microRNA Detection Research Articles

Exonuclease III-propelled DNAzyme walker: an electrochemical strategy for microRNA diagnostics.

MicroRNA detection is crucial for early infectious disease diagnosis and rapid cancer screening. However, conventional techniques like reverse transcription-quantitative polymerase chain reaction, requiring specialized training and intricate procedures, are less suitable for point-of-care analyses. To address this, we've developed a straightforward amplifier based on an exonuclease III (exo III)-propelled DNAzyme walker for sensitive and selective microRNA detection. This amplifier employs a specially designed hairpin probe with two exposed segments for strand recognition. Once the target microRNA is identified by the hairpin's extended single-strand DNA, exo III initiates its digestion, allowing microRNA regeneration and subsequent hairpin probe digestion cycles. This cyclical process produces a significant amount of DNAzyme, leading to a marked reduction in electrochemical signals. The biosensor exhibits a detection range from 10 fM to 100pM and achieves a detection limit of 5 fM (3σcriterion). Importantly, by integrating an "And logic gate," our system gains the capacity for simultaneous diagnosis of multiple microRNAs, enhancing its applicability in RNA-based disease diagnostics.

CRISPR/Cas13a-triggered entropy-driven amplification for colorimetric and fluorescent dual-mode detection of microRNA

MicroRNAs (miRNAs) are crucial biomarkers for the early detection and monitoring of disease progression of chronic obstructive pulmonary disease (COPD). Herein, we have devised a method for detecting miRNA using a combination of colorimetric and graphene oxide-based fluorescent techniques. The target miRNA in our design could precisely activate the trans-cleavage activity of the CRISPR-Cas13a system. The activated Cas13a enzyme cuts the “rUrU” section in the P1 probe, generating a nicking site to induce entropy-driven amplification (EDA). One of the available EDA products has the capability to unfold the hairpin probe, thereby initiating the catalytic hairpin assembly, exposing the G-quadruplex structure, facilitating the subsequent color response. The fuel strand labeled with Cy3 successfully established a double-stranded DNA structure with DNA3, and consequently the Cy3 would not be quenched by graphene oxide (GO). The implementation of the dual-mode technique in this method yields greater benefits in terms of improving the precision and consistency of the miRNA measurements. The developed method has the capability to fluorescently measure miRNA-21 levels down to a concentration of 5.8 fM. In addition, the analysis of miRNA targets from clinical samples using this method demonstrates its promising utility in the fields of biomedical research of COPD.

An integrated liposome-based microfluidic strategy for rapid colorimetric analysis: A case study of microRNA-21 detection

In this study, a novel integrated liposome-based microfluidic platform combined with a smartphone was designed for the rapid colorimetric detection of microRNA-21 (miRNA-21) in real samples. The flowing surface-functionalized liposomes were first captured by nucleic acid-functionalized Au nanoparticles in the microfluidic chip. In the presence of miRNA-21, the DNA strand modified on the surface of Au nanoparticles hybridized with the target to form double-stranded products and was cleaved by duplex-specific nuclease (DSN) enzyme, causing the liposomes to be re-released. Then, as the liposomes in the colorimetric module were lysed and the “cellular” contents were released, a step-by-step “glucose-glucose oxidase-3,3′,5,5′-tetramethylbenzidine (TMB)” colorimetric reaction process catalyzed by the G-quadruplex/hemin was triggered. The grayscale values were recorded and recognized by the smartphone camera for miRNA-21 analysis. The advantages of the present strategy included the portability of smartphone-based colorimetric assay, the encapsulation and transport of reactants by liposomes and the low solvent usage of microfluidic chip. Under optimal conditions, this assay exhibited a wide linear range from 1 pM to 1 nM (r2 = 0.9981), and the limit of detection of miRNA-21 was as low as 0.27 pM. Moreover, the high specificity of this strategy allowed its successful application to the rapid analysis of miRNA-21 in real blood serum samples of people with type 2 diabetes.

Catalytic Assembly of DNAzyme Integrates with Primer Exchange Reaction (CDiPER) for Highly Sensitive Detection of MicroRNA.

