miRNA Detection Research Articles

A Review of Nanotechnology in microRNA Detection and Drug Delivery.

MicroRNAs (miRNAs) are small, non-coding RNAs that play a crucial role in regulating gene expression. Dysfunction in miRNAs can lead to various diseases, including cancers, neurological disorders, and cardiovascular conditions. To date, approximately 2000 miRNAs have been identified in humans. These small molecules have shown promise as disease biomarkers and potential therapeutic targets. Therefore, identifying miRNA biomarkers for diseases and developing effective miRNA drug delivery systems are essential. Nanotechnology offers promising new approaches to addressing scientific and medical challenges. Traditional miRNA detection methods include next-generation sequencing, microarrays, Northern blotting, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Nanotechnology can serve as an effective alternative to Northern blotting and RT-qPCR for miRNA detection. Moreover, nanomaterials exhibit unique properties that differ from larger counterparts, enabling miRNA therapeutics to more effectively enter target cells, reduce degradation in the bloodstream, and be released in specific tissues or cells. This paper reviews the application of nanotechnology in miRNA detection and drug delivery systems. Given that miRNA therapeutics are still in the developing stages, nanotechnology holds great promise for accelerating miRNA therapeutics development.

Bioinspired superwettable electrode towards sensitive detection of myocardial infarction-specific miRNA

The development of a reliable and sensitive method for the detection of myocardial infarction (MI)-specific miRNAs is of great significance in the diagnosis and management of MI. Electrochemical biosensors have emerged as a promising tool for the detection of biomolecules due to their high sensitivity, selectivity, simplicity of operation, and miniaturization. In order to further improve the sensitivity and selectivity of DNA electrochemical biosensors and meet the requirements of microRNA detection in biological samples, we fabricated a patterned superwettable gold electrode (PS-Au electrode) by mimicking the superwettable micropatterns of the desert beetle. Combining this bioinspired electrode with a DSN-assisted target recycling amplification strategy, we achieved ultrasensitive detection of myocardial infarction-specific miRNA-483 ranging from 10 aM to 100 fM with a low detection limit of 5.32 aM for miRNA-483. The electrochemical biosensor based on PS-Au electrode and DSN-assisted target recycling amplification (PSE-DTRA-EB) has good stability and specificity. Our study provides a new method for the ultrasensitive detection of MI-specific miRNAs, which may have important clinical applications in the diagnosis and prognosis of MI.

Cu Single-Atom Nanozyme-Mediated Electrochemiluminescence Biosensor for Highly Sensitive Detection of MicroRNA-622.

MicroRNA (miRNA) detection is a critical aspect of disease diagnosis, and recent studies indicate that miRNA-622 could be a potential target for lung cancer. Herein, Cu single atoms were anchored on graphitic carbon nitride (Cu SAs@CN) as a coreaction accelerator applied in luminol-H2O2 system, thereby establishing an efficient and sensitive electrochemiluminescence (ECL) biosensor for miRNA-622 detection. Cu SAs@CN was explored to possess excellent enzyme-like activities that promote the generation of abundant reactive oxygen species, which amplified ECL emission. Meanwhile, in order to improve the accuracy and sensitivity for miRNA-622 detection, the highly specific trans-cleavage ability of CRISPR/Cas12a was combined with a catalytic hairpin assembly strategy. Therefore, an ECL biosensor for miRNA-622 detection was systematically constructed as a proof of concept, achieving an ultralow limit of detection of 1.09 fM, and the feasibility was demonstrated in human serum samples. The findings of this research provide a promising strategy to enhance the ECL response using versatile single-atom catalysts, thus advancing the development of ECL biosensing applications.

Target-induced multiregion MNAzyme nanowires for ultrasensitive homogeneous detection of microRNAs

A target-induced multiregion MNAzyme nanowire system is designed for the ultrasensitive and homogeneous detection of microRNAs (miRNAs). miRNA-21 and miRNA-375 are chosen as analytes, and a miRNA-induced primer exchange reaction (PER) is utilized to construct a long DNA strand with repetitive sequences. This innovative design enables the efficient anchoring of numerous MNAzymes. This unique architecture significantly boosts the effective local concentration of MNAzymes, thereby enhancing the sensitivity and efficiency of miRNA detection. Notably, the limit of detection (LOD) achieved with our target-induced multiregion MNAzyme nanowire approach is over an order of magnitude lower than most other MNAzyme-based methods, while the MNAzyme reaction time is reduced from several hours to 50 min. The method has demonstrated successful applications in quantitatively determining the expression levels of two miRNAs in cell lysates of MCF-7, HeLa and MCF-10 A cells, highlighting its potential for assaying miRNA biomarkers in clinical samples.

