Nucleic Acid Analysis Research Articles

Nucleic Acid Plate Culture: Label-Free and Naked-Eye-Based Digital Loop-Mediated Isothermal Amplification in Hydrogel with Machine Learning.

Digital nucleic acid amplification enables the absolute quantification of single molecules. However, due to the ultrasmall reaction volume in the digital system (i.e., short light path), most digital systems are limited to fluorescence signals, while label-free and naked-eye readout remain challenging. In this work, we report a digital nucleic acid plate culture method for label-free, ultrasimple, and naked-eye nucleic acid analysis. As simple as the bacteria culture, the nanoconfined digital loop-mediated isothermal amplification was performed by using polyacrylamide (PAM) hydrogel as the amplification matrix. The nanoconfinement of PAM hydrogel with an ionic polymer chain can remarkably accelerate the amplification of target nucleic acids and the growth of inorganic byproducts, namely, magnesium pyrophosphate particles (MPPs). Compared to that in aqueous solutions, MPPs trapped in the hydrogel with enhanced light scattering characteristics are clearly visible to the naked eye, forming white "colony" spots that can be simply counted in a label-free and instrument-free manner. The MPPs can also be photographed by a smartphone and automatically counted by a machine-learning algorithm to realize the absolute quantification of antibiotic-resistant pathogens in diverse real samples.

Coupling Exponential to Linear Amplification for Endpoint Quantitative Analysis.

Exponential DNA amplification techniques are fundamental in ultrasensitive molecular diagnostics. These systems offer a wide dynamic range, but the quantification requires real-time monitoring of the amplification reaction. Linear amplification schemes, despite their limited sensitivity, can achieve quantitative measurement from a single end-point readout, suitable for low-cost, point-of-care, or massive testing. Reconciling the sensitivity of exponential amplification with the simplicity of end-point readout would thus break through a major design dilemma and open a route to a new generation of massively scalable quantitative bioassays. Here a hybrid nucleic acid-based circuit design is introduced to compute a logarithmic function, therefore providing a wide dynamic range based on a single end-point measurement. CELIA (Coupling Exponential amplification reaction to LInear Amplification) exploits a versatile biochemical circuit architecture to couple a tunable linear amplification stage - optionally embedding an inverter function - downstream of an exponential module in a one-pot format. Applied to the detection of microRNAs, CELIA provides a limit of detection in the femtomolar range and a dynamic range of six decades. This isothermal approach bypasses thermocyclers without compromising sensitivity, thereby opening the way to applications in various diagnostic assays, and providing a simplified, cost-efficient, and high throughput solution for quantitative nucleic acid analysis.

Excimer Fluorescence of Acriflavine Dye in Glycerol and Ethylene Glycol.

This research investigates the excimerisation of acriflavine dye in ethylene glycol and glycerol solvents. Acriflavine, a member of the acridine dye family, exhibits unique fluorescence properties with applications in various fields, including cellular nucleus observation, nucleic acid analysis, and dye laser active media etc. The study explores the impact of solvent and concentration on acriflavine's emission properties, with a focus on excimer formation, which can influence its suitability as a dye laser active medium. UV-Visible absorption spectroscopy reveals concentration-dependent absorption profiles, with distinctive monomer bands. Steady-state fluorescence studies demonstrate the emergence of red-shifted excimer fluorescence bands as concentrations increase in both solvents. Temperature-dependent fluorescence studies reveal the dynamics of excimer formation, suggesting dynamic diffusion as the excimerisation mechanism. Time-resolved fluorescence spectroscopy confirms the singlet character of both monomer and excimer states, providing insights into the excimerisation process. Critical concentration values are determined, representing the equilibrium between monomeric and excimeric forms. The study also explores pH-induced spectral shifts, highlighting the influence of acidity on fluorescence properties. Overall, this research deepens our understanding of acriflavine's excimerisation in ethylene glycol and glycerol, offering insights that can enhance its diverse applications, especially in laser technologies.

