Microfluidic Integration of Parallel Solid-Phase Liquid Chromatography

We report the development of a fully integrated microfluidic chromatography system based on a recently developed column geometry that allows for robust packing of high-performance separation columns in poly­(dimethylsiloxane) microfluidic devices having integrated valves made by multilayer soft lithography (MSL). The combination of parallel high-performance separation columns and on-chip plumbing was used to achieve a fully integrated system for on-chip chromatography, including all steps of automated sample loading, programmable gradient generation, separation, fluorescent detection, and sample recovery. We demonstrate this system in the separation of fluorescently labeled DNA and parallel purification of reverse transcription polymerase chain reaction (RT-PCR) amplified variable regions of mouse immunoglobulin genes using a strong anion exchange (AEX) resin. Parallel sample recovery in an immiscible oil stream offers the advantage of low sample dilution and high recovery rates. The ability to perform nucleic acid size selection and recovery on subnanogram samples of DNA holds promise for on-chip genomics applications including sequencing library preparation, cloning, and sample fractionation for diagnostics

Fabrication of High-Quality Microfluidic Solid-Phase Chromatography Columns

Here we report a low-pressure bead packing technique for the robust integration of high-performance chromatography columns in poly­(dimethylsiloxane) microfluidic devices made by multilayer soft lithography (MSL). A novel column geometry featuring micrometer-sized bypass channels along the entire length of the separation channel is used to achieve rapid packing of multiple high-quality bead bed columns in parallel with near-perfect yield. Pulse tests show that these microfluidic columns achieve exceptional reproducibility and efficiency, with measured plate counts of 1 650 000/m ± 7%, corresponding to a reduced plate height of <i>h</i> = 0.12 ± 7%. The combination of high-performance chromatography columns and valve-based microfluidics offers new opportunities for the integration of sample processing with preparative and analytical separations for biology and chemistry

Ion Exchange in Hydroxyapatite with Lanthanides

Naturally occurring hydroxyapatite, Ca₅(PO₄)₃(OH) (HAP), is the main inorganic component of bone matrix, with synthetic analogues finding applications in bioceramics and catalysis. An interesting and valuable property of both natural and synthetic HAP is the ability to undergo cationic and anionic substitution. The lanthanides are well-suited for substitution for the Ca²⁺ sites within HAP, because of their similarities in ionic radii, donor atom requirements, and coordination geometries. We have used isothermal titration calorimetry (ITC) to investigate the thermodynamics of ion exchange in HAP with a representative series of lanthanide ions, La³⁺, Sm³⁺, Gd³⁺, Ho³⁺, Yb³⁺ and Lu³⁺, reporting the association constant (<i>K</i>_a), ion-exchange thermodynamic parameters (Δ<i>H</i>, Δ<i>S</i>, Δ<i>G</i>), and binding stoichiometry (<i>n</i>). We also probe the nature of the La³⁺:HAP interaction by solid-state nuclear magnetic resonance (³¹P NMR), X-ray diffraction (XRD), and inductively coupled plasma–optical emission spectroscopy (ICP-OES), in support of the ITC results

Lectin Interactions on Surface-Grafted Glycostructures: Influence of the Spatial Distribution of Carbohydrates on the Binding Kinetics and Rupture Forces

We performed quantitative analysis of the binding kinetics and affinity of carbohydrate–lectin binding and correlated them directly with the molecular and structural features of ligands presented at the nanoscale within the glycocalyx mimicking layers on surfaces. The surface plasmon resonance analysis identified that the mode of binding changed from multivalent to monovalent, which resulted in a near 1000-fold change in the equilibrium association constant, by varying the spatial distribution of carbohydrate ligands within the surface-grafted polymer layer. We identified, for the first time, that the manner in which the ligands presented on the surface has great influence on the binding at the first stage of bivalent chelating, not on the binding at the second stage. The rupture forces measured by atomic force microscope force spectroscopy also indicated that the mode of binding between lectin and ligands changed from multiple to single with variation in the ligand presentation. The dependence of lectin binding on the glycopolymer composition and grafting density is directly correlated with the nanoscale presentation of ligands on a surface, which is a determining factor in controlling the clustering and statistical effects contributing to the enhanced bindin

International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a <i>KRAS</i> fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics