Nucleic Acid Analysis Research Articles

Miniaturized electrophoresis: An integrated microfluidic cartridge with functionalized hydrogel-assisted LAMP for sample-to-answer analysis of nucleic acid.

Accurate detection of pathogenic nucleic acids is crucial for early diagnosis, effective treatment, and containment of infectious diseases. It facilitates the timely identification of pathogens, aids in monitoring disease outbreaks, and helps prevent the spread of infections within healthcare settings and communities. We developed a multi-layered, paper-based microfluidic and miniaturized electrophoresis system for rapid nucleic acid extraction, separation, amplification, and detection, designed for resource-limited settings. Constructed from acrylic, transparency film, pressure-sensitive adhesion, and Whatman paper using a CO2 laser, the setup simplifies traditional methods and eliminates the need for complex equipment. DNA extraction and purification are achieved using Zweifach-Fung bifurcation and Fahraeus effect principles, with detection via a hydrogel-assisted colorimetric isothermal reverse transcriptase-loop-mediated isothermal amplification technique. The system accurately identified the SARS-CoV-2 N-gene and β-actin human gene, validated by a compact electrophoresis setup. In clinical validation with 12 patient specimens, the system demonstrated a positive predictive agreement of 83.0% and a negative predictive agreement of 100%. The system achieves a limit of detection of 1 copy/μl and can potentially transform nucleic acid detection assays in healthcare settings. This study addresses key challenges in nucleic acid detection, such as ensuring sample quality and quantity, reducing reliance on sophisticated equipment, preventing contamination, simplifying procedures, and providing rapid and accurate diagnostics for emerging pathogens.

Highly Specific and Rapid Multiplex Identification of Candida Species Using Digital Microfluidics Integrated with a Semi-Nested Genoarray.

Candida species are the most common cause of fungal infections around the world, associated with superficial and even deep-seated infections. In clinical practice, there is great significance in identifying different Candida species because of their respective characteristics. However, current technologies have difficulty in onsite species identification due to long turnover time, high cost of reagents and instruments, or limited detection performance. We developed a semi-nested recombinase polymerase amplification (RPA) genoarray as well as an integrated system for highly specific identification of four Candida species with a simple design of primers, high detection sensitivity, fast turnover time, and good cost-effectiveness. The system constructed to perform the assay consists of a rapid sample processing module for nucleic acid release from fungal samples in 15 min and a digital microfluidic platform for precise and efficient detection reactions in 35 min. Therefore, our system could automatically identify specific Candida species, with a reagent consumption of only 2.5 μL of the RPA reaction mixture per target and no cross-reaction. Its detection sensitivity for four Candida species achieved 101-102 CFU/mL, which was 10-fold better than conventional RPA and even comparable to a common polymerase chain reaction. Evaluated by using cultured samples and 24 clinical samples, our system shows great applicability to onsite multiplex nucleic acid analysis.

Enzyme-Responsive DNA Condensates.

Membrane-less compartments and organelles are widely acknowledged for their role in regulating cellular processes, and there is an urgent need to harness their full potential as both structural and functional elements of synthetic cells. Despite rapid progress, synthetically recapitulating the nonequilibrium, spatially distributed responses of natural membrane-less organelles remains elusive. Here, we demonstrate that the activity of nucleic-acid cleaving enzymes can be localized within DNA-based membrane-less compartments by sequestering the respective DNA or RNA substrates. Reaction-diffusion processes lead to complex nonequilibrium patterns, dependent on enzyme concentration. By arresting similar dynamic patterns, we spatially organize different substrates in concentric subcompartments, which can be then selectively addressed by different enzymes, demonstrating spatial distribution of enzymatic activity. Besides expanding our ability to engineer advanced biomimetic functions in synthetic membrane-less organelles, our results may facilitate the deployment of DNA-based condensates as microbioreactors or platforms for the detection and quantitation of enzymes and nucleic acids.

Fully Integrated Microfluidic Digital Chip for Simple and Highly Quantitative Detection of Norovirus.

