miRNA Detection Research Articles

A self-powered sensor based on hollow bimetallic sulfide and double-strand TSDR amplification achieves sensitive miRNA-21 detection

MicroRNA (miRNA) sensitive detection is significant for cancer diagnosis. Currently, self-powered biosensors in clinical miRNA detection show the advantages of no external power supply, mild reaction conditions, low cost and portability. In this work, the bimetallic sulfide heterojunction hollow nanocubes are prepared as the substrate material, which can significantly improve the electrochemical reaction rate and increase the enzyme load. Meanwhile, we design the independent duplex-chain assisted toehold mediated strand displacement reaction (TSDR) strategy. The two substable double chains trigger a continuous chain displacement reaction, and the current signal amplification is realized based on molecular recognition. The constructed sensor has a linear response range of 0.05–10000 fM and a detection limit (LOD) of 8.3 aM. In addition, we use commercial apps to present signals, fundamentally changing the signal output mode of electrochemical sensors, and facilitating the construction of a portable real-time sensor platform. The self-powered biosensor has the characteristics of good biocompatibility, real-time monitoring and economic efficiency in miRNA detection, which provides a new idea for sensitive detection of miRNA. Moreover, it has far-reaching reference significance for the development of other sensing technologies, which is expected to extend from the medical field to environmental monitoring, food safety and other fields.

A self-powered theranostic DNA nanodevice for amplification of both intracellular microRNA imaging and photodynamic therapy

While numerous methods exist for diagnosing tumors through the detection of miRNA within tumor cells, few can simultaneously achieve both tumor diagnosis and treatment. In this study, a novel graphene oxide (GO)-based DNA nanodevice (DND), initiated by miRNA, was developed for fluorescence signal amplification imaging and photodynamic therapy in tumor cells. After entering the cells, tumor-associated miRNA drives DND to Catalyzed hairpin self-assembly (CHA). The CHA reaction generated a multitude of DNA Y-type structures, resulting in a substantial amplification of Ce6 fluorescence release and the generation of numerous singlet oxygen (1O2) species induced by laser irradiation, consequently inducing cell apoptosis. In solution, DND exhibited high selectivity and sensitivity to miRNA-21, with a detection limit of 11.47 pM. Furthermore, DND discriminated between normal and tumor cells via fluorescence imaging and specifically generated O21 species in tumor cells upon laser irradiation, resulting in tumor cells apoptosis. The DND offer a new approach for the early diagnosis and timely treatment of malignant tumors.

Dumbbell probe initiated multi-rolling circle amplification assisted CRISPR/Cas12a for highly sensitive detection of clinical microRNA

A novel miRNA detection technique named Dumbbell probe initiated multi-Rolling Circle Amplification assisted CRISPR/Cas12a (DBmRCA) was developed relying on the ligation-free dumbbell probe and the high-sensitivity CRISPR/Cas12a signal out strategy. This DBmRCA assay streamlines miRNA quantification within a mere 30-min timeframe and with exceptional analytical precision. The efficacy of this method was validated by assessing miRNA levels in clinical samples, revealing distinct expression panel of miR-200a and miR-126 in lung cancer/adjacent/normal tissue specimens. Moreover, a predictive model was established to classify benign and malignant tumor. Due to its time efficiency, enhanced sensitivity, and streamlined workflow, this assay would be a reliable tool for miRNA analysis in clinical settings, offering potential guidance for early diagnosis and treatment of lung cancer.

Panel miRNAs are potential diagnostic markers for chronic kidney diseases: a systematic review and meta-analysis

