microRNA Detection Research Articles

All-in-One Fusogenic Nanoreactor for the Rapid Detection of Exosomal MicroRNAs for Breast Cancer Diagnosis.

Molecular-profiling-based cancer diagnosis has significant implications for predicting disease prognosis and selecting targeted therapeutic interventions. The analysis of cancer-derived extracellular vesicles (EVs) provides a noninvasive and sequential method to assess the molecular landscape of cancer. Here, we developed an all-in-one fusogenic nanoreactor (FNR) encapsulating DNA-fueled molecular machines (DMMs) for the rapid and direct detection of EV-associated microRNAs (EV miRNAs) in a single step. This platform was strategically designed to interact selectively with EVs and induce membrane fusion under a specific trigger. After fusion, the DMMs recognized the target miRNA and initiated nonenzymatic signal amplification within a well-defined reaction volume, thus producing an amplified fluorescent signal within 30 min. We used the FNRs to analyze the unique expression levels of three EV miRNAs in various biofluids, including cell culture, urine, and plasma, and obtained an accuracy of 86.7% in the classification of three major breast cancer (BC) cell lines and a diagnostic accuracy of 86.4% in the distinction between patients with cancer and healthy donors. Notably, a linear discriminant analysis revealed that increasing the number of miRNAs from one to three improved the accuracy of BC patient discrimination from 78.8 to 95.4%. Therefore, this all-in-one diagnostic platform performs nondestructive EV processing and signal amplification in one step, providing a straightforward, accurate, and effective individual EV miRNA analysis strategy for personalized BC treatment.

A reverberating triple AND logic gate nanomachine for intracellular multi-enzyme tracking and MicroRNA detection

With the rapid advancement of molecular biology and nanotechnology, DNA logic gates have emerged as a method to simulate complex chemical reaction networks, which is crucial for distinguishing tumor cells from normal cells in early cancer diagnosis and treatment. The current challenges include the compositional differences in biomarkers between tumors and normal tissues and achieving high specific imaging in low-expression miRNAs within cells. We developed a triple AND logic-gate nanomachine that utilizes the high expression of matrix metalloproteinase (MMPs) in tumor metastasis and the specific overexpression of telomerase in cancer cells to initiate a first-stage signal and accurately identify tumor cells. This system then interacts with miRNA-21 to trigger a second-stage signal, enabling precise detection and signal amplification through the activity of purine-free/pyrimidine-free endonuclease 1 (APE1). This system can not only achieve efficient target recognition in complex bio-logical samples, but also provide a strong detection signal through continuous signal amplification, greatly improving the accuracy and reliability of diagnosis. This research provides a new molecular tool for cancer diagnosis, facilitating early detection and improving patient treatment outcomes and quality of life. Additionally, the principles and methods of this technique can be applied to the detection of other disease markers and tumor cell subtypes.

A microfluidic-based chemiluminescence biosensor for sensitive multiplex detection of exosomal microRNAs based on hybridization chain reaction

The analysis of microRNAs (miRNAs) in exosomes is of great importance for noninvasive early disease diagnosis. However, current techniques to detect exosomal miRNAs is hampered either by laborious exosome isolation or low abundance of miRNAs in exosomes. Here, we developed a microfluidic chemiluminescence (CL) analysis method for the multiplexed detection of exosomal miR-21 and miR-155. The microfluidic device contained three parts: a snake-shaped channel for fully mixing chemiluminescent reagents, a ship-shaped channel modified with CD63 protein aptamer for capturing exosomes, and another two parallel ship-shaped channels for hybridization chain reaction (HCR) amplification and CL detection. The multiple signal amplification was realized by Y-shaped arrays, HCR amplification, and poly-HRP catalyzed CL reaction. Using this multiple signal amplification method, our microfluidic CL biosensor achieves a limit of detection of miRNAs of 0.49 fM, with a linear range of 1 fM–10 pM, which is better or comparable to previously reported biosensors. What's more, the proposed microfluidic biosensor exhibits great specificity and selectivity to the target miRNA. Moreover, the microfluidic CL strategy exhibited excellent accuracy and could significantly distinguish different cancer subtypes as well as cancer patients and healthy people. These results suggest that this simple, high sensitive, and more accurate analytical strategy by analyzing different types of exosomal miRNAs has the potential applications in cancer diagnosis and stage monitoring.