Sensitive Multiplexed MicroRNA Spatial Profiling and Data Classification Framework Applied to Murine Breast Tumors.

MicroRNAs (miRNAs) are small RNAs that are often dysregulated in many diseases, including cancers. They are highly tissue-specific and stable, thus, making them particularly useful as biomarkers. As the spatial transcriptomics field advances, protocols that enable highly sensitive and spatially resolved detection become necessary to maximize the information gained from samples. This is especially true of miRNAs where the location their expression within tissue can provide prognostic value with regard to patient outcome. Equally as important as detection are ways to assess and visualize the miRNA's spatial information in order to leverage the power of spatial transcriptomics over that of traditional nonspatial bulk assays. We present a highly sensitive methodology that simultaneously quantitates and spatially detects seven miRNAs in situ on formalin-fixed paraffin-embedded tissue sections. This method utilizes rolling circle amplification (RCA) in conjunction with a dual scanning approach in nanoliter well arrays with embedded hydrogel posts. The hydrogel posts are functionalized with DNA probes that enable the detection of miRNAs across a large dynamic range (4 orders of magnitude) and a limit of detection of 0.17 zeptomoles (1.7 × 10-4 attomoles). We applied our methodology coupled with a data analysis pipeline to K14-Cre Brca1f/fTp53f/f murine breast tumors to showcase the information gained from this approach.

Synergy of Target-Induced Magnetic Network and Single-Drop Chromogenic System for Ultrasensitive "All-in-Tube" Detection of miRNA in Whole Blood.

The development of liquid biopsy methods for the accurate and reliable detection of miRNAs in whole blood is critical for the early diagnosis and monitoring of diseases. However, accurate quantification of miRNA expression levels remains challenging due to the complex matrix and low abundance of miRNAs in blood samples. Herein, we report a contactless signal output strategy with low background interference that ensures "zero-contact" between the reaction system and the colorimetry system. The designed target-induced magnetic ZnS/ZIF-90/ZnS network can serve as a unique signal amplifier and transducer. It releases hydrogen sulfide (H2S) gas in an acidic solution which can be concentrated in a droplet of only a few microliters in volume, etching the silver layer of Au@Ag nanostars (NSTs) in the droplet. This will lead to changes in the localized surface plasmon resonance signals of the NSTs. Finally, quantitative detection of let-7a is realized by measuring the offset value of the UV-vis absorption peak. Therefore, by virtue of the synergistic action of quadruple signal amplification methods, including catalytic hairpin assembly, ZnS/ZIF-90/ZnS, magnetic separation, and microextraction, the "All-in-Tube" ultrasensitive detection of low-abundance let-7a in whole blood is achieved with a detection limit as low as the aM level. In addition, the "zero-contact" signal output mode effectively solves the problem of complex matrix interference, demonstrating the great potential of this method for miRNA quantification in complex samples, such as whole blood.

Ultrasensitive Electrochemiluminescence Imaging Detection of Multiple miRNAs in Single Cells with a Closed Bipolar Electrode Array Chip.

In situ sensitive detection of multiple biomarkers in a single cell was highly necessary for understanding the pathogenesis mechanism and facilitating disease diagnosis. Herein, a bipolar electrode (BPE)-electrochemiluminescence (ECL) imaging chip was designed for ultrasensitive in situ detection of multiple miRNAs in single cells based on a dual-signal amplification strategy. A single cell was trapped and lysed within the microtrap of the cathode chamber and an HCR amplification process and nanoprobes (Fc/DNA/Fe3O4) were introduced, leading to a large number of electroactive molecules (Fc) being modified on the surface. Under a suitable potential, Fc+ in the cathodic chamber was reduced to Fc and L-012 was oxidized in the anodic chamber according to the electric neutrality principle of the bipolar electrode system, resulting in the ECL signal recorded by EMCCD. Ascribed to the dual-signal amplification, sensitive visual detection of miRNA-21 and miRNA-155 in single cells was achieved. For MCF-7 cells, miRNA-21 and miRNA-155 were calculated to be 4385 and 1932 copies/cell (median), respectively. For HeLa cells, miRNA-21 and miRNA-155 were calculated to be 1843 and 1012 copies/cell (median), respectively. The comprehensive evaluation of two kinds of miRNA could effectively eliminate error signals, and the detection precision was improved by 10%.