BackgroundAccurate detection of kidney damage is key to preventing renal failure, and identifying biomarkers is essential for this purpose. We aimed to assess the accuracy of miRNAs as diagnostic tools for chronic kidney disease (CKD).MethodsWe thoroughly searched five databases (MEDLINE, Web of Science, Embase, Scopus, and CENTRAL) and performed a meta-analysis using R software. We assessed the overall diagnostic potential using the pooled area under the curve (pAUC), sensitivity (SEN), and specificity (SPE) values and the risk of bias by using the QUADAS-2 tool. The study protocol was registered on PROSPERO (CRD42021282785).ResultsWe analyzed data from 8351 CKD patients, 2989 healthy individuals, and 4331 people with chronic diseases. Among the single miRNAs, the pooled SEN was 0.82, and the SPE was 0.81 for diabetic nephropathy (DN) vs. diabetes mellitus (DM). The SEN and SPE were 0.91 and 0.89 for DN and healthy controls, respectively. miR-192 was the most frequently reported miRNA in DN patients, with a pAUC of 0.91 and SEN and SPE of 0.89 and 0.89, respectively, compared to those in healthy controls. The panel of miRNAs outperformed the single miRNAs (pAUC of 0.86 vs. 0.79, p < 0.05). The SEN and SPE of the panel miRNAs were 0.89 and 0.73, respectively, for DN vs. DM. In the lupus nephritis (LN) vs. systemic lupus erythematosus (SLE) cohorts, the SEN and SPE were 0.84 and 0.81, respectively. Urinary miRNAs tended to be more effective than blood miRNAs (p = 0.06).ConclusionMiRNAs show promise as effective diagnostic markers for CKD. The detection of miRNAs in urine and the use of a panel of miRNAs allows more accurate diagnosis.

Hybrid chain reaction nanoscaffold-based functional nucleic acid nanomaterial cascaded with rolling circle amplification for signal enhanced miRNA let-7a detection.

Anovel functional nucleic acid (FNA) nanomaterial based on hybrid chain reaction (HCR) nanoscaffolds isproposed to solve the problemof time superposition and repeated primer designin sensitive miRND detectionusing cascade amplification technique. Rolling circle amplification (RCA) was cascaded with the prepared FNA nanomaterials for miRNA let-7a (as a model target) sensitive detection by lateral flow assay (LFA). Under the optimal conditions, the proposed RCA-FNA-LFA assay demonstrated the specificity and accuracy for miRNA let-7a detection with a detection limit of 1.07pM, which increased sensitivity by nearly 20 times compared with that of RCA -LFA assay. It is worth noting that the non-target-dependent self-assembly process of HCR nanoscaffolds does not take up the whole detection time, thus, less time is taken than that of the conventional cascaded method. Moreover, the proposed assay does not need to consider the system compatibility between two kinds of isothermal amplification techniques. As for detection of different miRNAs, only the homologous arm of the padlock probe of RCA needs to be changed, while the FNA nanomaterial does not need any change, which greatly simplifies the primer design of the cascaded amplification techniques. With further development, the proposed RCA-FNA-LFA assay might achieve more sensitive and faster results to better satisfy the requirements of clinical diagnosis combing with more sensitive labels or small strip reader.

Highly sensitive ECL detection of miRNA based on self-generated coreactant and target-induced catalyzed hairpin assembly

BackgroundRecently, various techniques have been developed to accurately and sensitively detect tumor biomarkers for the early diagnosis and effective therapy of cancer. The electrochemiluminescence (ECL) method holding outstanding features including high sensitivity, ease of operation, and spatiotemporal controllability exhibited great potential for DNA/RNA detection, immunoassay, cancer cell detection, and environmental analysis. However, a glaring problem of ECL approaches is that the layer-by-layer modification on the electrode leads to poor stability and sensitivity of the sensors. Therefore, new simple and efficient methods for electrode modification which can effectively improve the ECL signal have attracted more and more research interests. ResultsBased on the dual amplification strategy of target-induced CHA and nanocomposite probes leading to self-generated co-reactant (H2O2), we proposed a highly sensitive miRNA-ECL detection system. The introduction of the target miRNA-21 triggers the CHA cycle amplification of DNA1 and biotin-modified DNA2, releasing the target miRNA-21 sequence for the target cycle reaction. After the reaction, the newly introduced DNA2 was combined with Au NPs modified with SA and Glucose oxidase (GOD). In the presence of oxygen, glucose was decomposed by GOD to produce H2O2, and then H2O2 was immediately catalyzed by the Hemin/G-quadruplex at the double-stranded end of the CHA product to produce a large amount of O2−•. As a co-reactant of luminol, the ECL signal was significantly enhanced, thereby achieving highly sensitive detection of miRNA-21 content and obtaining a low detection limit of 0.65 fM. The high specificity of the ECL biosensor was also proved by base mismatch. SignificanceCompared with other current detection methods, this sensor can achieve quantitative analysis of other target analytes by flexibly changing the probe DNA sequence, and provide a new feasible solution for the detection of tumor-associated markers. Benefiting from the improved sensitivity and selectivity, the proposed biosensing platform is expected to provide a new strategy for biomarkers analysis and outstanding prospect for further clinical application.

