Rapid protein depletion from complex samples using a bead-based microfluidic device for the point of care.

Translation of sample preparation methods to point-of-care formats has remained a challenge. We present a plastic laminate microfluidic device for protein depletion from human plasma using ligand immobilized porous beads stored dry within a novel, pneumatically-driven mixer. The card design accelerated the protein depletion process from hours to minutes. Using immunoglobulin G as a model protein, we have successfully shown protein removal efficiency from spiked buffer between 70-80% and from diluted human plasma samples between 66-77%. Low non-specific binding of our downstream target ligand, immunoglobulin M, was observed with the spiked buffer and diluted human plasma samples. For future device optimization, the physical limitations to rapid protein removal on card were also explored. Bench-top experiments with improved mixing efficiency and a lower sample dilution factor achieved 99% IgG removal using the same amount of mixing time. This design can easily be adapted for depletion of other high abundance or interfering proteins by inclusion of other ligand immobilized beads.

Enabling a microfluidic immunoassay for the developing world by integration of on-card dry reagent storage.

As part of an effort to create a point-of-care diagnostic system for the developing world, we present a microfluidic flow-through membrane immunoassay with on-card dry reagent storage. By preserving reagent function, the storage and reconstitution of anhydrous reagents enables the devices to remain viable in challenging, unregulated environmental conditions. The assay takes place on a disposable laminate card containing both a porous membrane patterned with capture molecules and a fibrous pad containing an anhydrous analyte label. To conduct the assay, the card is placed in an external pumping and imaging instrument capable of delivering sample and rehydrated reagent to the assay membrane at controlled flow rates to generate quantitative results. Using the malarial antigen Plasmodium falciparum histidine-rich protein II (PfHRP2) as a model, we demonstrate selection of dry storage conditions, characterization of reagent rehydration, and execution of an automated on-card assay. Gold-antibody conjugates dried in a variety of sugar matrices were shown to retain 80-96% of their activity after 60 days of storage at elevated temperatures, and the release profile of the reconstituted reagent was characterized under flow in microfluidic channels. The system gave a detection limit in the sub-nanomolar range in under nine minutes, showing the potential to expand into quantitative, multi-analyte analysis of human blood samples.

A Rapid, Multiplexed, High-Throughput Flow-Through Membrane Immunoassay: A Convenient Alternative to ELISA.

This paper describes a rapid, high-throughput flow-through membrane immunoassay (FMIA) platform. A nitrocellulose membrane was spotted in an array format with multiple capture and control reagents for each sample detection area, and assay steps were carried out by sequential aspiration of sample and reagents through each detection area using a 96-well vacuum manifold. The FMIA provides an alternate assay format with several advantages over ELISA. The high surface area of the membrane permits high label concentration using gold labels, and the small pores and vacuum control provide rapid diffusion to reduce total assay time to ~30 min. All reagents used in the FMIA are compatible with dry storage without refrigeration. The results appear as colored spots on the membrane that can be quantified using a flatbed scanner. We demonstrate the platform for detection of IgM specific to lipopolysaccharides (LPS) derived from Salmonella Typhi. The FMIA format provides analytical results comparable to ELISA in less time, provides integrated assay controls, and allows compensation for specimen-to-specimen variability in background, which is a particular challenge for IgM assays.