A triple-amplified electrochemical-photoelectrochemical dual-mode biosensing platform for viral gene assay based on catalytic hairpin assembly, CRISPR-Cas12a and liposome-entrapped bifunctional methylene blue

Taking SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene as the model due to the great destructiveness of COVID-19 in the past years, a triple-amplified electrochemical-photoelectrochemical (EC-PEC) dual-mode biosensing platform was constructed for viral gene assay based on catalytic hairpin assembly (CHA), CRISPR-Cas12a and liposome-entrapped bifunctional methylene blue. Firstly, the SARS-CoV-2 RdRp gene triggered CHA nonenzymatic amplification process to produce large amounts of double-stranded DNA (dsDNA) that could activate CRISPR-Cas12a trans-cleavage ability. Subsequently, the activated CRISPR-Cas12a trans-cleaved nearby ssDNA carrying methylene blue (MB)-encapsulated liposome sphere (MELS) and streptavidin-modified magnetic bead (SMB), and lots of MELS were released. After magnetic separation, MELS was lysed by Triton X-100, releasing a large quantity of bifunctional MB molecules that not only generate an obvious EC signal originated from the electro-oxidation of MB, but also effectively induce the photocurrent-polarity-switching of the Sb2S3 NRs-modified photoelectrode from cathodic photocurrent to anodic photocurrent and generate a large anodic photocurrent. Based on the combination of the above triple amplification processes with MB-induced photocurrent-polarity-switching and EC-PEC dual-mode sensing strategies, the SARS CoV-2 RdRp gene was sensitively, selectively and accurately assayed with a linear response range of 0.1 fM – 10nM (1 aM – 10nM) and a detection limit of 69.6 aM (0.11 aM) for EC (PEC) method. The proposed EC-PEC dual-mode biosensor is also easily extended to the analysis of other viral nucleic acid by changing the related sequences of H1 and H2 DNAs, providing a new way in the early diagnosis and epidemic control of diseases caused by viruses.

Sensitivity, Specificity, Positive Rate and Consistency of Two Kits in SARS-CoV-2 Nucleic Acid Detection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was a highly transmissible and pathogenic coronavirus and it emerged in late 2019. SARS-CoV-2 had caused a pandemic of acute respiratory disease and its name was coronavirus disease 2019 (COVID-19). It threatened human health and public safety. This study would analyze and evaluate two different kinds of SARS-CoV-2 nucleic acid detection reagents which could provide value for accurate detection. 80 patients were randomly selected in the First Affiliated Hospital of Anhui Medical University in December 2022 and 80 oropharyngeal swabs were collected. Nucleic acid was extracted first, and then two real-time fluorescent quantitative RT-PCR nucleic acid amplification reagents were used to detect ORF1ab and N genes. Statistical software was used to compare and analyze the results. Among 80 patients, 57 were males and 23 were females with an age range of 5 - 90 years with an average age of 65.7 years. Most of the specimens were collected from Department of Infectious Diseases and Department of Respiratory and Critical care. Compared with regent Reference, the sensitivity of regent A and B was 88.3% and 93.3% and the specificity was 90.0% and 95.0%, respectively. The positive rates of the double target genes were 85.0% and 93.3%, the positive rates of ORF1ab gene were 86.7% and 95.0% and the positive rates of N gene were 88.3% and 96.7%, respectively. The Kappa values were all greater than 0.75, indicating high consistency. Between two nucleic acid detection reagents, reagent B had higher sensitivity and specificity. And the positive rate and consistency of reagent B were also higher than that of reagent A, with statistical significance. For weakly positive specimens with low viral load, it was recommended to use another reagent with higher sensitivity to retest and ensure the accuracy and repeatability of the results.

Photosensitive Nanoprobes for Rapid High Purity Isolation and Size‐Specific Enrichment of Synthetic and Extracellular Vesicle Subpopulations

AbstractThe biggest challenge in current isolation methods for lipid bilayer‐encapsulated vesicles, such as exosomes, secretory, and synthetic vesicles, lies in the absence of a unified approach that seamlessly delivers high purity, yield, and scalability for large‐scale applications. To address this gap, an innovative method is developed that utilizes photosensitive lipid nanoprobes for the efficient isolation of vesicles and sorting them into subpopulations based on size. The photosensitive component in the probe undergoes cleavage upon exposure to light, facilitating the release of vesicles in their near‐native form. The method demonstrates a superior ability in isolating high purity extracellular vesicles from complex biological media and separating them into size‐based subpopulations within 1 h, achieving more efficiency and purity than ultracentrifugation. Furthermore, this method's cost‐effectiveness and rapid enrichment of the vesicles align with demands for large‐scale isolation and downstream analyses of nucleic acids and proteins. The method opens new avenues in exploring, analyzing, and utilizing synthetic and extracellular vesicle subpopulations in various biomedical applications, including diagnostics, therapeutic delivery, and biomarker discovery.

