A microchip-based flow confinement method for rapid delivery of small sample volumes to sensor surfaces is described. For flow confinement, a sample flow is joined with a perpendicular makeup flow of water or sample medium. Under laminar flow conditions, the makeup flow confines the sample into a thin layer above the sensing area and increases its velocity. This can benefit mass transport limited processes such as DNA hybridization or heterogeneous immunoassays. For proof of concept, this method was applied to a high-affinity immunoassay with excess capture antibody. Rabbit IgG was immobilized onto a silicon nitride waveguide. Cy5-labeled anti-rabbit IgG was hydrodynamically pumped over the immobilized zone through an attached 3D-PDMS flow cell with 20-μm-deep microchannels. The degree of confinement was adjusted through the volume flow rate of the confining flow. Evanescent field-based fluorescence detection enabled monitoring of the binding event. Assays were allowed to reach equilibrium to enable sensorgram normalization for inter-run comparison. The corresponding assay completion times could be reduced from 55 min for static drop conditions to 13 min for 25:1 flow confinement (ratio of confining to sample flow). For typical analytical applications, where equilibrium formation is not required, the faster response should translate to very short analysis times. Concurrently with the faster binding, sample consumption was reduced by 96% compared to conventional whole-channel sample delivery. Diffusional loss of analyte into the confining layer was identified as the main limitation of flow confinement, particularly for long sensing pads.

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Cellulose nanocrystals (CNCs), which offer a high aspect ratio, large specific surface area, and large number of reactive surface groups, are well suited for the facile immobilization of high density biological probes. We here report functional high surface area scaffolds based on cellulose nanocrystals (CNCs) and poly(vinyl alcohol) (PVA) and demonstrate that this platform is useful for fluorescence-based sensing schemes. Porous CNC/PVA nanocomposite films with a thickness of 25-70 nm were deposited on glass substrates by dip-coating with an aqueous mixture of the CNCs and PVA, and the porous nanostructure was fixated by heat treatment. In a subsequent step, a portion of the scaffold's hydroxyl surface groups was reacted with 2-(acryloxy)ethyl (3-isocyanato-4-methylphenyl)carbamate to permit the immobilization of thiolated fluorescein-substituted lysine, which was used as a first sensing motif, via nucleophile-based thiol-ene Michael addition. The resulting sensor films exhibit a nearly instantaneous and pronounced change of their fluorescence emission intensity in response to changes in pH. The approach was further extended to the detection of protease activity by immobilizing a Förster-type resonance energy transfer chromophore pair via a labile peptide sequence to the scaffold. This sensing scheme is based on the degradation of the protein linker in the presence of appropriate enzymes, which separate the chromophores and causes a turn-on of the originally quenched fluorescence. Using a standard benchtop spectrometer to monitor the increase in fluorescence intensity, trypsin was detected at a concentration of 250 μg/mL, i.e., in a concentration that is typical for abnormal proteolytic activity in wound fluids.

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