Ultrasensitive Electrochemical Biosensor with Powerful Triple Cascade Signal Amplification for Detection of MicroRNA.

In this work, by ingeniously integrating catalytic hairpin assembly (CHA), double-end Mg2+-dependent DNAzyme, and hybridization chain reaction (HCR) as a triple cascade signal amplifier, an efficient concatenated CHA-DNAzyme-HCR (CDH) system was constructed to develop an ultrasensitive electrochemical biosensor with a low-background signal for the detection of microRNA-221 (miRNA-221). In the presence of the target miRNA-221, the CHA cycle was initiated by reacting with hairpins H1 and H2 to form DNAzyme structure H1-H2, which catalyzed the cleavage of the substrate hairpin H0 to release two output DNAs (output 1 and output 2). Subsequently, the double-loop hairpin H fixed on the electrode plate was opened by the output DNAs, to trigger the HCR with the assistance of hairpins Ha and Hb. Finally, methylene blue was intercalated into the long dsDNA polymer of the HCR product, resulting in a significant electrochemical signal. Surprisingly, the double-loop structure of the hairpin H could prominently reduce the background signal for enhancing the signal-to-noise ratio (S/N). As a proof of concept, an ultrasensitive electrochemical biosensor was developed using the CDH system with a detection limit as low as 9.25 aM, achieving favorable application for the detection of miRNA-221 in various cancer cell lysates. Benefiting from its enzyme-free, label-free, low-background, and highly sensitive characteristics, the CDH system showed widespread application potential for analyzing trace amounts of biomarkers in various clinical research studies.

Highlighting the impact of blocking monolayers on DNA electrochemical sensors. Theoretical and experimental investigations under flow conditions

A model is proposed to account for the influence of electrode surface modifications in electrochemical DNA sensors operating under flow conditions. With blocking self-assembled monolayers of double-stranded DNA, the performance of the electrochemical transduction signal in the presence of a redox mediator is generally attributed to the rate of long-range electron transfer or to the screening effects of the monolayers due to negatively charged DNA. The originality of this model is here to only consider the influence of non-linear mass transport at the free-remaining sites. The model was tested for the detection of microRNAs (21 nucleotides) by implementing microchannel electrodes in the presence of an equimolar mixture of Fe(CN)63− / Fe(CN)64-. Two operating conditions leading to distinct monolayer structures were investigated using Pt and Au electrodes. The model successfully simulated the voltammograms confirming the apparent decrease in the signal transduction kinetics due to altered mass transport. These results were supported by measurements in electrochemical impedance spectroscopy.

Enhancement of microRNA Detection Signal Based on Au-nanoparticle-catalyzed Ag Accumulation Observed by Electrochemical and Surface Plasmon Resonance Analyses

Enhancement of microRNA Detection Signal Based on Au-nanoparticle-catalyzed Ag Accumulation Observed by Electrochemical and Surface Plasmon Resonance Analyses

SERS and electrochemical dual-mode detection of miRNA-141 by using single Au@Ag nanowire as a new platform.

As biomarkers of cancer, the accurate and sensitive detection of microRNAs is of great significance. Therefore, we proposed a surface-enhanced Raman scattering (SERS)/electrochemical (EC) dual-mode nanosensor for sensitively detecting miRNA-141. The nanosensor uses Au@Ag nanowires as a novel SERS/EC sensing platform, which has the advantages of good biocompatibility, fast response, and high sensitivity. The dual-mode nanosensor can not only effectively overcome the problem of insufficient reliability of single signal, but also realize the amplification and stable output of the detection signal, to ensure the reliability and repeatability of miRNA detection. With this sensing strategy, the target miRNA-141 can be detected over a wide linear range (100 fM to 50 nM) (LOD of 18.4 fM for SERS and 16.0 fM for electrochemical methods). In addition, the process shows good selectivity and can distinguish miRNA-141 from other interfering miRNAs. The actual analysis of human serum samples also proves that our strategy has good reliability, repeatability, and has broad application prospects in the field of analysis and detection.