Modular probe-based colorimetric miRNA detection via polymerase/endonuclease assisted chain displacement.

Methods for sequence-specific microRNA (miRNA) analysis are crucial for miRNA research and guiding nursing strategies. We have devised a colorimetric technique for detecting miRNA using a dumbbell probe-based polymerase/endonuclease assisted chain displacement, along with silver ions (Ag+) aptamer assisted color reaction. The suggested approach enables precise measurement of miRNA-21 within the concentration range of 100fM-5nM, with a lowdetection limitof 45.32fM. Additionally, it exhibits exceptional capability in distinguishing variations at the level of individual nucleotides. Furthermore, the detection technique may be utilized to precisely measure the amount of miRNA-21 in serum samples, demonstrating a high level of concordance with the findings obtained from a commercially available miRNA detection kit.

DMF-DM-seq: Digital-Microfluidics Enabled Dual-Modality Sequencing of Single-Cell mRNA and microRNA with High Integration, Sensitivity, and Automation.

Multimodal measurement of single cells provides deep insights into the intricate relationships between individual molecular layers and the regulatory mechanisms underlying intercellular variations. Here, we reported DMF-DM-seq, a highly integrated, sensitive, and automated platform for single-cell mRNA and microRNA (miRNA) co-sequencing based on digital microfluidics. This platform first integrates the processes of single-cell isolation, lysis, component separation, and simultaneous sequencing library preparation of mRNA and miRNA within a single DMF device. Compared with the current half-cell measuring strategy, DMF-DM-seq enables complete separation of single-cell mRNA and miRNA via a magnetic field application, resulting in a higher miRNA detection ability. DMF-DM-seq revealed differential expression patterns of single cells of noncancerous breast cells and noninvasive and aggressive breast cancer cells at both mRNA and miRNA levels. The results demonstrated the anticorrelated relationship between miRNA and their mRNA targets. Further, we unravel the tumor growth and metastasis-associated biological processes enriched by miRNA-targeted genes, along with important miRNA-interaction networks involved in significant signaling pathways. We also deconstruct the miRNA regulatory mechanisms underlying different signaling pathways across different breast cell types. In summary, DMF-DM-seq offers a powerful tool for a comprehensive study of the expression heterogeneity of single-cell mRNA and miRNA, which will be widely applied in basic and clinical research.

CRISPR/Cas12a-coupled multiplexed strand displacement amplification for miRNA155 one-tube detection: via a dual-cavity PCR tube.

A CRISPR/Cas12a-coupled multiplexed strand displacement amplification (CMSDA) for the detection of miR155 has been developed. Non-specific amplification was avoided by designing a single-stranded DNA template with a hairpin structure. The detection target miR155 was used as a primer to initiate a multiple-strand displacement reaction to produce abundant ssDNA. ssDNA was recognized by the Cas12a/CrRNA binary complex, activating the trans-cleaving activity of Cas12a. The multiple-strand displacement reaction is more efficiently detected compared with a single-strand displacement reaction. The detection range is from 250pM to 1nM, and the limit of the detection is 6.5pM. The proposed method showed a good applicability in complex serum environments, indicating that the method has a broad prospect for disease detection and clinical application. In addition, we designed a dual-cavity PCR tube, which realized one-tube detection of miRNA155 and avoided open-cap contamination.

MiRglmm: a generalized linear mixed model of isomiR-level counts improves estimation of miRNA-level differential expression and uncovers variable differential expression between isomiRs.