(Invited) Aiot-Integrated Digital Imaging Sensor for Molecular-Fingerprint Profiling of Extracellular Vesicles in Liquid Biopsy

The advancement of liquid biopsy techniques for non-invasive tumor analysis holds immense potential in continuously tracking tumor status—recurrence, progression, and treatment response—in real-time. To comprehensively assess tumor evolution, cellular diversity, and drug resistance mechanisms, a pivotal step involves scrutinizing proteins and nucleic acids within extracellular vesicles (EVs) present in biofluids. Our focus lies in crafting an advanced digital bead-based sensor system that seamlessly evaluates the molecular profile of EVs in glioblastoma cases. Our innovative approach enriches tumor-relevant genetic data within EVs using antibody-specific magnetic beads, subsequently amplifying it via reverse transcription recombinase polymerase amplification system across forty thousand droplets. These results are scrutinized using a compact imaging device. The core strengths of our methodology include: (a) Streamlined Sample Processing: Our system facilitates ultra-sensitive miRNA detection in EVs within a rapid ninety-minute window, owing to optimized sample preparation and a compact detection mechanism; (b) Enhanced Signal Clarity: Employing a straightforward isothermal assay significantly boosts the fluorescent signal-to-noise ratio within each 70-µm droplet, achieving over 104 turnovers of fluorescent reporters on a chip; (c) Real-Time Data Analysis: Our system seamlessly captures real-time imaging data for the edge computing, utilizing trained YOLOv5 models for classification, and presents genetic results on the smartphone. Its user-friendly design enables utilization by healthcare workers with varying skill levels. This pioneering approach has the potential to offer affordable, rapid, and informative diagnostics particularly beneficial for underserved regions and healthcare systems facing financial constraints. Furthermore, its adaptability for diverse cancers could revolutionize the pace of diagnosis and treatment decision-making through liquid biopsy, potentially establishing its widespread application in global healthcare.

(Sensor Division Early Career Award) Portable Diagnostics for Cancer Management at Printed Strips

Point-of-care testing (PoC) is revolutionizing the healthcare sector improving patient care in daily hospital practice and allowing reaching even remote areas. In the frame of cancer management, the design and validation of PoC enabling the non-invasive, rapid detection of cancer markers is urgently required to implement liquid biopsy. However, despite substantial advances in sensing technologies, the development, preparation, and use of self-testing devices is still confined to specialist laboratories and users. Decentralized analytical devices will enormously impact daily lives, enabling people to analyze diverse clinical, environmental, and food samples, evaluate them and make predictions to improve quality of life, particularly in remote, resource-scarce areas. In recent years, paper-based analytical tools have attracted a great deal of attention; the well-known properties of paper, such as abundance, affordability, lightness, and biodegradability, combined with features of printed electrochemical sensors, have enabled the development of sustainable devices that drive (bio)sensors beyond the state of the art. Their blindness toward colored/turbid matrices (i.e., blood, soil), their portability, and the capacity of paper to autonomously filter/purge/react with target species make such devices powerful in establishing point-of-need tools for use by non-specialists. Depending on analytical requisites, different types of paper (filter, office) and configurations (1D, 2D, 3D) can be adopted. Focusing on stable blood-based markers with high-specificity, such as miRNAs, is of crucial importance. A wide overview regarding application ranging from DNA to miRNA detection will be provided, with the aim in showing the potentialities of portable electrochemical biosensors for improving society involvement in monitoring circulating nucleic acids associated to diverse conditions, i.e. cancer, Alzheimer’s disease, HIV.

High-Sensitivity Homogeneous Detection of miRNA-155 Governed by DNA Walker-Regulated Surface DNA Density of Magnetic Electrochemiluminescence Nanoparticles.