Ingenious dual-circle DNA walker-mediated electrochemical biosensor for rapid and ultrasensitive detection of microRNA

In this work, an ingenious dual-circle DNA walker (DCDW) with pretty fast walking speed and high amplification efficiency was developed for rapid and ultrasensitive electrochemical detection of microRNA-221 (miRNA-221) related to liver cancer, combined with the toehold-mediated strand-displacement reactions (TSDRs). Impressively, compared with the traditional DNA walker, the DCDW with unique double-stranded interlocked DNA nanostructure not only possesses higher stability, flexibility, and anti-entanglement ability, but also enables more functional domain in a smaller area, thereby enhancing the local concentration, which can greatly improve the working efficiency. As a validation, the electrochemical biosensor realized rapid and ultrasensitive detection of miRNA-221 with a reaction time of 15 min and detection limit down to 1.9 aM, and had been applied in MHCC97L and HeLa cancer cell lysates, thus providing an innovative insight to design intelligent functional interlocked DNA walkers for ultimate application in the construction of biosensing platform and miRNA detection in biological sample.

Robust Sequence Design Space for the Isothermal Exponential Amplification of Short Oligonucleotides.

Advances in isothermal amplification techniques have accelerated development in biosensing applications and the design of complex molecular devices. The exponential amplification reaction technique, or EXPAR, is uniquely positioned to process molecular information from short oligonucleotide strands (≈10nucleotides length) typically encountered in molecular computing or microRNA detection. Despite its conceptual simplicity (requiring only a template strand and two enzymes), the issue of nonspecific background amplification has hindered broader adoption. In this work, a new system configuration is established at 37 °C to achieve significantly improved performance. Critical sequence motifs responsible for the excellent signal-to-background profile are identified and generalized as a universal adapter design framework. Orthogonal template sequences generated from the framework are implemented for a triplex reaction and successfully evaluated mixtures of multiple-target inputs in a single-step, one-pot format without the need for exogenous agents.

A dual-targeted electrochemical aptasensor for neuroblastoma-related microRNAs detection

Neuroblastoma (NB) is a significant pediatric cancer associated with high mortality rates, demanding innovative and appropriate approaches for its accurate detection. This paper described the design of a dual-target electrochemical aptasensor capable of simultaneously detecting neuroblastoma-associated microRNAs (miRNA-181 and miRNA-184) with exceptional sensitivity. Screen-printed carbon electrodes (SPCEs) were utilized with gold nanorods (AuNRs), and aptamers functionalized gold nanoparticles (AuNPs) to improve sensitivity, specificity, and portable detection ability. The detection method employed in this study includes differential pulse voltammetry (DPV) and cyclic voltammetry (CV). Our aptasensor exhibited remarkable limits of detections (LODs) of 5.10 aM for miRNA-181 and 9.39 aM for miRNA-184, respectively, along with a broad linear range spanning from 0.1 fM to 100 pM for both miRNAs. The practical significance of neuroblastoma diagnosis was shown through the validation of serum samples and comparison with quantitative polymerase chain reaction (qPCR). Our electrochemical aptasensor is user-friendly, easy to engineer, and offers a promising approach for accurately and selectively detecting important miRNA biomarkers in cancer screening and diagnosis, showing potential application in various clinical scenarios.

Making Healthcare Accessible: A Rapid Clean-Room-Free Fabrication Strategy for Microfluidics-Driven Biosensors Based on Coupling Stereolithography and Hot Embossing.

Microfluidics offers transformative potential in healthcare by enabling miniaturized, user-friendly, and cost-effective devices for disease diagnostics among other biomedical applications; however, their meaningful adoption is severely hindered, especially in developing countries and resource-limited settings, by the cost, time, and complexity of their fabrication. To overcome this barrier of access, this work develops a novel approach for highly efficient (<4 h), cost-effective, and clean-room-free fabrication of functional polydimethylsiloxane (PDMS)-based microfluidic devices based on coupling stereolithography three-dimensional (3D) printing with hot embossing. The strategy exhibits high fidelity between the digital design and final device, remarkable transfer accuracy between the 3D print and poly(methyl methacrylate) (PMMA) mold, in addition to highly smooth surfaces (R a < 1 μm). To establish the versatility of the approach and performance quality of the fabricated devices, three advanced microfluidics-driven biosensing platforms are developed: a microsphere droplet generator, a stop-flow lithography-based hydrogel microparticle synthesizer, and a hydrogel postembedded microfluidic device for multiplexed biomarker detection. As a proof-of-concept, the latter platform was applied to the multiplexed detection of microRNA, a highly promising class of liquid biopsy biomarkers for many diseases including cancer. Notably, the ability to demonstrate multiplexed sensing of disease biomarkers within devices made through a facile, rapid, and clean-room-free strategy demonstrates the immense potential of this fabrication approach to accelerate the adoption and advancement of biomedical microfluidic devices in practice and in resource-limited settings.

