity. More importantly, the elaborated biosensor showed ultrasensitive and highly specific detection of the target miRNA-122 over a wide linear range from 10 aM to 100 pM with a low detection limit of 3.3 aM and exhibited enormous potential in the fields of bioanalysis and clinical diagnosis.

DNA Tetrahedron-Based MNAzyme for Sensitive Detection of microRNA with Elemental Tagging.

Heterogeneous immunoassay based on magnetic separation is commonly used in inductively coupled plasma-mass spectrometry (ICP-MS)-based biomedical analysis with elemental labeling. However, the functionalized magnetic beads (MBs) often suffer from non-specific adsorption and random distribution of the functional probes. To overcome these problems, DNA tetrahedron (DT)-functionalized MBs were designed and further conjugated with substrate modified Au NPs (Sub-AuNP). Based on the prepared MB-DT-AuNP probes, an MB-DT based multicomponent nucleic acid enzyme (MNAzyme) system involving Au NPs as the elemental tags was proposed for highly sensitive quantification of miRNA-155 by ICP-MS. Target miRNA would trigger the assembly of MNAzyme, and Sub-AuNP would be cleaved from the MB-DT-AuNP probe, resulting in a cyclic amplification. Single-stranded DNA-functionalized MB (MB-ssDNA)-AuNP probes were prepared as well. Comparatively, the amount of Au NPs grafted onto MB-ssDNA-AuNP probes was higher than that grafted onto MB-DT-AuNP probes. Meanwhile, a higher signal-to-noise ratio was obtained by using MB-DT-AuNP probes over MB-ssDNA-AuNP probes in the MNAzyme system. Under the optimal experimental conditions, the limit of detection for target miRNA obtained by using MB-DT-AuNP probes was 1.15 pmol L-1, improved by 23 times over that obtained by the use of MB-ssDNA-AuNP probes. The proposed MB-DT-MNAzyme-ICP-MS method was applied to the analysis of miRNA-155 in serum samples, and recoveries of 86.7-94.6% were obtained. This method is featured with high sensitivity, good specificity, and simple operation, showing a great application potential in biomedical analysis.

Tetrahedral structure supported two stages DSN-assisted amplification strategy for sensitive detection of lung cancer related MicroRNA

MicroRNA (miRNA) plays a pivotal role in the regulation of cancer developments and could potentially work as an important biomarker candidate for the early-diagnosis, therapy and prognosis of lung cancer. Accurate and sensitive quantification of miRNA extracted from clinical samples in a stable and label-free way remain a huge challenge. Herein, we propose here a novel fluorescence assay for accurate analysis of miRNA via duplex specific nuclease (DSN) signal amplification. Highlights of the methods could be depicted as: i) a favorable detection sensitivity through the DSN enzyme based signal amplification; ii) fluorescence signal generation in a label-free way based on MG/G-quadruplex complex; iii) a stable signal generation even in complicated experimental conditions based on the tetrahedral structure. Eventually, we believe that this proposed sensitive and specific assay has great potential as a miRNA quantification method for use in biomedical research and clinical diagnosis.

A persistent luminescence resonance energy transfer-based molecular beacon probe for the highly sensitive detection of microRNA in biological samples

Herein, a time-resolved luminescence resonance energy transfer (TR-LRET) molecular beacon (MB) probe employing persistent luminescence nanoparticles (PLNPs) as the energy donors was first constructed, and further designed for microRNA21 (miR21) sensing. This probe (named as PLNPs-MB) was facilely fabricated by covalent bioconjugation between poly-(acrylic acid) (PAA) modified near-infrared (NIR) emissive PLNPs i.e. ZnGa2O4:Cr3+ and functionalized MB oligonucleotide (5′-NH2 and 3′-BHQ3). Accordingly, PLNPs and BHQ3 were in close proximity to each other, leading to the occurrence of LRET and obvious persistent luminescence (PL) quenching. In the presence of miR21, loop of the PLNP-MB was hybridized, accompanying BHQ3 away from PLNPs and the restraint of LRET process. As a result, PL of the PLNPs was recovered, which built the foundation of miR21 quantification. The probe provided a linear response range from 0.1 to 10 nM for miR21 detection. Quantification limit of this probe was competitive and about 1–2 orders of magnitude lower than that of other reported MB probes for nucleic acid. Moreover, the proposed probe was successfully adopted for miR21 detection in biological fluids (human serum, cell extraction). This work also provided a sensitive detection nanoplatform for other targets through modifying diverse MBs onto the surface of PLNPs.

