Microcontact printing of proteins inside microstructures

Microfluidic devices are well suited for the miniaturization of biological assays, in particular when only small volumes of samples and reagents are available, short time to results is desirable, and multiple analytes are to be detected. Microfluidic networks (MFNs), which fill by means of capillary forces, have already been used to detect important biological analytes with high sensitivity and in a combinatorial fashion. These MFNs were coated with Au, onto which a hydrophilic, protein-repellent monolayer of thiolated poly(ethyleneglycol) (HS-PEG) was self-assembled, and the binding sites for analytes were present on a poly(dimethylsiloxane) (PDMS) sealing cover. We report here a set of simple methods to extend previous work on MFNs by integrating binding sites for analytes inside the microstructures of MFNs using microcontact printing (mu CP). First, fluorescently labeled antibodies (Abs) were microcontact-printed from stamps onto planar model surfaces such as glass, Si, Si/SiO2, Au, and Au derivatized with HS-PEG to investigate how much candidate materials for MFNs would quench the fluorescence of printed, labeled Abs. Au coated with HS-PEG led to a fluorescence signal that was similar to 65% weaker than that of glass but provided a convenient surface for printing Abs and for rendering the microstructures of the MFNs wettable. Then, proteins were inked from solution onto the surface of PDMS (Sylgard 184) stamps having continuous or discontinuous micropattems or locally inked onto planar stamps to investigate how the aspect ratio (depth:width) of microstructures and the printing conditions affected the transfer of protein and the accuracy of the resulting patterns. By applying a controlled pressure to the back of the stamp, Abs were accurately microcontact-printed into the recessed regions of MFNs if the aspect ratio of the MFN microstructures was lower than approximately 1:6. Finally, the realization of a simple assay between Abs (used as antigens) microcontact-printed in microchannels and Abs from solution suggests that this method could become useful to pattern proteins in microstructures for advanced bioanalytical purposes.