(Co, S)-Codoped BiOBr as a novel signal probe coupled with LAMP (H+)-pH responsive photoelectrochemistry biosensor for ultrasensitive detection of microRNA

Herein, Co/S-BiOBr as a novel photoelectric material with excellent photoelectric property was prepared by cation–anion co-doping method with LAMP (H+)-pH responsive as signal amplification to construct a photochemical (PEC) biosensor for ultrasensitive detection of microRNA-221 (miRNA-221) related to triple-negative breast cancer (TNBC). Notably, not only the lattice distortion caused by S doping could elevate the photo-generated electron-hole separation efficiency, but also the tune of electronic structure by Co doping promoted electron transfer and decreased recombination rate, thus significantly improving the photoelectric performance. Furthermore, in the aid of Loop-mediated isothermal amplification (LAMP), a few miRNA-221 could be converted to abundant LAMP(H+) for changing the configuration of the Fc-DNA probe, which made the ferrocene (Fc) approach to electrode via pH-responsive for hindering the transmission of electrons, resulting in a significantly reduced photocurrent signal to enhance the sensitivity of the biosensor. The proposed PEC biosensor performed a wide range from 1 fM to 10 nM with a detection limit of 0.24 fM. This work prepared a novel material with efficient photoelectric efficiency for sensitive detection of biomolecules and exhibited enormous potential in disease diagnosis.

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.

Quantitative Investigation of MicroRNA-32 in the Urine of Prostate Cancer Patients and Its Relationship With Clinicopathological Characteristics

IntroductionProstate cancer (PCa) is one of the most common cancers worldwide. PCa diagnosis is mostly based on solid biopsy and prostate-specific antigen (PSA), which have the disadvantages of being invasive and insensitive, respectively. Recently, the detection of microRNAs (miRNAs) in expressed prostatic secretions (EPS) has been a promising approach for PCa diagnosis. The aim of this study is to quantify transcriptional levels of miRNA-32 in the urine of prostate cancer patients. Materials and methodsIn this study, we evaluated the expression of miRNA-32 in the urine of 27 PCa patients, 48 benign prostatic hyperplasia (BPH) and 20 healthy controls, using quantitative real-time PCR (qPCR). The expression levels were then compared with the clinicopathological characteristics of patients. ResultsThe expression level of miRNA-32 in PCa patients was significantly higher than the control group (P < .01) and BPH cases (P < .01), and was associated with advanced tumor stage (P < .05). In addition, the expression of miRNA-32 had significant correlation with patients’ age (r = 0.39, P = .043). Area under ROC curve (AUC) for the discrimination of PCa samples from control and BPH samples were 0.93 (P < .0001) and 0.78 (P < .0001), respectively. We also used logistic regression analysis to integrate the results of PSA, prostate volume and miRNA-32, and presented a predictive model for distinguishing PCa from BPH, highlighting the clinical utility of miRNA-32 in cancer diagnosis and risk assessment. ConclusionsMeasurement of miRNA-32 expression in urine may have significance for the detection of PCa. Inclusion of miRNA-32 in logistic regression along with PSA and prostate volume increases the accuracy of cancer diagnosis.

Dual Detection of microRNAs by a Signal-Off Colorimetric Nanobiosensor Based on Novel Split DNAzyme Nanostructure.

Breast cancer is the most common cancer and the second cause of cancer-related death in women, especially in the age of 20-59years. It is very important to diagnose it in the early stages of development due to high chance of survival. In this research, the early detection of two microRNAs involved in breast cancer including miR-21 and miR-155 was performed simultaneously using a nanobiosensor based on a special G-quadruplex structure and a colorimetric manner. This nanobiosensor contains two probes (p1, p2) that play a role in the formation of a special structure called DNA-G4. This structure has peroxidase-like properties and can oxidize TMB and produce a blue color. The diagnostic method is designed as a signal off, where the hybridization of probes with microRNA sequences, no DNA-G4 structure is formed and no color change is observed. The results of this study showed that the linear range of response is in the range of 2 to 10nm and limit of detection in buffer, blood and urine samples was calculated as 0.43nM, 0.54nM, and 0.62nM (R2 = 0.98), respectively. Evaluation using the method for cancer monitoring can be a simple, fast and cost-effective technique.

