Improvement of limit of detection in primer extension-based multiplexed mutation assay using capillary electrophoresis.

One of the challenges in liquid biopsy for early cancer detection is ascribed to the fact that mutation DNA often represents an extremely small ratio of less than 1% compared to wild-type genes in blood. However, in conventional fragment analysis with capillary electrophoresis (CE), the detectable allele frequency could be about 5%. In this work, we developed an original reagent-based fragment analysis with single base extension (SBE) reactions for cancer-associated mutation assay using a commercially available CE device, and investigated on a possibility of improvement of limit of detection (LOD) for genetic mutation. First, after adjustment of reagent conditions for the SBE reactions, the linear relationship between gene template concentration and fluorescence intensity was obtained from 1 to 100 fmol of target genes. Next, from the results of an experiment to detect mutation EGFR L858R at abundance ratios of mutant type to wild type (100-fmol template) of 0, 1, 5, and 10%, it was shown that the target gene can be detected with LOD of 0.33%. This high sensitivity was realized in part by separating fluorescently labeled substrates into an individual tube for an each-colored SBE reaction. Moreover, mutations EGFR L858R and KRAS G12V were simultaneously detected at sensitivities equivalent to LODs of 0.57 and 0.47%, respectively. These results indicate that < 1% of mutations in multiplex gene mutations can be simultaneously detected, and that possibility suggests that the developed method can be used in clinical practice for detecting cancers.

Effects of serum matrix on molecular interactions between drugs and target proteins revealed by giant magneto-resistive bio-sensing techniques.

We demonstrated that effects of serum matrix on molecular interactions between drugs and target proteins can be investigated in real time using magnetic bio-sensing techniques. A giant magneto-resistive (GMR) sensor was used on which target proteins were fixed and superparamagnetic nanoparticles (diameter: 50 nm) conjugated with drug were used in phosphate buffer, with and without serum. In this study, the following drug-protein pairs were investigated: quercetin and cAMP-dependent protein kinase A (PKA), Infliximab and tumor necrosis factor alpha (TNFα), and Bevacizumab and vascular endothelial growth factor (VEGF). For the quercetin and PKA pair, the time profile of the signal from the GMR sensor due to binding between quercetin and PKA clearly changed before and after the addition of serum. Moreover, it was revealed that not only the association process, but also the dissociation process was influenced by the addition of serum, suggesting that the quercetin and PKA complex may partially contain serum proteins, which affect the formation and stability of the complex. For antibody drugs, little effects of serum matrix were observed on both the association and dissociation processes. These clear differences may be attributed to the hydrophobic and electrostatic character of the drug molecule, target protein, and serum proteins. The real-time monitoring of molecular interactions in a biological matrix enabled by the GMR bio-sensing technique is a powerful tool to investigate such complicated molecular interactions. Understanding the molecular interactions that occur in a biological matrix is indispensable for determining the mechanism of action of the drugs and pharmacokinetics/pharmacodynamics inside the body. Additionally, this method can be applied for the analysis of the influence of any kind of third molecule that may have some interaction between two molecules, for example, an inhibitor drug against the interaction between two kinds of proteins.

Size-Selective Harvesting of Extracellular Vesicles for Strategic Analyses Towards Tumor Diagnoses.

Extracellular vesicles (EV), typified by exosomes or microvesicles, are expected to be effective diagnostic markers for cancers. The sizes of the vesicles range from 20 to 1000 nm, but the size-dependent variations of the contents of EVs are still poorly understood. We succeeded in the size-selective harvesting of the vesicles by utilizing the molecular weight-dependent characteristics of a variety of polyethylene glycols (PEG) as precipitating reagents and analyzed the antigens displayed on the surfaces of the vesicles and the miRNAs included in the vesicles from each size group. As a result, the relatively larger (<100 nm) particles precipitated by PEG5k clearly exhibited the greatest amount of epithelial cell adhesion molecule (EpCAM), from both breast cancer (MCF-7) and colon cancer (HCT116) cells, and a larger quantity of microRNA (miRNA) specific to breast cancer cells (miRNA155 for MCF-7) seemed to be contained in the PEG-precipitated particles. The results demonstrated that the quantities of both the tumor-specific miRNA and protein were similarly distributed among the several classes of the size-sorted EVs and that the size-selective harvesting of EVs may be informative for strategic analyses towards the diagnoses of cancers.