Solid-Phase Microextraction Enables Isolation of BRAF V600E Circulating Tumor DNA from Human Plasma for Detection with a Molecular Beacon Loop-Mediated Isothermal Amplification Assay.

Circulating tumor DNA (ctDNA) is a promising biomarker that can provide a wealth of information regarding the genetic makeup of cancer as well as provide a guide for monitoring treatment. Methods for rapid and accurate profiling of ctDNA are highly desirable in order to obtain the necessary information from this biomarker. However, isolation of ctDNA and its subsequent analysis remains a challenge due to the dependence on expensive and specialized equipment. In order to enable widespread implementation of ctDNA analysis, there is a need for low-cost and highly accurate methods that can be performed by nonexpert users. In this study, an assay is developed that exploits the high specificity of molecular beacon (MB) probes with the speed and simplicity of loop-mediated isothermal amplification (LAMP) for the detection of the BRAF V600E single-nucleotide polymorphism (SNP). Furthermore, solid-phase microextraction (SPME) is applied for the successful isolation of clinically relevant concentrations (73.26 fM) of ctDNA from human plasma. In addition, the individual effects of plasma salts and protein on the extraction of ctDNA with SPME are explored. The performed work expands the use of MB-LAMP for SNP detection as well as demonstrates SPME as a sample preparation tool for nucleic acid analysis in plasma.

Defining Cell Cluster Size by Dielectrophoretic Capture at an Array of Wireless Electrodes of Several Distinct Lengths.

Clusters of biological cells play an important role in normal and disease states, such as in the release of insulin from pancreatic islets and in the enhanced spread of cancer by clusters of circulating tumor cells. We report a method to pattern cells into clusters having sizes correlated to the dimensions of each electrode in an array of wireless bipolar electrodes (BPEs). The cells are captured by dielectrophoresis (DEP), which confers selectivity, and patterns cells without the need for physical barriers or adhesive interactions that can alter cell function. Our findings demonstrate that this approach readily achieves fine control of cell cluster size over a broader range set by other experimental parameters. These parameters include the magnitude of the voltage applied externally to drive capture at the BPE array, the rate of fluid flow, and the time allowed for DEP-based cell capture. Therefore, the reported method is anticipated to allow the influence of cluster size on cell function to be more fully investigated.

Drug Partitioning in Micellar Media and Its Implications in Rational Drug Design: Insights with Streptomycin.

Oral bioavailability of a drug molecule requires its effective delivery to the target site. In general, majority of synthetically developed molecular entities have high hydrophobic nature as well as low bioavailability, therefore the need for suitable delivery vehicles arises. Self-assembled structures such as micelles, niosomes, and liposomes have been used as effective delivery vehicles and studied extensively. However, the information available in literature is mostly qualitative in nature. We have quantitatively investigated the partitioning of antibiotic drug streptomycin into cationic, nonionic, and a mixture of cationic and nonionic surfactant micelles and its interaction with the transport protein serum albumin upon subsequent delivery. A combination of calorimetry and spectroscopy has been used to obtain the thermodynamic signatures associated with partitioning and interaction with the protein and the resulting conformational changes in the latter. The results have been correlated with other class of drugs of different nature to understand the role of molecular features in the partitioning process. These studies are oriented toward understanding the physical chemistry of partitioning of a variety of drug molecules into suitable delivery vehicles and hence establishing structure-property-energetics relationships. Such studies provide general guidelines toward a broader goal of rational drug design.