U. Beutling, D. Schwab, S. Thiele; with S. Dübel (TU-Braunschweig)
Arrays of probe molecules are primarily applied in capture and enzymatic transformation assays with the probe molecules tethered to the surface. Beyond these applications, there are ample opportunities if the captured biological targets could be isolated from the array and transferred separately to subsequent other analysis methods. This can be regarded as multiple capture and affinity enrichment. Examples for these include separations of polyclonal antisera into monospecific epitope-directed sub-populations, protein-presenting bacteriophages into ligand-specific sub-populations or ligand-specific proteins from cell extracts. So far such experiments could be carried out with our SPOT membranes only after physically cutting the array into probe segments, transfer to separate tubes and individual elution which is detrimental to the array, tedious to handle and difficult to automate. We have developed an easy lithographic process to generate structured array surfaces carrying small hydrophilic chemically functionalised patches that are separated by hydrophobic barriers from the unmodified plastic body of the array substrate. This allows to add droplets of polar liquids to the patch areas that will not contact each other (see figure). This type of patch array can be used for spatially addressable parallel chemical synthesis of probe molecules, their simultaneous assay and the spatially addressable elution of bound targets. All steps can be easily automated with conventional pipetting robots. Currently, we apply this type of arrays to multiplex phage enrichment of scFv antibody fragments on peptide antigens within the Antibody-Factory project of the NGFN.
These self-forming arrays of separated droplets can be also exploited for simultaneous parallel cell-based screening of compounds that are synthesized on and then released from the patches post-synthesis upon addition of aqueous media (a respective linker was developed by us). Thus, suspended cells can be distributed over the whole array surface, separating into distinct droplets upon which the compounds become released and act on the cells. With synthetic DT-2 peptide we have shown that this process efficiently reproduces the complex phenotype of A-498 kidney cancer cells (see below) as observed under conventional culture conditions in microtiter plates.
We feel very encouraged by this result and will advance this screening tool to allow for the on demand synthesis and in situ cell-based screening, ultimately making expensive storage and logistics obsolete.