The prevalence of foodborne illnesses is a significant global concern, resulting in numerous illnesses, deaths, and substantial economic losses annually. Traditional detection methods for foodborne pathogens are often slow, limited, and impractical for field use, underscoring the need for rapid, sensitive, and portable assays. Microfluidic technology has emerged as a promising solution for sample preparation, reaction, and detection on a small scale. Our study introduces a novel microfluidic digital loop-mediated isothermal amplification (LAMP) assay platform, which employs digital microfluidic chips for absolute quantitative analysis of nucleic acids. This portable chip utilizes LAMP technology to achieve ultrasensitive detection of target nucleic acids within 30 min and reduces the detection limit to 1 fM without the need for complex instrumentation. By digitizing amplification signals directly from the target sample, our platform offers simplicity, affordability, portability, and quantitative molecular readouts. This innovation represents a crucial step toward the on-site detection of foodborne pathogens, thereby enhancing food safety and mitigating disease outbreaks.

LAMP-MS for Locus-Specific Visual Quantification of DNA 5mC and RNA m6A Using Ultra-Low Input.

Enhancing the effectiveness of utilizing circulating cell-free DNA (cfDNA) for disease screening remains a challenge, necessitating improved sensitivity, specificity, cost-efficiency, and patient adherence. We present here LAMP-MS, an innovative technology that integrates linear amplification with single-base quantitative nucleic acid mass spectrometry on silicon chips. This approach overcomes several limitations in utilizing cfDNA 5-methylcytosine (5mC) status for colorectal cancer (CRC) screening. LAMP-MS enables unbiased amplification of as little as 1 ng of cfDNA, site-specifically quantify methylation levels of tens to hundreds of 5mC sites, thereby facilitating cost-effective, high-resolution quantitative detection of cfDNA methylation markers. We have validated the accuracy of DNA methylation determination using DNA probes and cfDNA from patient plasma samples, confirmed by mass spectrometric peak areas. Additionally, we have further shown this Mass Array technology could be expanded to also quantify RNA m6A modification sites. Combining the ability to work with ultra-low input materials and a visually interpretable output, LAMP-MS stands out as a promising method for real-world applications in clinics and laboratories for nucleic acid methylation detection and quantification.

LAMP‐MS for Locus‐Specific Visual Quantification of DNA 5mC and RNA m6A Using Ultra‐Low Input

Enhancing the effectiveness of utilizing circulating cell‐free DNA (cfDNA) for disease screening remains a challenge, necessitating improved sensitivity, specificity, cost‐efficiency, and patient adherence. We present here LAMP‐MS, an innovative technology that integrates linear amplification with single‐base quantitative nucleic acid mass spectrometry on silicon chips. This approach overcomes several limitations in utilizing cfDNA 5‐methylcytosine (5mC) status for colorectal cancer (CRC) screening. LAMP‐MS enables unbiased amplification of as little as 1 ng of cfDNA, site‐specifically quantify methylation levels of tens to hundreds of 5mC sites, thereby facilitating cost‐effective, high‐resolution quantitative detection of cfDNA methylation markers. We have validated the accuracy of DNA methylation determination using DNA probes and cfDNA from patient plasma samples, confirmed by mass spectrometric peak areas. Additionally, we have further shown this Mass Array technology could be expanded to also quantify RNA m6A modification sites. Combining the ability to work with ultra‐low input materials and a visually interpretable output, LAMP‐MS stands out as a promising method for real‐world applications in clinics and laboratories for nucleic acid methylation detection and quantification.

Cryo-X-Ray Phase Contrast Imaging Enables Combined 3D Structural Quantification and Nucleic Acid Analysis of Myocardial Biopsies.

