On-line electroextraction in capillary electrophoresis: Application on the determination of glutamic acid in soy sauces.

We present an on-line, single step coupling between liquid-liquid extraction and capillary electrophoresis with capacitively coupled contactless conductivity detection, which allows an efficient analysis of complex food matrices with high sodium content. The sodium depletion was demonstrated using an aqueous two-phase system. The aqueous two-phase system enables the electrically driven extraction of the target compounds. The sample was prepared in Dextran-rich phase (8% w/v 500 kDa Dextran, DEX). The background electrolyte (acetic acid 5.0 mol/L) contained 6% w/v of 6 kDa PEG. As proof of applicability, we employed the developed method for glutamic acid quantification on soy sauces. The peak area of glutamic acid presents no significant difference (α = 0.05), while the peak area of the sodium presented a reduction of 11.7 ± 0.2 and 19 ± 3% for premium and low-cost soy sauce samples analyzed. The glutamic acid concentration for premium soy sauce sample was 2.7 ± 0.8 and 4.8 ± 0.4 g/L, and for low-cost soy sauce sample, the concentration was 9.9 ± 0.9 g/L, which agreed with those obtained by other analytical techniques.

Analytical Technologies for Liquid Biopsy of Subcellular Materials.

Liquid biopsy markers, which can be secured from a simple blood draw or other biological samples, are used to manage a variety of diseases and even monitor for bacterial or viral infections. Although there are several different types of liquid biopsy markers, the subcellular ones, including cell-free DNA, microRNA, extracellular vesicles, and viral particles, are evolving in terms of their utility. A challenge with liquid biopsy markers is that they must be enriched from the biological sample prior to analysis because they are a vast minority in a mixed population, and potential interferences may be present in the sample matrix that can inhibit profiling the molecular cargo from the subcellular marker. In this article, we discuss existing and developing analytical enrichment platforms used to isolate subcellular liquid biopsy markers, and discuss their figures of merit such as recovery, throughput, and purity.

Molecular Profiling of Liquid Biopsy Samples for Precision Medicine.

In the context of oncology, liquid biopsies consist of harvesting cancer biomarkers, such as circulating tumor cells, tumor-derived cell-free DNA, and extracellular vesicles, from bodily fluids. These biomarkers provide a source of clinically actionable molecular information that can enable precision medicine. Herein, we review technologies for the molecular profiling of liquid biopsy markers with special emphasis on the analysis of low abundant markers from mixed populations.

Microfluidic-based solid phase extraction of cell free DNA.

Cell-free DNA (cfDNA) is a liquid biopsy marker that can carry signatures (i.e., mutations) associated with certain pathological conditions. Therefore, the extraction of cfDNA from a variety of clinical samples can be an effective and minimally invasive source of markers for disease detection and subsequent management. In the oncological diseases, circulating tumor DNA (ctDNA), a cfDNA sub-class, can carry clinically actionable mutations and coupled with next generation sequencing or other mutation detection methods provide a venue for effective in vitro diagnostics. However, cfDNA mutational analyses require high quality inputs. This necessitates extraction platforms that provide high recovery over the entire ctDNA size range (50 → 150 bp) with minimal interferences (i.e., co-extraction of genomic DNA), and high reproducibility with a simple workflow. Herein, we present a novel microfluidic solid-phase extraction device (µSPE) consisting of a plastic chip that is activated with UV/O3 to generate surface-confined carboxylic acid functionalities for the µSPE of cfDNA. The µSPE uses an immobilization buffer (IB) consisting of polyethylene glycol and salts that induce cfDNA condensation onto the activated plastic microfluidic surface. The µSPE consists of an array of micropillars to increase extraction bed load (scalable to loads >700 ng of cfDNA) and can be produced at low-cost using replication-based techniques. The entire µSPE can be fabricated in a single molding step negating the need for adding additional extraction supports to the device simplifying production and keeping device and assay cost low. The µSPE allowed for recoveries >90% of model cfDNA fragments across a range of sizes (100-700 bp) and even the ability to extract efficiently short cfDNA fragments (50 bp, >70%). In addition, the composition of the IB allowed for reducing the interference of co-extracted genomic DNA. We demonstrated the clinical utility of the µSPE by quantifying the levels of cfDNA in healthy donors and patients with non-small-cell lung and colorectal cancers. µSPE extracted cfDNA from plasma samples was also subjected to a ligase detection reaction (LDR) for determining the presence of mutations in the KRAS gene for colorectal and non-small cell lung cancer patients.