Advances in miRNA detection.

A direct and multiplex digital PCR chip for EGFR mutation

PurposeThis study aims to show the inconsistency of the approach to the development of artificial intelligence as an independent tool (just one more tool that humans have developed); to describe the logic and concept of intelligence development regardless of its substrate: a human or a machine and to prove that the co-evolutionary hybridization of the machine and human intelligence will make it possible to reach a solution for the problems inaccessible to humanity so far (global climate monitoring and control, pandemics, etc.).Design/methodology/approachThe global trend for artificial intelligence development (has been) was set during the Dartmouth seminar in 1956. The main goal was to define characteristics and research directions for artificial intelligence comparable to or even outperforming human intelligence. It should be able to acquire and create new knowledge in a highly uncertain dynamic environment (the real-world environment is an example) and apply that knowledge to solving practical problems. Nowadays artificial intelligence overperforms human abilities (playing games, speech recognition, search, art generation, extracting patterns from data etc.), but all these examples show that developers have come to a dead end. Narrow artificial intelligence has no connection to real human intelligence and even cannot be successfully used in many cases due to lack of transparency, explainability, computational ineffectiveness and many other limits. A strong artificial intelligence development model can be discussed unrelated to the substrate development of intelligence and its general properties that are inherent in this development. Only then it is to be clarified which part of cognitive functions can be transferred to an artificial medium. The process of development of intelligence (as mutual development (co-development) of human and artificial intelligence) should correspond to the property of increasing cognitive interoperability. The degree of cognitive interoperability is arranged in the same way as the method of measuring the strength of intelligence. It is stronger if knowledge can be transferred between different domains on a higher level of abstraction (Chollet, 2018).FindingsThe key factors behind the development of hybrid intelligence are interoperability – the ability to create a common ontology in the context of the problem being solved, plan and carry out joint activities; co-evolution – ensuring the growth of aggregate intellectual ability without the loss of subjectness by each of the substrates (human, machine). The rate of co-evolution depends on the rate of knowledge interchange and the manufacturability of this process.Research limitations/implicationsResistance to the idea of developing co-evolutionary hybrid intelligence can be expected from agents and developers who have bet on and invested in data-driven artificial intelligence and machine learning.Practical implicationsRevision of the approach to intellectualization through the development of hybrid intelligence methods will help bridge the gap between the developers of specific solutions and those who apply them. Co-evolution of machine intelligence and human intelligence will ensure seamless integration of smart new solutions into the global division of labor and social institutions.Originality/valueThe novelty of the research is connected with a new look at the principles of the development of machine and human intelligence in the co-evolution style. Also new is the statement that the development of intelligence should take place within the framework of integration of the following four domains: global challenges and tasks, concepts (general hybrid intelligence), technologies and products (specific applications that satisfy the needs of the market).

MicroRNA (miRNA) microarrays have been successfully used for profiling miRNA expression in many physiological processes such as development, differentiation, oncogenesis, and other disease processes. Detecting miRNA by miRNA microarray is actually based on nucleic acid hybridization between target molecules and their corresponding complementary probes. Due to the small size and high degree of similarity among miRNA sequences, the hybridization condition must be carefully optimized to get specific and reliable signals. Previously, we reported a microarray platform to detect miRNA expression. In this study, we evaluated the sensitivity and specificity of our microarray platform. After systematic analysis, we determined an optimized hybridization condition with high sensitivity and specificity for miRNA detection. Our results would be helpful for other hybridization-based miRNA detection methods, such as northern blot and nuclease protection assay.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that act as pivotal post-transcriptional regulators of gene expression, thus involving in many fundamental cellular processes such as cell proliferation, migration, and canceration. The detection of miRNAs has attracted significant interest, as abnormal miRNA expression is identified to contribute to serious human diseases such as cancers. Particularly, miRNAs in peripheral blood have recently been recognized as important biomarkers potential for liquid biopsy. Furthermore, as miRNAs are expressed heterogeneously in different cells, investigations into single-cell miRNA expression will be of great value for resolving miRNA-mediated regulatory circuits and the complexity and heterogeneity of miRNA-related diseases. Thus, the development of miRNA detection methods, especially for complex clinic samples and single cells is in great demand. In this Account, we will present recent progress in the design and application of isothermal amplification enabling miRNA detection transition from the test tube to the clinical sample and single cell, which will significantly advance our knowledge of miRNA functions and disease associations, as well as its translation in clinical diagnostics. miRNAs present a huge challenge in detection because of their extremely short length (∼22 nucleotides) and sequence homology (even with only single-nucleotide variation). The conventional golden method for nucleic acid detection, quantitative PCR (qPCR), is not amenable to directly detecting short RNAs and hardly enables distinguishing between miRNA family members with very similar sequences. Alternatively, isothermal amplification has emerged as a powerful method for quantification of nucleic acids and attracts broad interest for utilization in developing miRNA assays. Compared to PCR, isothermal amplification can be performed without precise control of temperature cycling and is well fit for detecting short RNA or DNA. We and other groups are seeking methods based on isothermal amplification for detecting miRNA with high specificity (single-nucleotide resolution) and sensitivity (detection limit reaching femtomolar or even attomolar level). These methods have recently been demonstrated to quantify miRNA in clinical samples (tissues, serum, and plasma). Remarkably, attributed to the mild reaction conditions, isothermal amplification can be performed inside cells, which has recently enabled miRNA detection in single cells. The localized in situ amplification even enables imaging of miRNA at the single-molecule level. The single-cell miRNA profiling data clearly shows that genetically identical cells exhibit significant cell-to-cell variation in miRNA expression. The leap of miRNA detection achievements will significantly contribute to its full clinical adoption and translation and give us new insights into miRNA cellular functions and disease associations.