Snap-frozen biopsies serve as a valuable clinical resource of archival material for disease research, as they enable a comprehensive array of downstream analyses to be performed, including extraction and sequencing of nucleic acids. Obtaining three-dimensional (3D) structural information before multi-omics is more challenging but can potentially allow for better characterization of tissues and targeting of clinically relevant cells. Conventional histological techniques are limited in this regard due to their destructive nature and the reconstruction artifacts produced by sectioning, dehydration, and chemical processing. These limitations are particularly notable in soft tissues such as the heart. In this study, the feasibility of using synchrotron-based cryo-X-ray phase contrast imaging (cryo-X-PCI) of snap-frozen myocardial biopsies is assessed and 3D structure tensor analysis of aggregated myocytes, followed by nucleic acid (DNA and RNA) extraction and analysis. It is shown that optimal sample preparation is the key driver for successful structural and nucleic acid preservation which is unaffected by the process of cryo-X-PCI. It is proposed that cryo-X-PCI has clinical value for 3D tissue analysis of cardiac and potentially non-cardiac soft tissue biopsies before nucleic acid investigation.

Hydrogel interfaced glass nanopore for high-resolution sizing of short DNA fragments

Solid-state nanopores, known for their label-free detection and operational simplicity, face challenges in accurately sizing the short nucleic acids due to fast translocation and a lack of enzyme-based control mechanisms as compared to their biological counterparts with sizing resolutions still limited to ≥100 bp. Here, we present a facile polyethylene glycol-dimethacrylate (PEG-DMA) hydrogel interfaced glass nanopore (HIGN) system by inserting glass nanopore into the hydrogel to achieve sub-100 base pair (bp) resolution in short DNA sizing analysis. We systematically investigated the effects of hydrogel mesh size, spatial configurations of glass nanopores about the hydrogel, applied bias voltage, and analyte concentration on the transport dynamics of 200 bp double-stranded DNA (dsDNA). A 7.5 w/v% PEG-DMA hydrogel induced ∼11x increase in the mean dwell times compared with bare solution nanopore system. The insertion locations and depths of the glass nanopore into the hydrogel resulted in 7.16% and 5.28% coefficients of variation (CV) for mean normalized event frequencies. This enhancement of dwell times and invariability in translocation characteristics enables precise sizing of dsDNA fragments under 400 bp using HIGN, with an achieved size resolution of 50 bp with observable mean normalized peak amplitude (ΔI/Io) of ∼0.005. Furthermore, we have demonstrated the capability of HIGN to perform multiplex detection of influenza A virus (IAV) and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) through reverse transcriptase-polymerase chain reaction (RT-PCR). These results demonstrated the potential of HIGN as a versatile tool in nucleic acid analysis and multiplexed label-free molecular diagnostics.

Revisiting ELISA with in situ amplification of biomarkers to boost its sensitivity

The enzyme-linked immunosorbent assay (ELISA) stands as a pivotal instrument in diagnostics and biomedical research, widely used for the detection and quantification of specific proteins associated with diseases. Despite its critical role, ELISA's ability to identify low-concentration proteins remains a significant challenge, hindering its effectiveness in early diagnostics, an area of growing importance. This study introduces a novel approach termed nucleic acid-templated amplification (NATA) to enhance ELISA's sensitivity without significantly modifying the common practices of the present ELISA. Drawing inspiration from viral proliferation mechanisms, our approach integrates a cell-free protein synthesis step using DNA sequences that encode the target biomarkers. This approach enables an in situ 'amplification' of target proteins during ELISA, boosting their concentrations to detectable levels. Our results demonstrate a substantial enhancement in sensitivity, with ELISA now being able to detect protein biomarkers at femtomolar concentrations—approaching the sensitivity range typically reserved for PCR in nucleic acid analysis. This breakthrough not only broadens ELISA’s applicability in early disease diagnosis and monitoring therapeutic interventions but also marks a significant advancement in protein biomarker detection technology. With its modular design, our method also offers high versatility and sensitivity, offering an advanced approach for protein detection in clinical diagnostics and research.