MicroRNAs (miRNAs) have significant regulatory functions in the modulation of gene expression, making them essential biomarkers for the diagnosis and prognosis of diseases. Nevertheless, the identification of miRNA poses significant difficulty in terms of its low abundance, necessitating sensitive and reliable approaches. Herein, we develop a simple approach, termed Catalytic assembly of DNAzyme integrates with Primer Exchange Reaction (CDiPER), for reliable and sensitive miRNA detection through the target recognition-triggered DNAzyme assembly and primer exchange reaction (PER) strategy. In this method, target miRNA can precisely bind with a specifically designed hairpin probe (H probe) to induce the conformation changes of the H probe, releasing DNAzyme sections to activate the PER process for signal amplification and fluorescence signal production. The established method displays a high dynamic range of over 6 orders of magnitude and a low detection limit of 312 aM. The created method has a number of unique advantages, such as (i) a better sensitivity than existing systems using PER for signal amplification as a result of its integration with the target recognition-triggered DNAzyme assembly and (ii) streamlined operating procedures. Further, the technology was used to detect the expression of miRNA in collected clinical samples from diabetes mellitus patients, revealing that miRNA was decreased in patients and demonstrating the significant clinical promise of the method.

A versatile upconversion-based multimode lateral flow platform for rapid and ultrasensitive detection of microRNA towards health monitoring

MicroRNAs are small single-stranded RNA molecules associated with gene expression and immune response, suggesting their potential as biomarkers for health monitoring. Herein, we designed a novel upconversion-based multimode lateral flow assay (LFA) system to detect microRNAs in body fluids by simultaneously producing three unique signals within a detection strip. The core-shell Au-DTNB@Ag nanoparticles act as both the Raman reporters and acceptors, quenching fluorescence from upconversion nanoparticles (UCNPs, NaYF4: Yb3+, Er3+) via the Förster resonance energy transfer mechanism. Using microRNA-21 as a representative analyte, the LFA system offers remarkable detection range from 2 nM to 1 fM, comparable to outcomes from signal amplification methods, due to the successful single-layer self-assembly of UCNPs on the NC membrane, which greatly enhances both the convenience and sensitivity of the LFA technique. Additionally, our proprietary fluorescence-Raman detection platform simplifies result acquisition by reducing procedural intricacies. The biosensor, when evaluated with diverse bodily fluids, showed remarkable selectivity and sustained stability. Importantly, our LFA biosensor effectively identified periodontitis and lung cancer patients from healthy subjects in genuine samples, indicating significant potential for disease prediction, early diagnosis, and progression tracking. This system holds promise as a multifunctional tool for various biomarker assays.

Simultaneous Detection of Exosomal microRNAs Isolated from Cancer Cells Using Surface Acoustic Wave Sensor Array with High Sensitivity and Reproducibility.

We present a surface acoustic wave (SAW) sensor array for microRNA (miRNA) detection that utilizes photocatalytic silver staining on titanium dioxide (TiO2) nanoparticles as a signal enhancement technique for high sensitivity with an internal reference sensor for high reproducibility. A sandwich hybridization was performed on working sensors of the SAW sensor array that could simultaneously capture and detect three miRNAs (miRNA-21, miRNA-106b, and miRNA-155) known to be upregulated in cancer. Sensor responses due to signal amplification varied depending on the concentration of synthetic miRNAs. It was confirmed that normalization (a ratio of working sensor response to reference sensor response) screened out background interferences by manipulating data and minimized non-uniformity in the photocatalytic silver staining step by suppressing disturbances to both working sensor signal and reference sensor signal. Finally, we were able to successfully detect target miRNAs in cancer cell-derived exosomal miRNAs with performance comparable to the detection of synthetic miRNAs.

Photocleavable DNA Nanotube-Based Dual-Amplified Resonance Rayleigh Scattering System for MicroRNA Detection Incorporating Molecular Computing-Cascaded Keypad Lock Functionality.