Imaging and detecting microRNAs (miRNAs) is of central importance in tumor cell analysis. It stays challenging to establish simple, accurate, and sensitive analytical assays for imaging and detection of miRNA in a single living cell, because of intracellular complex environment and miRNA sequence similarity. Herein, we designed a dual-signal twinkling probe (DSTP) with triplex-stem structure which employed a fluorescence-SERS signal reciprocal switch. The spatiotemporal dynamics of the miRNA molecular and intracellular uptake of the probe are monitored by fluorescence-SERS signal switch of the DSTP. Meanwhile, using the surface-enhanced Raman scattering (SERS) signals of DSTP, the measure of absolute value coupling of reciprocal signals is first used to real-time detection of miRNA. Through simultaneous enhancing the target response signal value and reducing blank value, this work deducted the background effect, and showed high sensitivity and reproducibility. Moreover, the probe shows excellent reversibility and specificity in real quantitative detection of intracellular miRNA. miR-203 was successfully monitored in MCF-7, in accord with the results in vitro as well as in cell lysates. We anticipate that this new dual-signal twinkling and dual-spectrum switch method will be generally useful to image and detect various types of biomolecules in single living cell.

MicroRNAs (miRNAs) play vital roles in a plethora of biological and cellular processes. The levels of miRNAs can be useful biomarkers for cellular events or disease diagnosis, thus the method for sensitive and selective detection of miRNAs is imperative to miRNA discovery, study, and clinical diagnosis. Here we develop a novel method to quantify miRNA expression levels as low as attomolar sensitivity by target-assisted isothermal exponential amplification coupled with fluorescent DNA-scaffolded AgNCs and demonstrated its feasibility in the application of detecting miRNA in real samples. The method reveals superior sensitivity with a detection limit of miRNA of 2 aM synthetic spike-in target miRNA under pure conditions (approximately 15 copies of a miRNA molecule in a volume of 10 μL) and can detect at least a 10 aM spike-in target miRNA in cell lysates. The method also shows the high selectivity for discriminating differences between miRNA family members, thus providing a promising alternative to standard approaches for quantitative detection of miRNA. This simple and cost-effective strategy has a potential of becoming the major tool for simultaneous quantitative analysis of multiple miRNAs (biomarkers) in tissues or cells and supplies valuable information for biomedical research and clinical early diagnosis.

A simple and highly sensitive electrochemical biosensor for microRNA (miRNA) detection was successfully developed by integrating a target‐assisted isothermal exponential amplification reaction (EXPAR) with enzyme‐amplified electrochemical readout. The binding of target miRNA with the immobilized linear DNA template generated a part duplex and triggered primer extension reaction to form a double‐stranded DNA. Then one of the DNA strands was cleaved by nicking endonuclease and extended again. The short fragments with the same sequence as the target miRNA except for the replacement of uridines and ribonucleotides with thymines and deoxyribonucleotides could be displaced and released. Hybridization of these released DNA fragments with other amplification templates and their extension on the templates led to target exponential amplification. Integrating with enzyme‐amplified electrochemical readout, the electrochemical signal decreases with the increasing target microRNA concentration. The method could detect miRNA down to 98.9 fM with a linear range from 100 fM to 10 nM. The fabrication and binding processes were characterized with cyclic voltammetry and electrochemical impedance spectroscopy. The specificity of the method allowed single‐nucleotide difference between miRNA family members to be discriminated. The established biosensor displayed excellent analytical performance toward miRNA detection and might present a powerful and convenient tool for biomedical research and clinic diagnostic application.