MicroRNA-seq data is produced by aligning small RNA sequencing reads of different miRNA transcript isoforms, called isomiRs, to known microRNAs. Aggregation to microRNA-level counts discards information and violates core assumptions of differential expression (DE) methods developed for mRNA-seq data. We establish miRglmm, a DE method for microRNA-seq data, that uses a generalized linear mixed model of isomiR-level counts, facilitating detection of miRNA with differential expression or differential isomiR usage. We demonstrate that miRglmm outperforms current DE methods in estimating DE for miRNA, whether or not there is significant isomiR variability, and simultaneously provides estimates of isomiR-level DE.

SERS biosensors based on catalytic hairpin self-assembly and hybridization chain reaction cascade signal amplification strategies for ultrasensitive microRNA-21 detection.

A highly sensitive surface-enhanced Raman scattering (SERS) biosensor has been developedfor the detection of microRNA-21 (miR-21) using an isothermal enzyme-free cascade amplification method involving catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). The CHA reaction is triggered by the target miR-21, which causes hairpin DNA (C1 and C2) to self-assemble into CHA products. After AgNPs@Capture captures the resulting CHA product, the HCR reaction is started, forming long-stranded DNA on the surface of AgNPs. A strong SERS signal is generated due to the presence of a large amount of the Raman reporter methylene blue (MB) in the vicinity of the SERS "hot spot" on the surface of AgNPs. The monitoring of the SERS signal changes of MB allows for the highly sensitive and specific detection of miR-21. In optimal conditions, the biosensor exhibits a satisfactory linear range and a low detection limit for miR-21 of 42.3 fM. Additionally, this SERS biosensor shows outstanding selectivity and reproducibility. The application of this methodology to clinical blood samples allows for the differentiation of cancer patients from healthy controls. As a result, the CHA-HCR amplification strategy used in this SERS biosensor could be a useful tool for miRNA detection and early cancer screening.

Rapid enzyme-free detection of miRNA-21 in human ovarian cancerous cells using a fluorescent nanobiosensor designed based on hairpin DNA-templated silver nanoclusters

BackgroundCancer is known as one of the main non-communicable diseases and the leading cause of death in the new era. Early diagnosis of cancer requires the identification of special biomarkers. Currently, microRNAs (miRNAs) have attracted the attention of researchers as useful biomarkers for cancer early detection. Hence, various methods have been recently developed for detecting and monitoring miRNAs. Among all miRNAs, detection of miRNA-21 (miR-21) is important because it is abnormally overexpressed in most cancers. Here, a new biosensor based on silver nanoclusters (AgNCs) is introduced for detecting miR-21. ResultsAs a fluorescent probe, a rationally designed hairpin sequence containing a poly-cytosine motif was used to facilitate the formation of AgNCs. A guanine-rich sequence was also employed to enhance the sensing signal. It was found that in the absence of miR-21, adding a guanine-rich sequence to the detecting probe caused only a slight change in the fluorescence emission intensity of AgNCs. While in the presence of miR-21, the emission signal enhanced. A direct correlation was observed between the increase in the fluorescence of AgNCs and the concentration of miR-21. The performance of the proposed biosensor was characterized thoroughly and confirmed. The biosensor detected miR-21 in an applicable linear range from 9 pM to 1.55 nM (LOD: 2 pM). SignificanceThe designed biosensor was successfully applied for detecting miR-21 in human plasma samples and also in human normal and lung and ovarian cancer cells. This biosensing strategy can be used as a model for detecting other miRNAs. The designed nanobiosensor can measure miR-21 without using any enzymes, with fewer experimental steps, and at a low cost compared to the reported biosensors in this field.

Ultrasensitive detection of miRNA 21 achieved through DNA structure-mediated self-assembly of Au NCs array functionalized surface-enhanced Raman scattering biosensor