Advancements in Early Detection and Screening Strategies for Pancreatic Cancer: From Genetic Susceptibility to Novel Biomarkers.

Pancreatic cancer is a rare but lethal cancer due to its biologically aggressive nature, advanced stage at the time of diagnosis, and poor response to oncologic therapies. The risk of pancreatic cancer is significantly higher to 5% in certain high-risk individuals with inherited genetic susceptibility. Screening for pancreatic cancer in these individuals from high-risk groups can help with the early detection of pancreatic cancer as well as the detection of precursor lesions leading to early surgical resection and improved overall outcomes. The advancements in radiological imaging as well as advanced endoscopic procedures has made a significant impact on the early diagnosis, surveillance, and staging of pancreatic cancer. There is also a significant advancement in the development of biomarkers for the early detection of pancreatic cancer, which has also led to the development of liquid biopsy, allowing for microRNA detection in serum and circulating tumor cells. Various societies and organizations have provided guidelines for pancreatic cancer screening and surveillance in high-risk individuals. In this review, we aim to discuss the hereditary risk factors for developing pancreatic cancer, summarize the screening recommendations by different societies, and discuss the development of novel biomarkers and areas for future research in pancreatic cancer screening for high-risk individuals.

(Invited) Single-Walled Carbon Nanotube Functionalization Strategies for Monitoring Enzymatic Activity and Inhibition

Semiconducting single-walled carbon nanotubes (SWCNTs) fluoresce in the near-infrared range, which overlaps with the transparency window of biological samples, and they do not photobleach or blink. Moreover, they benefit from biocompatibility and the large surface area available for functionalization. Using tailored surface functionalization, SWCNTs can be rendered optical nanosensors, such that surface binding events or changes in the local proximity of the nanotubes translate to a modulation of the emitted fluorescence. This approach was successfully used to demonstrate the detection of small molecules, volatiles, bacteria, microRNA, proteins, metals, self-assembly processes, and active processes in vivo. We will discuss different strategies for monitoring enzymatic activity and inhibition using SWCNT-based sensors, including the incorporation of the enzyme’s target bond within a synthetic molecular complex used for suspending the SWCNTs, or by the detection of the product of the enzyme hydrolytic activity. We will also highlight recent applications of these techniques in clinically relevant samples. These findings not only showcase the potential of near-infrared fluorescent SWCNT in tracking enzymatic activity but also underscore their promising role in advancing biomedical research and applications.

Dual-Function DNA Nanowires with Self-Feedback Amplification and Efficient Signal Transduction for Intracellular Imaging of MicroRNA-155.

Intracellular detection and imaging of microRNAs (miRNAs) with low expression usually face the problem of unsatisfactory sensitivity. Herein, a novel dual-function DNA nanowire (DDN) with self-feedback amplification and efficient signal transduction was developed for the sensitive detection and intracellular imaging of microRNA-155 (miRNA-155). Target miRNA-155 triggered catalytic hairpin assembly (CHA) to generate plenty of double-stranded DNA (dsDNA), and a trigger primer exposed in dsDNA initiated a hybridization chain reaction (HCR) between four well-designed hairpins to produce DDN, which was encoded with massive target sequences and DNAzyme. On the one hand, target sequences in DDN acted as self-feedback amplifiers to reactivate cascaded CHA and HCR, achieving exponential signal amplification. On the other hand, DNAzyme encoded in DDN acted as signal transducers, successively cleaving Cy5 and BHQ-2 labeled substrate S to obtain a significantly enhanced fluorescence signal. This efficient signal transduction coupling self-feedback amplification greatly improved the detection sensitivity with a limit of detection of 160 aM for miRNA-155, enabling ultrasensitive imaging of low-abundance miRNA-155 in living cells. The constructed DDN creates a promising fluorescence detection and intracellular imaging platform for low-expressed biomarkers, exhibiting tremendous potential in biomedical studies and clinical diagnosis of diseases.

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.