A deployable film method to enable replicable sampling of low-abundance environmental microbiomes

Urbanizing global populations spend over 90% of their time indoors where microbiome abundance and diversity are low. Chronic exposure to microbiomes with low abundance and diversity have demonstrated negative long-term impacts on human health. Sequencing-based analyses of environmental nucleic acids are critical to understanding the impact of the indoor microbiome on human health, however low DNA yields indoors, alongside sample collection and processing inconsistencies, currently challenge study replicability. This study presents a comparative assessment of a novel, passive, easily replicable sampling strategy using polydimethylsiloxane (PDMS) sheets alongside a representative swab-based collection protocol. Deployable, customizable PDMS films designed for whole-sample insertion into standardized extraction kits demonstrated 43% higher DNA yields per sample, and 76% higher yields per cm2 of sampler over swab-based protocols. These results indicate that this accessible, scalable method enables sufficient DNA collection to comprehensively evaluate indoor microbiome exposures and potential human health impacts using smaller, more space efficient samplers, representing an attractive alternative to swab-based collection. In addition, this process reduces the manual steps required for microbiome sampling which could address inter-study variability, transform the current microbiome sampling paradigm, and ultimately benefit the replicability and accessibility of microbiome exposure studies.

Co-freezing localized CRISPR-Cas12a system enables rapid and sensitive nucleic acid analysis

Rapid and sensitive nucleic acid detection is vital in disease diagnosis and therapeutic assessment. Herein, we propose a co-freezing localized CRISPR-Cas12a (CL-Cas12a) strategy for sensitive nucleic acid detection. The CL-Cas12a was obtained through a 15-minute co-freezing process, allowing the Cas12a/crRNA complex and hairpin reporter confined on the AuNPs surface with high load efficiency, for rapid sensing of nucleic acid with superior performance to other localized Cas12a strategies. This CL-Cas12a based platform could quantitatively detect targets down to 98 aM in 30 min with excellent specificity. Furthermore, the CL-Cas12a successful applied to detect human papillomavirus infection and human lung cancer-associated single-nucleotide mutations. We also achieved powerful signal amplification for imaging Survivin mRNA in living cells. These findings highlight the potential of CL-Cas12a as an effective tool for nucleic acid diagnostics and disease monitoring.Graphical abstract

Nucleic Acid-Based Biological Nanopore Sensing Strategies for Tumor Marker Detection.

Cancer, which is characterized by high mortality rates, poses a significant threat to global human health. Early diagnosis is of paramount importance in managing cancer, and tumor markers have emerged as crucial indicators for achieving this goal. The advent of precision medicine has further emphasized the need for the effective detection of these markers. However, traditional detection methods are hampered by numerous limitations. In recent years, nanopore technology has emerged as a promising alternative, due to its unique physical and chemical properties, which facilitate rapid, label-free, and amplification-free detection. This Review focuses on the direct detection of tumor markers through nucleic acid analysis and indirect detection mediated by nucleic acids and facilitated by biological nanopores. Furthermore, it also discusses the challenges and prospects of applying biological nanopore sensing technology in early cancer diagnosis, underscoring its potential to revolutionize tumor marker detection.

Microfluidic Encapsulation of DNAs in Liquid Beads for Digital Loop‐Mediated Isothermal Amplification

Digital nucleic acid analysis has emerged as a prominent tool for the detection and absolute quantification of diverse pathogens. Digital loop‐mediated isothermal amplification (dLAMP) offers highly sensitive, specific, time‐efficient, and cost‐effective nucleic acid amplification. However, existing dLAMP techniques face challenges such as droplet merging, reliance on surfactants, restricted partition capacities, and the potential for sample loss during heating. Herein, these issues are addressed by introducing liquid beads for sample partitioning. Compared to microwells, our approach overcomes the limitations of chamber dimensions, enabling the analysis of an unlimited number of digitized targets. Furthermore, our novel approach effectively addresses sample loss and merging during thermal processing and eliminates the need for surfactants. Accurate and reproducible the quantitative detection of the gene cluster XALB1 of leaf scald disease is conducted using dLAMP based on liquid beads to verify its availability. The results demonstrate a high correlation between target concentration and positive signals, indicating the robust performance of our technique. A comparative analysis is then performed between dLAMP using liquid beads and using single droplets. Benchmarking these two techniques highlights the effectiveness of our innovative technique in overcoming existing challenges in dLAMP.