The involvement of microRNA (miRNA) in the transcriptional and regulatory processes of gene expression in organisms, along with its aberrant expression patterns in various malignant diseases such as tumors, underscores its significance. Surface-enhanced Raman scattering (SERS) biosensing technology offers distinct advantages for miRNA detection, including simplified procedures, non-destructive analysis, fingerprint spectroscopy capabilities, and rapid detection times. This study presents the development of a novel SERS-based biosensor utilizing miRNA-21 stimulated-responsive DNA-functionalized nanomaterials. The DNA structure within the the catalyzed hairpin amplification (CHA) was utilized to modify Fe3O4 and Au nanocubes (Au NCs), and subsequently, miRNA21 served as a key to trigger the CHA reaction between these two materials. Notably, during this process, the signal probe Cy3 underwent directed migration and accumulation from Fe3O4 to Au NCs, resulting in the unidirectional enrichment of beacon probe from Fe3O4 to Au NCs, which was facilitated by the progression of the CHA cycle. This facilitated robust generation of SERS signals for precise quantification of miRNA-21. The sensor utilized Au NCs enriched with Cy3 to form highly stable and well-ordered two-dimensional arrays at the water-cyclohexane interface, demonstrating exceptional stability and reproducibility. It achieved an impressive detection limit of 0.81 fM across a dynamic range from 1 to 1 × 10⁶ fM. This work advances practical methods for amplification analysis in the sensing field, demonstrating significant potential for clinical medicine and early cancer diagnosis.

DSN signal amplification strategy based nanochannels biosensor for the detection of miRNAs

MiRNA-21 is recognized as an important biological marker for the diagnosis, treatment, and prognosis of breast cancer. Here, we have created a nanochannel biosensor utilizing the duplex-specific nuclease (DSN) signal amplification strategy to achieve the detection of miRNAs. In this system, DNA as the capture probe was covalently immobilized on the surface of nanochannels, which hybridized with the target miRNA and forms RNA/DNA duplexes. DSN could cleave the probe DNA in RNA/DNA duplexes, recycling target miRNA, which may again hybridized with other DNA probes. After N cycles, most of the DNA probes had been cleaved, and the content of miRNA could be quantified by detecting changes in surface charge density. This biosensor can distinguish miR-21 from non-complementary miRNAs and one-base mismatched miRNAs, with reliable detection limits as low as 1 fM in PBS. In addition, we had successfully applied this method to analysis of total RNA samples in MCF-7 cells and HeLa cells, and the nanochannels had also shown excellent responsiveness and strong anti-interference ability. This new method is expected to contribute to miRNA detection in clinical diagnostics, providing a unique approach to detecting and distinguishing disease-associated molecules.

Assembly of ligation chain reaction and DNA triangular prism for miRNA diagnostics

Controllable assembly of DNA nanostructure provides a powerful way for quantitative analysis of various targets including nucleic acid molecules. In this study, we have designed detachable DNA nanostructures at electrochemical sensing interface and constructed a ligation chain reaction (LCR) strategy for amplified detection of miRNA. A three-dimensional DNA triangular prism nanostructure is fabricated to provide suitable molecule recognition environment, which can be further regenerated for additional tests via convenient pH adjustment. Target triggered LCR is highly efficient and specific towards target miRNA. Under optimal experimental conditions, this approach enables ultrasensitive exploration in a wide linear range with a single-base resolution. Moreover, it shows excellent performances for the analysis of cell samples and clinical serum samples.

Ultrasensitive Photoelectrochemical Biosensor for Dual-miRNAs Detection Based on Molecular Logic Gates and Methylene Blue Sensitized ZnO@CdS@Au Nanorods.

The occurrence of cancer is often closely related to multiple tumor markers, so it is important to develop multitarget detection methods. By the proper design of the input signals and logical operations of DNA logic gates, detection and diagnosis of cancer at different stages can be achieved. For example, in the early stages, specific input signals can be designed to correspond to early specific tumor markers, thereby achieving early cancer detection. In the late stage, logic gates for multitarget detection can be designed to simultaneously detect multiple biomarkers to improve diagnostic accuracy and comprehensiveness. In this work, we constructed a dual-target-triggered DNA logic gate for anchoring DNA tetrahedra, where methylene blue was embedded in the DNA tetrahedra to sensitize ZnO@CdS@Au, achieving ultrasensitive detection of the target substance. We tested the response of AND and OR logic gates to the platform. For AND logic gates, the sensing platform only responds when both miRNAs are present. In the concentration range of 10 aM to 10 nM, the photoelectric signal gradually increases with an increase of the target concentration. Subsequently, we used OR logic gates for miRNA detection. Even if only one target exists, the sensing platform exhibits excellent performance. Similarly, within the concentration range of 10 aM to 10 nM, the photoelectric signal gradually increases with an increase of the target concentration. The minimum detection limit is 1.10 aM. Whether it is the need to detect multiple targets simultaneously or only one of them, we can achieve it by selecting the appropriate logic gate. This strategy holds promising application prospects in fields such as biosensing, medical diagnosis, and environmental monitoring.

