Gravitational field-flow fractionation with chemiluminescence detection for a flow-assisted point-of-care testing device for analysis of biological fluids

The “Point-Of-Care Testing" (POCT) approach is based on the development of portable analytical platforms suitable to perform analysis directly in any required place, through the use of simple analytical tools addressed to operators with limited technical skills. A POCT device should combine portability, minimum sample pre-treatment and allow performing highly sensitive multiplexed assays in a short assay time. Microfluidic integrated systems relying on biospecific recognition reactions (e.g., immunological reactions, nucleic acids hybridization) and ultrasensitive bio-chemiluminescence detection techniques represent one of the most promising options. To enable their use outside of a well-controlled environment like a central-traditional laboratory, POCT devices should also comprise an integrated module for on-line sample pre-analytical treatment and/or clean-up to achieve high sensitivity in complex matrices. With this respect, field-flow fractionation (FFF) techniques, a family of flow-assisted separation techniques which can separate analytes based on their morphologic characteristics (size, shape and superficial properties) can be exploited to develop pre-analytical modules for cells or macromolecules (e.g., proteins, protein complexes or adducts) fractionation, thus providing a selectively enriched fraction for the analysis with an increase of overall analytical output. In particular, gravitational FFF (GrFFF), exploiting the Earth gravitational field to structure the separation, appears to be particularly suited for integration in POCT devices, thanks to the simplicity of its separative device, amenable to miniaturization. In this work, we propose the implementation of GrFFF as a pre-analytical module of a POCT device, thus providing a selectively enriched fraction for the analysis with an increase of overall analytical output. We present the use of GrFFF to prepare whole blood samples for the automatic on-line analysis of alkaline phosphatase activity in serum, as a biomarker of obstructive liver diseases and bone disorders. Serum alkaline phosphatase is commonly assayed after a preliminary blood sample centrifugation to obtain serum, by employing spectrophotometric techniques. In this work, the GrFFF device is employed to separate plasma from cells components which elute with different retention times. After the injection of heparin-treated whole blood in the GrFFF device, plasma is separated from cells and then directly addressed by means of a microfluidic system and valves to the analytical module, where a chemiluminescent substrate is added and the enzyme activity is measured by means of an on-line flow-through luminometer. The diagnostic test gave quantitative results with low sample (50 uL) and reagents consumption, short analysis time (10 minutes) and high reproducibility. In addition, the use of an array of FFF devices will be considered to increase enrichment productivity and to allow the development of multiplexed systems based on the same separative principle.