Integrated Droplet-Based Digital Loop-Mediated Isothermal Amplification Microfluidic Chip with Droplet Generation, Incubation, and Continuous Fluorescence Detection.

This study integrated sample partition, incubation, and continuous fluorescence detection on a single microfluidic chip for droplet-based digital Loop-Mediated Isothermal Amplification (LAMP) of nucleic acids. This integration eliminated the need to transfer reactions between different platforms, avoiding sample contamination and loss. Prior to the reaction, filling the channels with an oil phase and adding a glass cover slip on top of the chip overcame the problem of bubble generation in the channels during the LAMP reaction due to heating.

Introducing Triplex Forming Oligonucleotide into Loop-Mediated Isothermal Amplification for Developing a Lateral Flow Biosensor for Streptococci Detection.

Loop-mediated isothermal amplification (LAMP) technology is extensively utilized for the detection of infectious diseases owing to its rapid processing and high sensitivity. Nevertheless, conventional LAMP signaling methods frequently suffer from a lack of sequence specificity. This study integrates a triplex-forming oligonucleotide (TFO) probe into the LAMP process to enhance sequence specificity.

Development and optimization of a DNA aptamer to delay β-bungarotoxin-induced lethality in a rodent model.

Current treatment of snakebite relies on immunoglobulin-rich antivenoms. However, production of these antivenoms is complicated and costly. Aptamers - single-stranded DNAs or RNAs with specific folding structures that bind to specific target molecules - represent excellent alternatives or complements to antibody-based therapeutics.

Methods for Collection of Extracellular Vesicles and Their Content RNA as Liquid Biopsy for Lung Cancer Detection: Application of Differential Centrifugation and Annexin A5 Coated Beads.

Extracellular vesicles (EVs) contain abundant extracellular RNA (exRNA), which can be a valuable source of liquid biopsy. However, as various RNA species exist in different types of EVs, lack of detailed characterization of these RNA species and efficient collection methods limits the clinical application of exRNA. In the present study, we measured two mRNAs, CK19 and PCTK1; one lncRNA, MALAT1; and two miRNAs, miR21 and miR155, in different EV fractions separated by differential centrifugation or captured by magnetic beads coated with annexin A5 (ANX beads).

Ultrafast DNA Amplification Using Microchannel Flow-Through PCR Device.

Polymerase chain reaction (PCR) is limited by the long reaction time for point-of-care. Currently, commercial benchtop rapid PCR requires 30−40 min, and this time is limited by the absence of rapid and stable heating and cooling platforms rather than the biochemical reaction kinetics. This study develops an ultrafast PCR (<3 min) platform using flow-through microchannel chips.

Targeted Sequencing of Circulating Cell Free DNA Can Be Used to Monitor Therapeutic Efficacy of Tyrosine Kinase Inhibitors in Non-small Cell Lung Cancer Patients.

Background/aim: Circulating tumor DNA (ctDNA) bears specific mutations derived from tumor cells. The amount of mutant ctDNA may reflect tumor burden. In this study, we detected epidermal growth factor receptor (EGFR) mutations in ctDNA as a monitoring marker for the response of non-small cell lung cancer (NSCLC) patients to tyrosine kinase inhibitors (TKIs).

Transcriptomic Analysis in Liquid Biopsy Identifies Circulating PCTAIRE-1 mRNA as a Biomarker in NSCLC.

Background/aim: Circulating mRNA can be a useful source of cancer biomarkers. We took advantage of direct transcriptomic analysis in plasma RNA to identify novel mRNA markers for non-small cell lung cancer (NSCLC).

Patients And Methods: Plasma RNA from NSCLC patients and healthy individuals was profiled with cDNA-mediated annealing, selection, extension and ligation (DASL) microarrays.

A primer and probe set for detecting multiple types of EGFR exon 19 deletions.

EGFR exon 19 deletion is an important indicator for tyrosine kinase inhibitor treatment in non-small cell lung cancer. However, detection of exon 19 deletions faces a challenge: there are more than 30 types of mutations reported at the hotspot. Moreover, considering the application in body fluid samples, assays with high sensitivity and specificity are necessary for the detection of rare mutant alleles.

A Sensitive Peptide Nucleic Acid Probe Assay for Detection of BRAF V600 Mutations in Melanoma.

Mutated v-Raf murine sarcoma viral oncogene homolog B (BRAF) is an important biomarker for the prediction of therapeutic efficacy of several anticancer drugs. The detection of BRAF mutation faces two challenges: Firstly, there are multiple types of mutations, and secondly, tumor samples usually contain various amounts of wild-type, normal tissues. Here, we describe a newly established method for sensitive detection of multiple types of BRAF V600 mutations in excess wild-type background.