Cascade molecular events in complex systems are of vital importance for enhancing molecular diagnosis and information processing. However, the conversion of a cascaded biosensing system into a multilayer encrypted molecular keypad lock remains a significant challenge in the development of molecular logic devices. In this study, we present a photocleavable DNA nanotube-based dual-amplified resonance Rayleigh scattering (RRS) system for detecting microRNA-126 (miR-126). The cascading dual-amplification biosensing system provides a multilayer-encrypted prototype with the functionality of a molecular computing cascade keypad lock. RRS signals were greatly amplified by using photocleavable DNA nanotubes and enzyme-assisted strand displacement amplification (SDA). In the presence of miR-126, enzyme-assisted SDA produced numerous identical nucleotide fragments as the target, which were then specifically attached to magnetic beads through the DNA nanotube by using a Y-shaped DNA scaffold. Upon ultraviolet irradiation, the DNA nanotube was released into the solution, resulting in an increase in the intensity of the RRS signal. This strategy demonstrated a low limit of detection (0.16 fM) and a wide dynamic range (1 fM to 1 nM) for miR-126. Impressively, the enzyme-assisted SDA offers a molecular computing model for generating the target pool, which serves as the input element for unlocking the system. By cascading the molecular computing process, we successfully constructed a molecular keypad lock with a multilevel authentication technique. The proposed system holds great potential for applications in molecular diagnosis and information security, indicating significant value in integrating molecular circuits for intelligent sensing.

Smartphone-Interfaced Electrochemical Biosensor for microRNA Detection Based on Laser-Induced Graphene with π–π Stacked Peptide Nucleic Acid Probes

Smartphone-Interfaced Electrochemical Biosensor for microRNA Detection Based on Laser-Induced Graphene with π–π Stacked Peptide Nucleic Acid Probes

Classification of Molecular Binding Traces for Dynamic Single-Molecule Sensing.

Interference from nonspecific binding imposes a fundamental limit in the sensitivity of biosensors that is dependent on the affinity and specificity of the available sensing probes. The dynamic single-molecule sensing (DSMS) strategy allows ultrasensitive detection of biomarkers at the femtomolar level by identifying specific binding according to molecular binding traces. However, the accuracy in classifying binding traces is not sufficient from separate features, such as the bound lifetime. Here, we establish a DSMS workflow to improve the sensitivity and linearity by classifying molecular binding traces in surface plasmon resonance microscopy with multiple kinetic features. The improvement is achieved by correlation analysis to select key features of binding traces, followed by unsupervised k-clustering. The results show that this unsupervised classification approach improves the sensitivity and linearity in microRNA (hsa-miR155-5p, hsa-miR21-5p, and hsa-miR362-5p) detection to achieve a limit of detection at the subfemtomolar level.

Detection of miR-155 Using Peptide Nucleic Acid at Physiological-like Conditions by Surface Plasmon Resonance and Bio-Field Effect Transistor.

MicroRNAs are small ribonucleotides that act as key gene regulators. Their altered expression is often associated with the onset and progression of several human diseases, including cancer. Given their potential use as biomarkers, there is a need to find detection methods for microRNAs suitable for use in clinical setting. Field-effect-transistor-based biosensors (bioFETs) appear to be valid tools to detect microRNAs, since they may reliably quantitate the specific binding between the immobilized probe and free target in solution through an easily detectable electrical signal. We have investigated the detection of human microRNA 155 (miR-155) using an innovative capturing probe constituted by a synthetic peptide nucleic acid (PNA), which has the advantage to form a duplex even at ionic strengths approaching the physiological conditions. With the aim to develop an optimized BioFET setup, the interaction kinetics between miR-155 and the chosen PNA was preliminarily investigated by using surface plasmon resonance (SPR). By exploiting both these results and our custom-made bioFET system, we were able to attain a low-cost, real-time, label-free and highly specific detection of miR-155 in the nano-molar range.