Homogeneous electrochemiluminescence (ECL) has gained attention for its simplicity and stability. However, false positives due to solution background interference pose a challenge. To address this, magnetic ECL nanoparticles (Fe3O4@Ru@SiO2 NPs) were synthesized, offering easy modification, magnetic separation, and stable luminescence. These were utilized in an ECL sensor for miRNA-155 (miR-155) detection, with locked DNAzyme and substrate chain (mDNA) modified on their surface. The poor conductivity of long-chain DNA significantly impacts the conductivity and electron transfer capability of Fe3O4@Ru@SiO2 NPs, resulting in weaker ECL signals. Upon target presence, unlocked DNAzyme catalyzes mDNA cleavage, leading to shortened DNA chains and reduced density. In contrast, the presence of short-chain DNA has minimal impact on the conductivity and electron transfer capability of Fe3O4@Ru@SiO2 NPs. Simultaneously, the material surface's electronegativity decreases, weakening the electrostatic repulsion with the negatively charged electrode, resulting in the system detecting stronger ECL signals. This sensor enables homogeneous ECL detection while mitigating solution background interference through magnetic separation. Within a range of 100 fM to 10 nM, the sensor exhibits a linear relationship between ECL intensity and target concentration, with a 26.91 fM detection limit. It demonstrates high accuracy in clinical sample detection, holding significant potential for clinical diagnostics. Future integration with innovative detection strategies may further enhance sensitivity and specificity in biosensing applications.

ZnS/MXene/CdS heterostructure modified electrode for ultrasensitive miRNA-21 assay with signal amplified photoelectrochemical strategy

The early-stage detection of microRNA (miRNA) holds significant value for clinical diagnosis of tumor. In this study, a highly sensitive miRNA detection platform is developed using a photoelectrochemical sensing system based on layer-by-layer (LBL) assembled ZnS/MXene/CdS (ZMC) heterostructure electrode. By incorporating ultrathin and highly conductive MXene, the ZMC electrode exhibits robust photocurrent signals upon exposure to light. The electrode is further modified HS-H1 DNA via disulfide bonds.With the help of auxiliary H2 DNA, a target miRNA-21 triggered catalytic hairpin assembly (CHA) amplification reaction is realized on the electrode surface through complementary base pairing. Then, manganese porphyrin (MnPP) is introduced to the formed double-stranded DNA after CHA reaction to catalyze the conversion of 4-chloro-1-naphthol (4-CN) to benzo-4-chloro-hexanedione (4-CD) precipitate in the presence of hydrogen peroxide, which leads to a significant decrease in photocurrent signals. As a result, the sensor demonstrates a reliable linear correlation in miRNA-21 detection with detection limits as low as 0.36 fM. The recoveries of miRNA-21 in human serum samples ranged from 98.27 % to 101.35 % with RSD values below 3.67 % suggest the reliability and practicality of this method. Therefore it shows great potential for the construction and application of highly sensitive photoelectrochemical biosensors in clinical sample detection.

Recent Strategies for MicroRNA Detection: A Comprehensive Review of SERS-Based Nanobiosensors

With advances in technology, diagnostic techniques have become more sophisticated and efficient at detecting biomarkers rapidly. Biomarkers such as microRNA (miRNA), which exhibit exceptional specificity and sensitivity compared with other biomarkers, have garnered particular interest. Composed of 21–24 nucleotides, miRNAs constitute a noncoding RNA group that regulates gene expression, immune system activation, apoptosis, and other cellular processes; hence, they are frequently used as biomarkers for various diseases. This has sparked significant interest regarding the identification of the specific miRNAs implicated in many diseases. Presently, miRNA detection methods include northern blots, reverse transcription-quantitative polymerase chain reaction, and next-generation sequencing. While these methods are all sensitive, they are time-consuming, complex, and expensive, which renders them unsuitable for on-site detection. Surface-enhanced Raman scattering (SERS) can overcome these limitations to enable the sensitive and rapid detection of miRNA. This technique amplifies Raman signals, with signal enhancement levels changing sensitively depending on the distance between the target molecule and substrate. Therefore, this review covers the principle of SERS as a method for detecting miRNAs using nanomaterials, along with examples of nanomaterials and SERS applications. Based on the available literature, SERS is anticipated to enable the convenient, early diagnosis of various diseases, potentially lowering mortality rates. This review could therefore contribute significantly to the advancement of medical and diagnostic technologies.

Combined Analysis of Multi-Study miRNA and mRNA Expression Data Shows Overlap of Selected miRNAs Involved in West Nile Virus Infections.