GeneGazer: A Toolkit Integrating Two Pipelines for Personalized Profiling and Biosignature Identification.

Background: Next-generation sequencing provides useful information about gene mutations, gene expression, epigenetic modification, microRNA expression, and copy number variations. More and more computing tools have been developed to analyze this large quantity of information. However, to test and find suitable analytical tools and integrate their results is tedious and challenging for users with little bioinformatics training.

Programming a nonvolatile memory-like sensor for KRAS gene sensing and signal enhancement.

A programmable field effect-based electrolyte-insulator-semiconductor (EIS) sensor constructed with a nonvolatile memory-like structure is proposed for KRAS gene DNA hybridization detection. This programmable EIS structure was fabricated with silicon oxide (SiO2)/silicon nitride (Si3N4)/silicon oxide on a p-type silicon wafer, namely electrolyte-oxide-nitride-oxide-Si (EONOS). In this research, voltage stress programming from 4 to 20V was applied to trigger holes confinement in the nitride-trapping layer that, consequently, enhances the DNA attachment onto the sensing surface due to additional electrostatic interaction.

Spin-coated Au-nanohole arrays engineered by nanosphere lithography for a Staphylococcus aureus 16S rRNA electrochemical sensor.

The nanopatterning of gold nanoparticle (AuNP) arrays on an indium tin oxide (ITO) electrode using efficient and low-cost methods is described. This process used nanosphere lithography (NSL) encompassing the deposition of monolayered Polystyrene (PS) followed by a convective self-assembly drop coating protocol onto the ITO substrate that further acted as the mask after the AuNP assembly. The results showed that spin-coating allowed AuNPs to follow the contour and adhere to the PS nanospheres.

Circulating Messenger RNA Profiling with Microarray and Next-generation Sequencing: Cross-platform Comparison.

Background: Circulating mRNA is a less invasive and more easily accessed source of samples for biomedical research and clinical applications. However, it is of poor quality. We explored and compared the ability of two high-throughput platforms for the profiling of circulating mRNA regarding their ability to retrieve useful information out of this type of samples.

Development of a magnetic bead-based method for the collection of circulating extracellular vesicles.

Cells release different types of extracellular vesicles (EVs). These EVs contain biomolecules, including proteins and nucleic acids, from their parent cells, which can be useful for diagnostic applications. The aim of this study was to develop a convenient procedure to collect circulating EVs with detectable mRNA or other biomolecules.

Integrating solid-state sensor and microfluidic devices for glucose, urea and creatinine detection based on enzyme-carrying alginate microbeads.

A solid-state sensor embedded microfluidic chip is demonstrated for the detection of glucose, urea and creatinine in human serum. In the presented device, magnetic powder-containing enzyme-carrying alginate microbeads are immobilized on the surface of an electrolyte-insulator-semiconductor (EIS) sensor by means of a step-like obstacle in the microchannel and an external magnetic force. The sample is injected into the microchannel and reacts with the enzyme contained within the alginate beads; prompting the release of hydrogen ions.

Sensitive detection of unlabeled oligonucleotides using a paired surface plasma waves biosensor.

Detection of unlabeled oligonucleotides using surface plasmon resonance (SPR) is difficult because of the oligonucleotides' relatively lower molecular weight compared with proteins. In this paper, we describe a method for detecting unlabeled oligonucleotides at low concentration using a paired surface plasma waves biosensor (PSPWB). The biosensor uses a sensor chip with an immobilized probe to detect a target oligonucleotide via sequence-specific hybridization.

Monitoring triplex DNA formation with fluorescence resonance energy transfer between a fluorophore-labeled probe and intercalating dyes.

Triplex-forming oligonucleotides (TFOs) are sequence-dependent DNA binders that may be useful for DNA targeting and detection. A sensitive and convenient method to monitor triplex formation by a TFO and its target DNA duplex is required for the application of TFO probes. Here we describe a novel design by which triplex formation can be monitored homogeneously without prelabeling the target duplex.

Single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe for the detection of rare mutations.

The detection of rare mutant DNA from a background of wild-type alleles usually requires laborious manipulations, such as restriction enzyme digestion and gel electrophoresis. Here, we describe a protocol for homogeneous detection of rare mutant DNA in a single tube. The protocol uses a peptide nucleic acid (PNA) as both PCR clamp and sensor probe.

Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics.

Reactive oxygen species (ROS) produced either endogenously or exogenously can attack lipid, protein and nucleic acid simultaneously in the living cells. In nuclear and mitochondrial DNA, 8-hydroxydeoxyguanosine (8-OHdG), an oxidized nucleoside of DNA, is the most frequently detected and studied DNA lesion. Upon DNA repair, 8-OHdG is excreted in the urine.