Encoded hydrogel microparticles mounting DNA probes are powerful tools for high-performance microRNA (miRNA) detection in terms of sensitivity, specificity, and multiplex detection capability. However, several particle rinsing steps in the assay procedure present challenges for rapid and efficient detection. To overcome this limitation, we encapsulated dense magnetic nanoparticles to reduce the rinsing steps and duration via magnetic separation. A large number of magnetic nanoparticles were encapsulated into hydrogel microparticles based on a discontinuous dewetting technique combined with degassed micromolding lithography. In addition, we attached DNA probes targeting three types of miRNAs related to preeclampsia to magnetically encoded hydrogel microparticles by post-synthesis conjugation and achieved sensitivity comparable to that of conventional nonmagnetic encoded hydrogel microparticles. To demonstrate the multiplex capability of magnetically encoded hydrogel microparticles while maintaining the advantages of the simplified rinsing process when addressing multiple samples, we conducted a triplex detection of preeclampsia-related miRNAs. In conclusion, the introduction of magnetically encoded hydrogel microparticles not only allowed efficient miRNA detection but also provided comparable sensitivity and multiplexed detectability to conventional nonmagnetic encoded hydrogel microparticles.

MicroRNAs (miRNAs) are important biomarkers for liquid biopsy, with extensive applicability to diverse diseases. Among diverse miRNA sensing platforms, graphically encoded hydrogel-based miRNA detection technology is a highly promising diagnostic tool, in terms of sensitivity, specificity, and multiplexing capability. However, the conventional hydrogel-based miRNA detection process suffers from a long assay time (more than 3 h) and redundant assay steps, limiting the practical applicability to actual clinical fields. In this study, we develop a hydrogel-based in situ DNA extension assay for rapid, simple, and multiplexed miRNA detection. Unlike typical hydrogel-based assays, the target hybridization and biotinylation for fluorophore labeling are integrated into a single step via target miRNA-primed DNA extension in hydrogel microparticles. Therefore, multiple microRNA targets can be quantitatively detected within 45 min by two assay steps composed of (1) target capture/biotinylation and (2) fluorophore labeling via streptavidin-biotin interaction. We validate robust sensitivities (down to the low picomolar level) and specificities (single-nucleotide level) by conducting singleplex assays for breast cancer-related miRNA markers (miR-16, miR-92a, and let-7a). Furthermore, multiplexed detection of these miRNA markers is conducted to validate robust multiplexing capacity with negligible nonspecific signal expression. Finally, multiple types of miRNAs in the lysate of breast cancer cells (MCF-7) are successfully detected using the developed assay. We expect the developed hydrogel-based assay can contribute to biomedical and omic fields, enabling high-throughput profiling of multiple miRNAs.

Design considerations for point-of-need devices based on nucleic acid amplification for COVID-19 diagnostics and beyond.

There have been rising interests in ultra-sensitive biosensing technologies for early diagnosis and prognosis monitoring of infectious diseases, cancers, and neurodegenerative diseases. Digital signal readout strategy represented by digital ELISA or digital PCR, advanced biosensing field enormously, which enables detection of biomolecules under the detection limit of conventional biosensing methods. However, due to the need for compartmentalization and limited multiplex capability, it has been hurdled for utilization in applications requiring hierarchical resolution analysis such as sub-cellular molecules or molecular cargo of single cells or single extracellular vesicles (EVs). Rolling circle amplification (RCA), an isothermal DNA amplification method enabling localization of an amplified signal, can eliminate the need for compartmentalization and increase multiplex capability. It also has potential to expand applications of single molecule counting assay for understanding hierarchy of biological systems. In this review, recent advances in RCA-based single molecule counting assay are overviewed and their applications in single cells and single EVs quantitative analysis are discussed. Furthermore, the limitations and outlook of RCA-based single molecule counting assay are highlighted.

Comparison of Real-Time PCR and Droplet Digital PCR for the Determination of JAK2V617F Mutation in Ph’-Negative Myeloproliferative Neoplasms

Colorimetric detection of African swine fever (ASF)-associated microRNA based on rolling circle amplification and salt-induced gold nanoparticle aggregation

Next-generation diagnostics is expected to use the abundant data on living bodies and provide sufficiently useful healthcare information. A significant portion of the data are considered to be collected from microRNAs (miRNAs), which play crucial roles in various activities inside the body. Here, we demonstrate single-miRNA detection using metasurface fluorescence (FL) biosensors, which are optimized all-dielectric nanostructured surfaces featuring excellent FL detection capability. Ultimate high-sensitivity discrimination of one miRNA from zero miRNA is achieved at the subattomolar level by employing optimized reverse transcription (RT) of miRNAs, polymerase chain reaction (PCR) suppressing false reactions, and highly efficient and target-selective FL detection of the miRNA amplicons on the metasurface biosensors using appropriately designed oligo DNA probes. This degree of precision has never been obtained using any other technique, such as digital PCR, which is currently one of the most efficient techniques. Furthermore, we demonstrate the specific detection of a cancer-correlated miRNA that is deeply mixed with another miRNA. We also examine and discuss other methods that possibly work for miRNA detection at femtomolar or lower concentrations, such as chromatography and different amplification methods, including handy one-step RT-PCR.

Novel Chip-Based Digital Real-Time PCR Platform with Lab on an Array for the Sensitive Detection of BCR::ABL1 Transcripts in Chronic Myeloid Leukemia