Kineto-optical fingerprinting for multiplexed detection of micro-RNAs

Kineto-optical fingerprinting for multiplexed detection of micro-RNAs

Rigidifying AIEgens in covalent organic framework nanosheets for electrochemiluminescence enhancement: TABE-PZ-CON as a novel emitter for microRNA-21 detection

Enhancing electrochemiluminescence (ECL) properties of luminophores is a hot direction in the current ECL field. Herein, we found that covalent rigidification of the aggregation-induced emission luminogens (AIEgens) TABE (TABE = tetra-(4-aldehyde-(1,1-biphenyl))ethylene) into covalent organic framework nanosheets (TABE-PZ-CON, PZ = piperazine) could result in stronger ECL emission than those of TABE aggregates and TABE monomers. We termed the interesting phenomenon “covalent rigidification-triggered electrochemiluminescence (CRT-ECL) enhancement”. The superior ECL performance of TABE-PZ-CON not only because massive TABE luminogens were covalently assembled into the rigid TABE-PZ-CON network, which limited the intramolecular motions of TABE and hampered the radiationless transition, but also because the ultrathin porous TABE-PZ-CON significantly reduced the transportation distance of ions, electrons, and coreactants, which enabled the electrochemical excitation of more TABE luminogens and thus enhanced the ECL efficiency. Bearing in mind the exceptional ECL performance of TABE-PZ-CON, it was utilized as a high-efficient ECL indicator in combination with the DNA walker and duplex-specific nuclease-assisted target recycling amplification strategies to design an “off-on” ECL biosensor for the ultrasensitive assay of microRNA-21, exhibiting a favorable response range (100 aM–1 nM) with an ultralow detection limit of 17.9 aM. Overall, this work offers a valid way to inhibit the intramolecular motions of AIEgens for ECL enhancement, which gives a new vision for building high-performance AIEgen-based ECL materials, thus offering more chances for assembling hypersensitive ECL biosensors.

A Simple and Robust Exponential Amplification Reaction (EXPAR)-Based Hairpin Template (exp-Hairpin) for Highly Specific, Sensitive, and Universal MicroRNA Detection.

Specific and sensitive detection of microRNAs continues to encounter significant challenges, especially in the development of rapid and efficient isothermal amplification strategies for point-of-care settings. The exponential amplification reaction (EXPAR) has garnered significant attention owing to its simplicity and rapid amplification of signals within a short period. However, a substantial loss of amplification efficiency, difficulty in distinguishing closely related homologous sequences, and adapting the designed templates to other targets seriously hamper the practical application of the EXPAR. In this work, a hairpin template tailored for the EXPAR system (exp-Hairpin) was constructed by adding identical trigger sequences and enzyme cleavage sites on two arms of the hairpin, achieving theoretically more than 2n amplification efficiency and minimal background amplification of EXPAR. Modulating the stability of the exp-Hairpin template by increasing the stem length, the specificity of detecting target miRNA in highly homologous sequences could be significantly improved. Using miRNA let-7a as a target model, the exp-Hairpin with 8 bp stem length for EXPAR amplification curves could effectively distinguish target let-7a and nontarget let-7b/7c/7f/7g/7i homologous sequences. This strategy enabled the sensitive and accurate analysis of let-7a in diluted human serum with satisfactory recoveries. By simply replacing the loop recognition sequence of exp-Hairpin, the specific detection of miR-200b was also achieved, demonstrating the universality of this strategy. The exp-Hairpin EXPAR accelerates simple and rapid molecular diagnostic applications for short nucleic acids.

Single-molecule assay guided crRNA optimization enhances specific microRNA detection by CRISPR-Cas12a

CRISPR-Cas12a is a promising tool for nucleic acid detection. However, due to the protein flexibility, Cas12a tolerates mismatches, which limits its specificity. In this study, the single-molecule assay revealed that the length of crRNA regulates the association kinetics between crRNA/Cas12a complex and target DNA. Short-crRNA/Cas12 associates the target dsDNA 2-fold faster than the single-nucleotide mismatched dsDNA, whereas the long-crRNA/Cas12a binds to the fully matched and single-nucleotide mismatched DNA targets with similar rates. These findings are further corroborated by electrophoretic mobility shift assay (EMSA) and double-stranded DNA (dsDNA) cleavage results. Inspired by these findings, we established stem-loop amplification conjugated short crRNA CRISPR-Cas12a (SlashCas12a) detection. In this approach, stem-loop-mediated microRNA reverse transcription was harnessed to enhance the amplification efficiency of the short single-stranded RNA. The short-crRNA Cas12a specifically distinguishes the single mismatched miRNA homologs, i.e. let-7a family members. The introduction of PAM containing stem-loop can break through the limitation of PAM for Cas12a and efficiently amplify the miRNAs. Multiple miRNAs, including miR-122b, miR-21, and let-7a, can be efficiently detected, and the limit of detection is up to 7.8 aM. Furthermore, the distinct expression of miR-21 and let-7a can be detected in the lung adenocarcinoma and breast samples using this method. These results demonstrate the appropriate crRNA engineering will extend the application of Cas12a in the specific molecule diagnosis.