Ratiometric electrochemical biosensor based on silver nanoparticles coupled with walker amplification for sensitive detection of microRNA

A novel ratiometric electrochemical biosensor for sensitive detection of microRNA was developed by using walker amplification technique. Ag nanoparticles (NPs) and methylene blue (MB) were used as signal probe and as reference, respectively. Firstly, the present target (miR-155) hybridized with DNA2 to release walker (DNA1), and then the walker hybridized with DNA3 to form activated DNA enzyme, inducing cycling shearing of DNA3 in the presence of Mn2+, thus a large number of product chains (S1) were produced. Then, S1 opened hairpin DNA (HP1) labeled with MB on the electrode, initiating enzyme-assisted cyclic clipping to generate abundant capture DNA. Thus numerous signal probes (DNA HP2-Ag NPs) were linked for electrochemical detection of target. As the electrochemical signal of MB at capture DNA was constant, so it was used as internal reference. Thus the signal ratio of Ag NPs to MB was used for detection, which can greatly reduce the background. The relation between the IAg/IMB and the concentration of target was 10−13-10−6 M, showing wide linear detection range. The biosensor displayed simple design, excellent sensitivity and high specificity for microRNA, showing enormous application prospect in bioanalysis and disease diagnosis fields.

Zinc-Metal Organic Frameworks: A Coreactant-free Electrochemiluminescence Luminophore for Ratiometric Detection of miRNA-133a.

Developing a coreactant-free ratiometric electrochemiluminescence (ECL) strategy based on a single luminophore to achieve more accurate and sensitive microRNA (miRNA) detection is highly desired. Herein, utilizing zinc-metal organic frameworks (Zn-MOFs) as the single luminophore, a novel dual-potential ratiometric ECL biosensor was constructed for ultrasensitive detection of miRNA-133a. The as-prepared Zn-MOFs exhibited simultaneous cathode and anode ECL emission. Furthermore, the Zn-MOFs were confirmed to be a multichannel ECL sensing platform with excellent annihilation and coreactant ECL emission. The corresponding ECL behaviors were investigated in detail. Benefiting from the hybridization chain reaction (HCR) amplification technology, N,N-diethylethylenediamine (DEAEA) was modified on hairpin DNA, and the gained products loaded with quantities of DEAEA enhanced the anodic ECL intensity of Zn-MOFs. In the presence of miRNA-133a, the ECL intensity ratio of anode to cathode (Ia/Ic) was significantly increased, which realized the ultrasensitive ratiometric detection of miRNA-133a. In addition, without an exogenous coreactant, the biosensor revealed superb accuracy and stability. Under optimal conditions, the detection linearity of miRNA-133a was from 50 aM to 50 fM with a low detection limit of 35.8 aM (S/N = 3). This is the first work to use Zn-MOFs as a single emitter for reliable ratiometric ECL bioanalysis, which provides a new perspective for fabricating a ratiometric ECL biosensor platform.

A photocathode based on BiOI-Bi/CNTs for microRNA detection coupling with target recycling strand displacement amplification

A novel cathode photoelectrochemical (CPEC) biosensor by integrating BiOI-Bi/CNTs photocathode with target recycling strand displacement amplification (TRSDA) was proposed for selective and sensitive detection of microRNA (miRNA). Herein, an innovative strategy of two-site hybridization in a hairpin loop to specifically recognize the target miRNA was designed. Strand displacement amplification (SDA) reaction was triggered by target let-7a miRNA with the assistance of polymerase and nicking enzyme. TRSDA was then realized by using short complementary sequence hidden in the 3′-terminal region of the hairpin probe stem, which produced large amount of product DNA (pDNA). The as-generated pDNA fragments were collected by capture probe (CP) on BiOI-Bi/CNTs platform. Since K3[Fe(CN)6] could take in photoelectrons from BiOI-Bi/CNTs effectively, the photocurrent was largely decreased because DNA layer on CPEC platform inhibited photoelectron transfer towards electron acceptor. Under optimized conditions, the photocurrent response of biosensor was linear with let-7a concentration from 0.2 to 2000 pM, and the detection limit (S/N = 3) was as low as 0.1 pM. The proposed approach opened up new opportunity for developing robust biochemical detection systems.

Paper-Based Electrochemical Biosensors for Voltammetric Detection of miRNA Biomarkers Using Reduced Graphene Oxide or MoS2 Nanosheets Decorated with Gold Nanoparticle Electrodes.

Paper-based biosensors are considered simple and cost-efficient sensing platforms for analytical tests and diagnostics. Here, a paper-based electrochemical biosensor was developed for the rapid and sensitive detection of microRNAs (miRNA-155 and miRNA-21) related to early diagnosis of lung cancer. Hydrophobic barriers to creating electrode areas were manufactured by wax printing, whereas a three-electrode system was fabricated by a simple stencil approach. A carbon-based working electrode was modified using either reduced graphene oxide or molybdenum disulfide nanosheets modified with gold nanoparticle (AuNPs/RGO, AuNPs/MoS2) hybrid structures. The resulting paper-based biosensors offered sensitive detection of miRNA-155 and miRNA-21 by differential pulse voltammetry (DPV) in only 5.0 µL sample. The duration in our assay from the point of electrode modification to the final detection of miRNA was completed within only 35 min. The detection limits for miRNA-21 and miRNA-155 were found to be 12.0 and 25.7 nM for AuNPs/RGO and 51.6 and 59.6 nM for AuNPs/MoS2 sensors in the case of perfectly matched probe-target hybrids. These biosensors were found to be selective enough to distinguish the target miRNA in the presence of single-base mismatch miRNA or noncomplementary miRNA sequences.