The emerging zoonotic West Nile virus (WNV) has serious impact on public health. Thus, understanding the molecular basis of WNV infections in mammalian hosts is important to develop improved diagnostic and treatment strategies. In this context, the role of microRNAs (miRNAs) has been analyzed by several studies under different conditions and with different outcomes. A systematic comparison is therefore necessary. Furthermore, additional information from mRNA target expression data has rarely been taken into account to understand miRNA expression profiles under WNV infections. We conducted a meta-analysis of publicly available miRNA expression data from multiple independent studies, and analyzed them in a harmonized way to increase comparability. In addition, we used gene-set tests on mRNA target expression data to further gain evidence about differentially expressed miRNAs. For this purpose, we also studied the use of target information from different databases. We detected a substantial number of miRNA that emerged as differentially expressed from several miRNA datasets, and from the mRNA target data analysis as well. When using mRNA target data, we found that the targetscan databases provided the most useful information. We demonstrated improved miRNA detection through research synthesis of multiple independent miRNA datasets coupled with mRNA target set testing, leading to the discovery of multiple miRNAs which should be taken into account for further research on the molecular mechanism of WNV infections.

Primer exchange reaction assisted CRISPR/Cas9 cleavage for detection of dual microRNAs with electrochemistry method.

Anelectrochemical sensor assisted by primer exchange reaction (PER) and CRISPR/Cas9 system (PER-CRISPR/Cas9-E) was established for the sensitive detection of dual microRNAs (miRNAs). Two PER hairpin (HP) were designed to produce a lot of extended PER products, which could hybridize with two kinds of hairpin probes modified on the electrode and initiate the cleavage of two CRISPR/Cas9 systems guided by single guide RNAs (sgRNAs) with different recognition sequences. The decrease of the two electrochemical redox signals indicated the presence of dual-target miRNAs. With the robustness and high specificity of PER amplification and CRISPR/Cas9 cleavage system, simultaneous detection of two targets was achieved and the detection limits for miRNA-21 and miRNA-155 were 0.43 fM and 0.12 fM, respectively. The developed biosensor has the advantages of low cost, easy operation, and in-situ detection, providing a promising platform for point-of-care detection of multiple miRNAs.

Al-doped ZnO Nanostars for Electrochemical miRNA-21 Biosensors

The accurate and cost-effective detection of miRNAs, which are strongly associated with numerous diseases and disorders, poses significant technical challenges due to their small size, low concentration, and sequence similarity. In this study, we address these challenges by developing an Al-doped ZnO nanostar working electrode for electrochemical biosensors targeting miRNA-21 detection, employing a simple hydrothermal growth method. The introduction of Al doping modifies the nanostructure of the nanostars, enhancing their affinity for DNA probing and enabling tunability of the working electrode’s conductivity. This enhancement is achieved through the formation of a high-density, uniformly distributed nanostar structure, leading to improved electron transfer rates and increased surface area for miRNA binding. The nanostar biosensors, optimized with the appropriate Al content, exhibit satisfactory analytical properties, including a wide linear range from 1 pM to 10 nM, a low detection limit of 3.98 pM, reproducibility, and excellent selectivity for miRNA assays. Notably, real-time detection is achievable, demonstrating promising potential for point-of-care testing.

Integration of RCA-Based DNA Nanoscaffold with Target Triggered RNA-Cleaving DNAzyme for Sensitive Detection of miRNA21.

Cascaded amplification showed promising potential for detection of trace target miRNAs in molecular diagnosis and prevention of many diseases. In this study, miRNA21 was chosen as the target, and rolling circle amplification (RCA)-based DNA nanoscaffold was integrated with target triggered RNA-cleaving DNAzyme for sensitive detection of miRNA21. That is, the H1 probe was bound with the long-chain product of RCA to self-assemble into DNA nanoscaffold. Target miRNA21 triggered the hybridization chain reaction (HCR) located on the nanoscaffold, and led to rapid proximity of DNAzyme fragments modified at both ends of the H2 probe, which realized the cyclic cleavage of self-quenching substrate probe efficiently, and the fluorescence signal was restored. The results demonstrated that the proposed assay was sensitive, 0.76pM of miRNA21 can be detected. The proposed assay was specific; only one-base mismatched miRNA21 can be effectively recognized, other nucleic acid sequence and the serum matrix did not cause any interference. The proposed assay was accurate; recoveries from 82.1 to 115.0% can be obtained in the spiked fetal bovine serum (FBS). The flexible and programmable characteristics of DNA nanoscaffold and DNAzyme provide a confident and robust strategy for more sensitive nucleic acid detection, and can be developed to be a universal sensing platform for detecting other miRNAs just needing modification on the corresponding sequence of H1 probe in HCR.

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.