Experimental investigation of confinement effect in single molecule amplification via real-time digital PCR on a multivolume droplet array SlipChip

BackgroundDigital polymerase chain reaction (digital PCR) is an important quantitative nucleic acid analysis method in both life science research and clinical diagnostics. One important hypothesis is that by physically constraining a single nucleic acid molecule in a small volume, the relative concentration can be increased therefore further improving the analysis performance, and this is commonly defined as the confinement effect in digital PCR. However, experimental investigation of this confinement effect can be challenging since it requires a microfluidic device that can generate partitions of different volumes and an instrument that can monitor the kinetics of amplification. (96). ResultsHere, we developed a real-time digital PCR system with a multivolume droplet array SlipChip (Muda-SlipChip) that can generate droplet of 125 nL, 25 nL, 5 nL, and 1 nL by a simple “load-slip” operation. In the digital region, by reducing the volume, the relative concentration is increased, the amplification kinetic can be accelerated, and the time to reach the fluorescence threshold, or Cq value, can be reduced. When the copy number per well is much higher than one, the relative concentration is independent of the partition volume, thus the amplification kinetics are similar in different volume partitions. This system is not limited to studying the kinetics of digital nucleic acid amplification, it can also extend the dynamic range of the digital nucleic acid analysis by additional three orders of magnitude by combining a digital and an analog quantification algorithm. (140). SignificanceIn this study, we experimentally investigated for the first time the confinement effect in the community of digital PCR via a new real-time digital PCR system with a multivolume droplet array SlipChip (Muda-SlipChip). And a wider dynamic range of quantification methods compared to conventional digital PCR was validated by this system. This system provides emerging opportunities for life science research and clinical diagnostics. (63).

Abstract 3706: Single cell and cell-free nucleic acid analyses of liquid biopsy specimens from a blood collection tube using a new droplet digital PCR system

Abstract Introduction: Liquid biopsy-based testing has increasingly been adopted by oncologists for biomarker-driven diagnostics, therapy selection, and patient monitoring. Unlike tissue-based approaches, the target analytes in blood specimens are often rare events that must be discriminated from a background of non-tumor derived material. Droplet digital PCR (ddPCR) is an ideal platform for detection of rare nucleic acid markers in the clinic, because it is highly sensitive and specific, robust, and quantitative. Additionally, the workflow is simple to adopt and execute. The QX600 System is a new ddPCR instrument designed for advanced multiplexing which enables simultaneous detection of up to 6 unique fluorophores in a single well. This capability can decrease the sub-sampling needed for detection of multiple analytes. The objective of our study was to develop multiplexed ddPCR assays for detection of cell-based and cell-free circulating biomarkers from a single tube of whole blood. Methods: Tumor cells were enriched from the buffy coat fraction of whole blood collected in fixative-containing cfDNA BCT based on size and morphology using a microfluidics-based system. Enriched tumor cells were enumerated by immunofluorescence (DAPI+/Pan-CK+/CD45-), and gene expression was measured using the QX600 for a panel of cytokeratins (CK) and PD-L1 in CTC negative, NSCLC donor specimens (n=8). Results were compared to CTC positive, contrived specimens (whole blood spiked with breast tumor cell line MCF7 cells; n=5). For ctDNA analysis of the plasma fraction, a multiplexed ddPCR assay was designed to test for five actionable mutations prevalent in NSCLC, including EGFR del19, L858R, T790M, KRAS G12C, BRAF V600E, and wildtype EGFR. To evaluate performance of the multiplexed assay, concordance evaluation was conducted using cfDNA from 8 mutation-positive NSCLC donors on the QX600 and compared to single assays run on the QX200 system. Results: CK8 and CK19 were differentially expressed in the cells enriched from the CTC negative NSCLC cohort as compared to the contrived positives (p=0.0058, and p=0.0049, respectively). All the NSCLC cfDNA samples were positive for the reference variant, and linear regression analysis of the minor variant frequency for ctDNA biomarkers showed good concordance between QX systems (R2=0.8847). Conclusions: These studies demonstrate proof of concept for single cell and cell-free analysis from a single blood draw into a cfDNA BCT using the QX600 system. Liquid biopsy analysis of CTCs and ctDNA can enable minimally invasive, comprehensive decision making in the diagnostic workup of patients. Future studies will focus on further optimizing the cell enrichment methodology from cfDNA BCT and development of additional multiplexed ddPCR assays for NSCLC-relevant biomarkers on the QX600. Citation Format: Leisa Jackson, Colin Cochran, Claire Gould, Sarah Kreston, Jazmin Bello, Brittany D’Alessio, Janice Riley, Alisa Tubbs, Gary A. Pestano. Single cell and cell-free nucleic acid analyses of liquid biopsy specimens from a blood collection tube using a new droplet digital PCR system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3706.

