Date

2011-11-28

Author

Bat, Erhan
Huskens, Jurriaan

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Protein microarrays have recently gained great interest as they are suited for performing bioanalytical applications such as in vitro diagnostics, proteomics, antibody characterization and drug screening. Several techniques such as ink-jet printing, dip-pen nanolithography and microcontact printing have been used to generate protein microarrays. Inkjet printing and dip pen nanolithography allow printing of multiple proteins however these are serial techniques, control over the spot geometry/size and reproducibility is challenging. Moreover the most commonly applied technique of inkjet printing does not allow printing of proteins with less than 50 µm resolution. Microcontact printing is a soft lithography technique that allows parallel arraying of a single biomolecule over a large surface area with high resolution. Microarrays with smaller feature sizes are desired as they would allow rapid, highly sensitive, and direct determination of analyte concentration using a small amount of sample. Microcontact printing is a low cost, simple and reproducible method but usually re-inking of the stamp is required in each replication step, which is time consuming and costly and creating a multiplexed array is still a challenge. In this project, we aim at addressing the multiplexicity and inking challenges of microcontact printing. We present hydrogel-filled silicon stamps having individually addressable ink reservoirs. With this design, multiplexicity can be achieved while avoiding the need for re-inking. To prepare the stamps, we fabricated silicon microstructures having separate wells (320×320×380 µm) and each well having a 25 µm thick membrane (144 microchannels measuring 5 µm in diameter) on the printing side. The reservoirs and the microchannels of the silicon microstructures were filled with macroporous poly(2-hydroxyethyl methacrylate-co-ethylene glycol dimethacrylate) hydrogels. Methacryloxypropyl silane functionalized wells allowed covalent binding of hydrogels to the surface of the silicon. Using these hydrogel-filled stamps, we printed arrays of fluorescently labeled immunoglobulins on polydimethylsiloxane substrates reproducibly at least up to twenty times without re-inking. The spot sizes were very close to 5 µm in agreement with the stamp design. The fluorescence intensities of the printed spots were comparable for each printing step indicating that the amount of transferred protein did not vary significantly. With the help of an inkjet spotter, we addressed three immunoglobulins each labelled with a different fluorophore to separate reservoirs of the stamp. In this manner, multiplexed protein microarrays were obtained. We have also evaluated this printing system in an antibody-antigen assay. These hydrogel-filled silicon stamps hold great promise for multiplexed bioanalytical applications as well as for patterning of bioactive molecules for tissue engineering applications.

URI

https://hdl.handle.net/11511/87003
https://www.mrs.org/fall2011/symposium-sessions/?code=II

Collections

Department of Chemical Engineering, Conference / Seminar