SERS-electrochemical dual-mode detection of microRNA on same interface assisted by exonuclease III signal transformation

Dual-mode sensing has attracted more attentions which provide more accurate and reliable approach of cancer-related biomarkers. Herein, we developed a novel SERS/electrochemical dual-mode biosensor for miRNA 21 detection based on Exo III-assisted signal transformation. Firstly, the Au NPs were deposited on electrode as SERS substrate and Mn3O4/S4(DNA signal strand) was modified on Au NPs/S5 by the DNA strands S5-S4 pairing principle as hydrogen peroxide catalyst, leading to an obviously high DPV electrical signal without Raman signal. Subsequently, the presence of miRNA 21 will activate the Mn3O4/S4 to be decomposed under exonuclease III-assisted process, then the S3′ chains modified with Raman molecular Cy3(Cy3-S3′) is continuously connected to the Au NPs/S5 by DNA stands S5-S3′ pairing principle, leading to the Raman signal response and DPV signal reduction. The biosensor shows good linear calibration curves of both SERS and electrochemical sensing modes with the detection limit of 3.98 × 10−3 nM and 6.89 × 10−5 nM, respectively. This work finds an ingenious mode for dual detection of microRNA on a same interface, which opens a new strategy for SERS and electrochemical analysis.

Photoresponsive Hydrogen-Bonded Organic Frameworks-Enabled Organic Photoelectrochemical Transistors for Sensitive Bioanalysis.

A facile route for exponential magnification of transconductance (gm) in an organic photoelectrochemical transistor (OPECT) is still lacking. Herein, photoresponsive hydrogen-bonded organic frameworks (PR-HOFs) have been shown to be efficient for gm magnification in a typical poly(ethylene dioxythiophene):poly(styrenesulfonate) OPECT. Specifically, 450 nm light stimulation of 1,3,6,8-tetrakis (p-benzoic acid) pyrene (H4TBAPy)-based HOF could efficiently modulate the device characteristics, leading to the considerable gm magnification over 78 times from 0.114 to 8.96 mS at zero Vg. In linkage with a DNA nanomachine-assisted steric hindrance amplification strategy, the system was then interfaced with the microRNA-triggered structural DNA evolution toward the sensitive detection of a model target microRNA down to 0.1 fM. This study first reveals HOFs-enabled efficient gm magnification in organic electronics and its application for sensitive biomolecular detection.

Endogenously Gated DNA Walking Machine for Prescreened MicroRNA Detection in Extracellular Vesicles.

Tumor-derived extracellular vesicle (T-EV) microRNAs have been investigated as promising biomarkers in clinical diagnosis as well as disease progression monitoring. However, the expression profiles of microRNA in different tissues vary widely, the precise monitoring of microRNA levels in EVs originating from diseased tissues is susceptible to background interference, thus remains a challenge. Conventional assays require extensive processing, such as EV isolation and even sample lysis, which is both slow and laborious, and the cumbersome pretreatment could spoil the downstream analysis. To address this issue, we developed a generalizable strategy for T-EVs-selective activation and therefore specific amplified microRNA imaging. The conditional signal amplification is established by integrating a traditional DNA walker system with endogenously activated motif to achieve sensitized microRNA imaging in T-EVs. The preorganized endogenous activation with additional sensing criteria narrowed the scope against the complex specimens, and the amplified sensing with reduced off-target signals was supposed to be sensitive to monitor the tiny changes of microRNA expression during the disease course, which holds great potential for accurate diagnosis and prognosis.