Assumption-free analysis for amplification-based quantitative nucleic acid detection.

The accurate detection and quantification of biological species that are rarely present but potentially devastating is of paramount importance for the life sciences, biosecurity, food safety, and environmental monitoring. Consequently, there has been significant interest in the sensitive and accurate detection of nucleic acids, leveraging both chemical and biological methods. Among these, quantitative polymerase chain reaction (qPCR) is regarded as the gold standard due to its sensitivity and precision in identifying specific nucleic acid targets. Despite the widespread adoption of qPCR for nucleic acid detection, the analysis of qPCR data typically depends on the use of calibrated standard curves and a threshold method to interpret signal measurements. In this study, we use a stochastic simulation to show the limitations of the threshold method due to its assumptions on amplification kinetics. We propose a new approach for the absolute quantification of nucleic acids that overcomes these limitations by reconstructing the efficiency profile across amplification cycles and using cumulative amplification folds to build a standard curve, thus avoiding the constant efficiency assumption. Our method, validated through experiments with nucleic acid amplification in the presence of potent inhibitors, demonstrates improved accuracy in quantifying nucleic acids, avoiding the systematic errors of the threshold method. This innovation enhances the reliability of nucleic acid quantification, especially where traditional methods struggle with kinetic variability.

Field-effect transistor biosensor with signal amplification by ternary initiation complexes for detection of wide-range RNA concentration

Electrical detection of RNAs using transistor-based biosensors has attracted attention as a strategy for medical diagnosis and environmental monitoring. Herein, we demonstrated a proof-of-concept for specific, sensitive, and label-free RNA detection using a field-effect transistor (FET) biosensor with signal amplification by ternary initiation complexes (SATIC), which is an isothermal one-step nucleic acid amplification initiated by the combination of target RNA, circular DNA template and DNA primer. The SATIC system-applied FET biosensor specifically and quantitatively detected the target RNA with a single-nucleotide difference via the negative charges derived from the amplification products formed by a nucleic acid amplification reaction with φ29 DNA polymerase on the gate surface. In particular, the control of the amplification time allowed the detection of target RNA molecules over a wide concentration range, resulting in a detection limit of up to 6 copies/μL. Therefore, a transistor-based bioassay using the SATIC system could be useful for simple and sensitive nucleic acid analysis.

Novel Anti-CRISPR-Assisted CRISPR Biosensor for Exclusive Detection of Single-Stranded DNA (ssDNA).

Nucleic acid analysis plays an important role in disease diagnosis and treatment. The discovery of CRISPR technology has provided novel and versatile approaches to the detection of nucleic acids. However, the most widely used CRISPR-Cas12a detection platforms lack the capability to distinguish single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA). To overcome this limitation, we first employed an anti-CRISPR protein (AcrVA1) to develop a novel CRISPR biosensor to detect ssDNA exclusively. In this sensing strategy, AcrVA1 cut CRISPR guide RNA (crRNA) to inhibit the cleavage activity of the CRISPR-Cas12a system. Only ssDNA has the ability to recruit the cleaved crRNA fragment to recover the detection ability of the CRISPR-Cas12 biosensor, but dsDNA cannot accomplish this. By measuring the recovered cleavage activity of the CRISPR-Cas12a biosensor, our developed AcrVA1-assisted CRISPR biosensor is capable of distinguishing ssDNA from dsDNA, providing a simple and reliable method for the detection of ssDNA. Furthermore, we demonstrated our developed AcrVA1-assisted CRISPR biosensor to monitor the enzymatic activity of helicase and screen its inhibitors.