Rapid and unbiased enrichment of extracellular vesicles via a meticulously engineered peptide

Extracellular vesicles (EVs) have garnered significant attention in biomedical applications. However, the rapid, efficient, and unbiased separation of EVs from complex biological fluids remains a challenge due to their heterogeneity and low abundance in biofluids. Herein, we report a novel approach to reconfigure and modify an artificial insertion peptide for the unbiased and rapid isolation of EVs in 20 min with ∼80% recovery in neutral conditions. Moreover, the approach demonstrates exceptional anti-interference capability and achieves a high purity of EVs comparable to standard ultracentrifugation and other methods. Importantly, the isolated EVs could be directly applied for downstream protein and nucleic acid analyses, including proteomics analysis, exome sequencing analysis, as well as the detection of both epidermal growth factor receptor (EGFR) and V-Ki-ras2 Kirsten Rat Sarcoma Viral Oncogene Homologue (KRAS) gene mutation in clinical plasma samples. Our approach offers great possibilities for utilizing EVs in liquid biopsy, as well as in various other biomedical applications.

Functional gold nanoparticles for analysis and delivery of nucleic acids.

Gold nanoparticles (AuNPs) have become the rising stars in the field of nanotechnology and made a revolution in exploiting the profundity of genomics due to their distinguished properties such as stability, ease in preparation and conjugation, biocompatibility, and unique optical properties. These characteristics have greatly expanded their applications such as sensitive and selective quantitation of nucleic acids and as effective carriers for specifically delivering various important molecules/biomolecules to various targets, which are the cornerstone in treating genetic disorders. This review comprehensively discusses the most recent progress in utilization of AuNPs in quantitation and delivery of nucleic acids. The future prospects and challenges of various methods have also been illustrated. It is believed that researchers will continue to overcome the limitations in previous approaches and AuNPs will still play vital roles in the development of diagnosis and treatment of gene-related diseases.

Bioinformatics Goes Viral: I. Databases, Phylogenetics and Phylodynamics Tools for Boosting Virus Research.

Computer-aided analysis of proteins or nucleic acids seems like a matter of course nowadays; however, the history of Bioinformatics and Computational Biology is quite recent. The advent of high-throughput sequencing has led to the production of "big data", which has also affected the field of virology. The collaboration between the communities of bioinformaticians and virologists already started a few decades ago and it was strongly enhanced by the recent SARS-CoV-2 pandemics. In this article, which is the first in a series on how bioinformatics can enhance virus research, we show that highly useful information is retrievable from selected general and dedicated databases. Indeed, an enormous amount of information-both in terms of nucleotide/protein sequences and their annotation-is deposited in the general databases of international organisations participating in the International Nucleotide Sequence Database Collaboration (INSDC). However, more and more virus-specific databases have been established and are progressively enriched with the contents and features reported in this article. Since viruses are intracellular obligate parasites, a special focus is given to host-pathogen protein-protein interaction databases. Finally, we illustrate several phylogenetic and phylodynamic tools, combining information on algorithms and features with practical information on how to use them and case studies that validate their usefulness. Databases and tools for functional inference will be covered in the next article of this series: Bioinformatics goes viral: II. Sequence-based and structure-based functional analyses for boosting virus research.

Simplified Electrochemical Approach for End-Point Yet Quantitative Detection of Nucleic Acids in Resource-Limited Settings.

Nucleic acid detection plays a crucial role in various aspects of health care, necessitating accessible and reliable quantification methods, especially in resource-limited settings. This work presents a simplified electrochemical approach for end-point yet quantitative nucleic acid detection. By elevating the concentration of redox species and choosing potential as the signals, we achieved enhanced signal robustness, even in the presence of interfering substances. Leveraging this robustness, we accurately measured pH-induced redox potential changes in methylene blue solution for end-point nucleic acid detection after loop-mediated isothermal amplification (LAMP). Our method demonstrated quantitative detection of the SARS-CoV-2 N gene and human ATCB gene and successful discrimination of the human BRAF V600E mutation, comparable in sensitivity to commercial kits. The developed user-friendly electrochemical method offers a simplified and reliable approach for end-point yet quantitative detection of nucleic acids, potentially expanding the benefits of nucleic acid testing in resource-limited settings.

