Development of an integrated Point-Of-Care Testing device based on Field-Flow Fractionation as pre-analytical treatment 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. To meet analytical and diagnostic requirements, a POCT device should combine portability, minimum sample pre-treatment, and the possibility to perform highly sensitive simultaneous detection of several biomarkers (multiplexing) in a short assay time. POCT devices should also comprise an integrated module for on-line sample pre-analytical treatment and/or clean-up to achieve high sensitivity and specificity even 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. Thanks to its “soft” separation mechanism, FFF has been successfully applied to a wide range of bioanalytes, from relatively small biomolecules to living cells in complex biological samples, which after separation keep their native characteristics such as the enzymatic activity, cell vitality, or quaternary protein structure [1]. In FFF, separation is achieved within a capillary empty channel in which a laminar flow of mobile phase sweeps sample components down the channel. A field is applied perpendicularly to the parabolic flow to make the analytes be driven into different laminar flows due to their differences in physical properties. As a consequence, the different analytes will elute from the separation device with a characteristic velocity. Gravitational FFF (GrFFF), exploiting the Earth gravitational field to structure the separation, is the humblest FFF technique which appears to be particularly suited for its implementation in POCT devices, thanks to the simplicity of its separative device, amenable to miniaturization, and the potential easy integration with specific analytical modules [2]. 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.