Characterisation and engineering of a thermophilic RNA ligase from Palaeococcus pacificus.

RNA ligases are important enzymes in molecular biology and are highly useful for the manipulation and analysis of nucleic acids, including adapter ligation in next-generation sequencing of microRNAs. Thermophilic RNA ligases belonging to the RNA ligase 3 family are gaining attention for their use in molecular biology, for example a thermophilic RNA ligase from Methanobacterium thermoautotrophicum is commercially available for the adenylation of nucleic acids. Here we extensively characterise a newly identified RNA ligase from the thermophilic archaeon Palaeococcus pacificus (PpaRnl). PpaRnl exhibited significant substrate adenylation activity but low ligation activity across a range of oligonucleotide substrates. Mutation of Lys92 in motif I to alanine, resulted in an enzyme that lacked adenylation activity, but demonstrated improved ligation activity with pre-adenylated substrates (ATP-independent ligation). Subsequent structural characterisation revealed that in this mutant enzyme Lys238 was found in two alternate positions for coordination of the phosphate tail of ATP. In contrast mutation of Lys238 in motif V to glycine via structure-guided engineering enhanced ATP-dependent ligation activity via an arginine residue compensating for the absence of Lys238. Ligation activity for both mutations was higher than the wild-type, with activity observed across a range of oligonucleotide substrates with varying sequence and secondary structure.

Synthesis and Enzymatic Incorporation of a Dual-App Nucleotide Probe That Reports Antibiotics-Induced Conformational Change in the Bacterial Ribosomal Decoding Site RNA.

Natural nucleosides are nonfluorescent and do not have intrinsic labels that can be readily utilized for analyzing nucleic acid structure and recognition. In this regard, researchers typically use the so-called "one-label, one-technique" approach to study nucleic acids. However, we envisioned that a responsive dual-app nucleoside system that harnesses the power of two complementing biophysical techniques namely, fluorescence and 19F NMR, will allow the investigation of nucleic acid conformations more comprehensively than before. We recently introduced a nucleoside analogue by tagging trifluoromethyl-benzofuran at the C5 position of 2'-deoxyuridine, which serves as an excellent fluorescent and 19F NMR probe to study G-quadruplex and i-motif structures. Taking forward, here, we report the development of a ribonucleotide version of the dual-app probe to monitor antibiotics-induced conformational changes in RNA. The ribonucleotide analog is derived by conjugating trifluoromethyl-benzofuran at the C5 position of uridine (TFBF-UTP). The analog is efficiently incorporated by T7 RNA polymerase to produce functionalized RNA transcripts. Detailed photophysical and 19F NMR of the nucleoside and nucleotide incorporated into RNA oligonucleotides revealed that the analog is structurally minimally invasive and can be used for probing RNA conformations by fluorescence and 19F NMR techniques. Using the probe, we monitored and estimated aminoglycoside antibiotics binding to the bacterial ribosomal decoding site RNA (A-site, a very important RNA target). While 2-aminopurine, a famous fluorescent nucleic acid probe, fails to detect structurally similar aminoglycoside antibiotics binding to the A-site, our probe reports the binding of different aminoglycosides to the A-site. Taken together, our results demonstrate that TFBF-UTP is a very useful addition to the nucleic acid analysis toolbox and could be used to devise discovery platforms to identify new RNA binders of therapeutic potential.

A High-Throughput Workflow for Mass Spectrometry Analysis of Nucleic Acids by Nanoflow Desalting

A High-Throughput Workflow for Mass Spectrometry Analysis of Nucleic Acids by Nanoflow Desalting

Stability of BK polyomavirus DNA in urine over time and analytical evaluation of an automated BKV quantitative nucleic acid test.