Polysaccharide as a Separation Medium for Gel Electrophoresis

Gel electrophoresis and size exclusion chromatography (SEC) are vital techniques in biochemical research, employing gel matrix structures made of polysaccharides or synthetic polymers like polyacrylamide for the analysis and separation of macromolecules. Polysaccharides, such as agarose, offer safer alternatives to acrylamide. Polysaccharide gels, notably agarose, facilitate the analysis and purification of proteins and nucleic acids through a molecular sieving mechanism. Gel electrophoresis for proteins is mainly divided into denaturing and native methods. Denaturing electrophoresis with sodium dodecyl sulfate (SDS) simplifies protein migration but disrupts molecular interactions. Conversely, native gel electrophoresis, without SDS, allows proteins to migrate based on the running pH and the isoelectric point of the proteins, while nucleic acids consistently migrate toward the anode. The electrophoresis of proteins with variable charges presents complexes. This review focuses on the use of polysaccharides, particularly agarose, for native gel electrophoresis, highlighting their applications in separating macromolecules. It also discusses the applications and limitations of agarose gels when used as a matrix for electrophoresis. Such information should help in designing electrophoresis experiments using polysaccharides.

The detection of Salmonella in food based on PCR combined with Pyrococcus furiosus Argonaute

Salmonella is a common zoonotic foodborne bacterial pathogen. In poultry farms, Salmonella can cause pullorum disease, which characterizes decreased appetite and diarrhea in chicks, accompanied by a high mortality rate in the first week of life itself. In pig farms, Salmonella mainly infects piglets and causes porcine paratyphoid, characterizing septicemia and necrotic enteritis and sometimes leading to severe symptoms such as encephalitis. This bacterium poses significant challenges to global public health and food safety. PfAgo (Pyrococcus furiosus Argonaute) is an Argonaute enzyme from the Pyrococcus furiosus, which is utilized in highly sensitive and accurate nucleic acid detection and analysis methods based on the polymerase chain reaction (PCR) combined with PfAgo. In this study, targeting the hilA gene, and can detect as low as 3 × 100 copies and 2.7 × 10°CFU (cells) with no cross-reactivity with other pathogens, it enhances the sensitivity of PCR. The PCR-PfAgo method was successfully applied to detect in clinical food samples including chicken and milk, and the results were consistent with those of traditional bacterial culture. The findings suggest that the PCR-PfAgo method has the potential to replace traditional detection methods and has broad application prospects in the detection of Salmonella pathogen.

Increased levels of circulating cell-free double-stranded nucleic acids in the plasma of glioblastoma patients.

Circulating cell-free nucleic acids are considered a promising source of biomarkers for diseases and cancer. Liquid biopsy biomarkers for brain tumours represent a major, still unmet, clinical need. In plasma, nucleic acids can be free or be associated with extracellular vesicles (EVs). Here we report an easy and reproducible method to analyse cell-free nucleic acids in plasma and EVs by conventional flow cytometry easy to translate into the clinics. Nucleic acids associated with the EVs or present in plasma samples are stained by Pyronin Y, which is a fluorescent dye that is preferably binding double-stranded nucleic acids. Fluorescent staining of EVs isolated from cell-conditioned media is suitable for DNA and RNA detection by flow cytometry. The nucleic acids are partially protected from degradation by the EVs' membrane. Additionally, DNA and RNA can be stained in plasma samples and plasma-derived EVs. Remarkably, analysis of plasma from patients and healthy individuals reveals a difference in their nucleic acid profiles. Taken together, our results indicate that the proposed methodology, which is based on conventional direct flow cytometry, is a promising easy tool for plasma nucleic acid analysis.