Live-Cell Imaging of MicroRNA Expression via Photoinduced Electron Transfer Controlled by Catalytic Hairpin Assembly.

MicroRNAs (miRNAs) serve as emerging biomarkers for a range of diseases, and their quantitative analysis draws increasing attention. Yet, current invasive methods limit continuous tracking within living cells. To overcome this, a nonenzymatic DNA-based nanoprobe is developed for dynamic, noninvasive miRNA tracking via live-cell imaging. This probe features a unique hairpin DNA structure with five guanines that act as internal quenchers, suppressing fluorescence from an attached fluorophore via photoinduced electron transfer. Target miRNA initiates toehold-mediated strand displacement, restoring, and amplifying the fluorescence signal. Additionally, by introducing a single mismatch to the hairpin DNA, the nanoprobe's sensitivity is significantly enhanced, lowering the detection limit to about 60 pM without compromising specificity. To optimize intracellular delivery for prolonged monitoring, the nanoprobe is encapsulated within multilamellar lipid nanovesicles, fluorescently labeled for dual-wavelength ratiometric analysis. The proposed nanoprobe demonstrates a significant advance in live-cell miRNA detection, promising enhanced in situ analysis for a better understanding of miRNAs' pathophysiological function.

A cancer-targeted glutathione-gated probe for self-sufficient ST/CDT combination therapy and FRET-based miRNA imaging.

Acancer-targeted glutathione (GSH)-gated theranostic probe (CGT probe) for intracellular miRNA imaging and combined treatment of self-sufficient starvation therapy (ST) and chemodynamic therapy (CDT) was developed. TheCGT probe is constructed using MnO2 nanosheet (MS) as carrier material to adsorb the elaborately designed functional DNAs. It can be internalized by cancer cells via specific recognition between the AS1411 aptamer and nucleolin.After CGT probe entering the cancer cells, the overexpressed GSH, as gate-control, can degrade MS to Mn2+ which can be used for CDT by Fenton-like reaction. Simultaneously, Mn2+-mediated CDT can further cascade with the enzyme-like activities (catalase-like activity and glucose oxidase-like activity) of CGT probe, achieving self-sufficient ST/CDT synergistic therapy. Meanwhile, the anchored DNAs are released, achieving in situ signal amplification via disubstituted-catalytic hairpin assembly (DCHA) and FRET (fluorescence resonance energy transfer) imaging of miR-21. The in vitro and in vivo experiments demonstrated that accurate and sensitive miRNA detection can be achieved using the CGT probe. Overall, the ingenious CGT probe opens a new avenue for the development of early clinical diagnosis and cancer therapy.

Single Particle Analysis-Enhanced DNA Walking Machine for Sensitive miRNA Detection.

DNA walking machines have achieved significant breakthroughs in areas such as biosensing, bioimaging, and early cancer diagnosis, facilitated by the self-assembly of DNA or its combination with other materials, such as magnetic beads and metal nanoparticles. However, current DNA walking machine strategies are constantly challenged by inadequate analytical sensitivity, while sophisticated signal amplification procedures are often indispensable. Single-particle inductively coupled plasma mass spectrometry (SP-ICPMS) provides superior sensitivity and can effectively discriminate between background noise and detected signals due to the large number of metal atoms in a nanoparticle and the concentrating effect of single nanoparticle detection. In this study, we present a novel approach utilizing single nanoparticle counting and duplex-specific nuclease (DSN)-assisted signal amplification to construct a 3D DNA walking machine for detecting the aggressive prostate cancer (PCa) biomarker miRNA-200c. The proposed strategy showed an improvement in sensitivity with a detection limit (LOD) of 0.93 pM (28 amol) and was successfully applied in human serum samples. To the best of our knowledge, this is the first report of the DNA walking machine with single nanoparticle counting study.