Introduction. BK polyomavirus (BKPyV) quantitative testing is an important screening tool post-transplantation, although interpretation can be challenging due to lack of standardization, assay heterogeneity and variability of BKPyV DNA over time (in urine).Methods. Remnant clinical EDTA plasma and urine samples were tested by the cobas BKV test and a validated laboratory-developed test (LDT). Accuracy [positive and negative percent agreement (PPA and NPA), Pearson's correlation, Bland-Altman analysis] and reproducibility were evaluated. To assess BKPyV DNA stability in urine, prospective urine samples were maintained at two different storage temperatures and tested in triplicate over 7 days.Results. Overall PPA was 95.6 % (43/45) and NPA was 94.4 % (170/180). For plasma, Pearson's correlation (0.950) and Bland-Altman analysis (0.113±0.22 log10 IU ml-1) showed high agreement. For neat urine, Pearson's correlation (0.842) and Bland-Altman analysis (0.326±0.80 log10 IU ml-1) showed somewhat higher variability. Reproducibility was high for the cobas BKV versus the LDT. BKPyV DNA levels in neat urine remained relatively stable over 7 days at both storage temperatures, although outlier results were intermittently detected.Conclusion. The cobas BKV test showed high agreement and reproducibility compared to the reference LDT. BKPyV viral load testing in urine has known limitations, but neat urine can be processed by the cobas BKV.

Rapid and sensitive detection of nucleic acids using an RAA-CRISPR/Cas12b one-pot detection assay (Rcod)

Rapid, sensitive and specific methods are crucial for nucleic acid detection. CRISPR/Cas12b has recently been widely used in nucleic acid detection. However, due to its thermophagic property, DNA isothermal recombinase-aided amplification (RAA) and subsequent CRISPR/Cas12b detection require two separate reactions, which is cumbersome and inconvenient and may cause aerosol pollution. In this study, we propose an RAA-CRISPR/Cas12b one-pot detection assay (Rcod) for Bordetella pertussis detection without additional amplification product transfer steps. The time from sample processing to response time was less than 30 min using nucleic acid extraction-free method, and the sensitivity reached 0.2 copies/μL. In this system, Alicyclobacillus acidoterrestris Cas12b protein (AacCas12b) exhibited strong and specific trans-cleavage activity at a constant temperature of 37 °C, while the cis-cleavage activity was weak. This characteristic reduces the interference of AacCas12b with nucleic acids in the system. Compared with real-time PCR, our Rcod system detected B. pertussis in 221 clinical samples with a sensitivity and specificity of 97.96 % and 99.19 %, respectively, with nucleic acid extraction-free method. The rapid, sensitive and specific Rcod system provides ideas for the establishment of CRISPR-based one-step nucleic acid detection and may aid the development of reliable point-of-care nucleic acid tests. ImportancePertussis is an acute respiratory infection caused by B. pertussis that is highly contagious and potentially fatal, and early diagnosis is essential for the treatment of whooping cough. In this study, we found that AacCas12b has high and strongly specific trans-cleavage activity at lower temperatures. A RAA-CRISPR/Cas12b one-step detection platform (Rcod) without interference with amplification was developed. In addition, the combination of Rcod and nucleic acid extraction-free method can quickly and accurately detect the qualitative detection of B. pertussis, and the detection results are visualized, which makes the pathogen nucleic acid detection and analysis process simpler, and provides a new method for the rapid clinical diagnosis of B. pertussis.

Combination-Lock SlipChip Integrating Nucleic Acid Sample Preparation and Isothermal LAMP Amplification for the Detection of SARS-CoV-2.

Nucleic acid analysis with an easy-to-use workflow, high specificity and sensitivity, independence of sophisticated instruments, and accessibility outside of the laboratory is highly desirable for the detection and monitoring of infectious diseases. Integration of laboratory-quality sample preparation on a hand-held system is critical for performance. A SlipChip device inspired by the combination lock can perform magnetic bead-based nucleic acid extraction with several clockwise and counterclockwise rotations. A palm-sized base station was developed to assist sample preparation and provide thermal control of isothermal nucleic acid amplification without plug-in power. The loop-mediated isothermal amplification reaction can be performed with a colorimetric method and directly analyzed by the naked eye or with a mobile phone app. This system achieves good bead recovery during the sample preparation workflow and has minimal residue carryover from the lysis and elution buffers. Its performance is comparable to that of the standard laboratory protocol with real-time qPCR amplification methods. The entire workflow is completed in less than 35 min and the device can achieve 500 copies/mL sensitivity. Thirty clinical nasal swab samples were collected and tested with a sensitivity of 95% and a specificity of 100% for SARS-CoV-2. This combination-lock SlipChip provides a promising fast, easy-to-use nucleic acid test with bead-based sample preparation that produces laboratory-quality results for point-of-care settings, especially in home use applications.

