Lab in a tube: Purification, amplification, and detection of DNA using poly(2-oxazoline) multilayers

Fast and easy purification and amplification of DNA are prerequisites for the development of point-of-care diagnostics. For this reason covalent coatings of amine containing poly(2-oxazoline)s (POx) on glass and poly(propylene) surfaces are prepared, to reversibly bind genetic material directly from biological samples. The polymer is deposited in a layer-by-layer process, whereas initial immobilization of macromolecules on the surface is accomplished by the use of an epoxy functionalized siloxane monolayer. Alternating treatment with polymer and cross-linker leads to the construction of amine containing POx multilayers on the substrates. Successful deposition is investigated by confocal laser scanning microscopy (using labeled polymers), contact angle measurements, as well as reflectometric interference spectroscopy. The interaction of these layer systems with DNA regarding binding and temperature dependent release is studied using labeled genetic material. Finally, polymerase chain reaction (PCR) vessels are coated with POx layers on the inside, and used for quantitative real-time PCR (qPCR) experiments. It is possible to bind genetic material directly from cell lysates to perform qPCR assays from surface adsorbed DNA within the same tube including amplification, as well as detection. The presented system displays an easy to use device for a point of care diagnostic. Detection of DNA directly from biological material is performed by the use of covalently bound poly(2-oxazoline) multilayers on polypropylene. Layer-by-layer assembly and interaction with genetic material is investigated in detail, and the amplification and detection of surface adsorbed DNA is performed by quantitative real-time polymerase chain reaction using coated reaction vessels.