Non-invasive molecular biomarkers for monitoring solid organ transplantation: A comprehensive overview.

Solid organ transplantation is a life-saving intervention for individuals with end-stage organ failure. Despite the effectiveness of immunosuppressive therapy, the risk of graft rejection persists in all viable transplants between individuals. The risk of rejection may vary depending on the degree of compatibility between the donor and recipient for both human leucocyte antigen (HLA) and non-HLA gene-encoded products. Monitoring the status of the allograft is a critical aspect of post-transplant management, with invasive biopsies being the standard of care for detecting rejection. Non-invasive biomarkers are increasingly being recognized as valuable tools for aiding in the detection of graft rejection, monitoring graft status and evaluating the efficacy of immunosuppressive therapy. Here, we focus on the importance of molecular biomarkers in solid organ transplantation and their potential role in clinical practice. Conventional molecular biomarkers used in transplantation include HLA typing, detection of anti-HLA antibodies, killer cell immunoglobulin-like receptor genotypes, and anti-MHC class 1-related chain A antibodies, which are important for assessing the compatibility of the donor and recipient. Emerging molecular biomarkers include the detection of donor-derived cell-free DNA, microRNAs (regulation of gene expression), exosomes (small vesicles secreted by cells), and kidney solid organ response test, in the recipient's blood for early signs of rejection. This review highlights the strengths and limitations of these molecular biomarkers and their potential role in improving transplant outcomes.

SERS microfluidic chip integrated with double amplified signal off-on strategy for detection of microRNA in NSCLC.

In this work, based on Fe3O4@AuNPs and double amplified signal Off-On strategy, a simple and sensitive SERS microfluidic chip was constructed to detect microRNA associated with non-small cell lung cancer (NSCLC). Fe3O4@AuNPs have two advantages of SERS enhanced and magnetic adsorption, the introduction of microfluidic chip can realize double amplification of SERS signal. First, the binding of complementary ssDNA and hpDNA moved the Raman signaling molecule away from Fe3O4@AuNPs, at which point the signal was turned off. Second, in the presence of the target microRNA, they were captured by complementary ssDNA and bound to them. HpDNA restored the hairpin conformation, the Raman signaling molecule moved closer to Fe3O4@AuNPs. At this time, the signal was turned on and strong Raman signal was generated. And last, through the magnetic component of SERS microfluidic chip, Fe3O4@AuNPs could be enriched to realize the secondary enhancement of SERS signal. In this way, the proposed SERS microfluidic chip can detect microRNA with high sensitivity and specificity. The corresponding detection of limit (LOD) for miR-21 versus miR-125b was 6.38 aM and 7.94 aM, respectively. This SERS microfluidic chip was promising in the field of early detection of NSCLC.

Integration of catalytic hairpin assembly probes into microneedles for detection of MicroRNA in plants

MicroRNAs (miRNAs) are crucial in regulating plant growth and their response to abiotic stress. Traditional miRNA detection methods are techniques which are often criticized for being expensive, labor-intensive, and time-consuming. In our research, we present a novel method that combines absorbent microneedles with a catalytic hairpin assembly (CHA) detection system. This integration facilitates swift plant sap extraction and miRNA detection. The CHA system, which consists of two hairpin probes, showcases exceptional sensitivity in identifying target miRNAs, boasting a notable detection limit of 89 pM. By modulating the photo-crosslinking duration, we controlled the swelling attributes of the methacrylic acid-modified hyaluronic acid microneedles. These microneedles proved efficient in rapidly absorbing solutions and maintained sturdy mechanical properties, allowing easy penetration into tobacco leaves for plant sap absorption. Additionally, after absorbing the miRNA solution, the fluorescence intensity of the integrated microneedles correlated linearly with concentration. It's noteworthy that while the microneedles responded to miRNA within plant tissues, it was predominantly at elevated miRNA concentrations. Our findings underscore the potential of hydrophilic microneedle arrays in both extracting and detecting miRNA in plant leaves, emphasizing their promising role in the rapid identification of vital plant compounds. This innovative method paves the way for groundbreaking research and analysis in plant studies.