Context-dependent accuracy of the cobas plasma separation card for HCV RNA viral load measurement.

Collection and preservation of plasma are challenging in remote or under-resourced settings. The cobas® Plasma Separation Card (PSC) is an alternative specimen type for blood-borne pathogen nucleic acid quantitation. We assessed PSC as a specimen type for HCV RNA quantitation in Pakistan. Plasma from venous blood and PSC from finger prick blood were prepared at two sites: Site 1 (in Lahore, n = 199) consisted of laboratory-based outpatient clinics. Specimens were prepared in the same facility and stored frozen. Site 2 was a catchment area within a resource-limited, semi-urban locality of Islamabad with limited access to healthcare services (n = 151). Community public health outreach staff collected blood and prepared the PSC in the participants' homes. Specimens were transported to the central hepatitis laboratory in Lahore to be stored frozen until tested. HCV RNA testing was performed using the cobas HCV RNA test in a central laboratory. Concordance with respect to RNA detectability was high at Site 1 (97.4%), but lower at Site 2 (82.4%). At Site 1, HCV viral load in plasma and PSC were well correlated across the linear range with a 0.21 log10 IU/mL mean bias toward higher concentrations in PSC. At Site 2, HCV viral load in plasma and PSC were poorly correlated. There was a 0.11 log10 IU/mL mean bias toward higher concentrations in PSC. PSC performance can be excellent in underserved settings where refrigerated transport of traditional specimens is difficult. In very challenging field settings, extra support must be provided to ensure correct specimen collection and handling.

Direct Determination of the Structure of Single Biopolymer Molecules Using Nanopore Sequencing.

This review highlights operational principles, features, and modern aspects of the development of third-generation sequencing technology of biopolymers focusing on the nucleic acids analysis, namely the nanopore sequencing system. Basics of the method and technical solutions used for its realization are considered, from the first works showing the possibility of creation of these systems to the easy-to-handle procedure developed by Oxford Nanopore Technologies company. Moreover, this review focuses on applications, which were developed and realized using equipment developed by the Oxford Nanopore Technologies, including assembly of whole genomes, methagenomics, direct analysis of the presence of modified bases.

Deciphering biomolecular complexities: the indispensable role of surface-enhanced Raman spectroscopy in modern bioanalytical research.

Surface-enhanced Raman spectroscopy (SERS) has emerged as an indispensable analytical tool in biomolecular research, providing unmatched sensitivity critical for the elucidation of biomolecular structures. This review presents a thorough examination of SERS, outlining its fundamental principles, cataloging its varied applications within the biomolecular sphere, and contemplating its future developmental trajectories. We begin with a detailed analysis of SERS's mechanistic principles, emphasizing both the phenomena of surface enhancement and the complexities inherent in Raman scattering spectroscopy. Subsequently, we delve into the pivotal role of SERS in the structural analysis of diverse biomolecules, including proteins, nucleic acids, lipids, carbohydrates, and biochromes. The remarkable capabilities of SERS extend beyond mere detection, offering profound insights into biomolecular configurations and interactions, thereby enriching our comprehension of intricate biological processes. This review also sheds light on the application of SERS in real-time monitoring of various bio-relevant compounds, from enzymes and coenzymes to metal ion-chelate complexes and cellular organelles, thereby providing a holistic view and empowering researchers to unravel the complexities of biological systems. We also address the current challenges faced by SERS, such as enhancing sensitivity and resolution, developing stable and reproducible substrates, and conducting thorough analyses in complex biological matrices. Nonetheless, the continual advancements in nanotechnology and spectroscopy solidify the standing of SERS as a formidable force in biomolecular research. In conclusion, the versatility and robustness of SERS not only deepen our understanding of biomolecular intricacies but also pave the way for significant developments in medical research, therapeutic innovation, and diagnostic approaches.