title: Fast and reliable protein microarray production by a new drop-in-drop technique.
abstract: In contrast to DNA microarrays, production of protein microarrays is an immense technological challenge due to high complexity and diversity of proteins. In this paper we investigate three essential aspects of protein microarray fabrication based on the highly parallel and non-contact TopSpot technology: evaporation of probes during long lasting production times, optimization of protein immobilization and improvement of protein microarray reproducibility. Evaporation out of the printhead reservoirs was reduced to a minimum by sealing the reservoirs with gas permeable foils or PDMS frames. This led to dramatically lowered setup times through the possibility of long-term, ready-to-print storage of filled printheads. To optimize immobilization efficiency 128 printing buffers were tested by printing two different proteins onto seven different microarray slide types. This way we were able to reduce the CV of spot diameter on the microarray slide below 1.14%. To remarkably increase protein immobilization efficiency on microarray slides the commonly used EDC-NHS system (a laboratory method for immobilization of proteins) was miniaturized by using a new drop-in-drop printing technique. Additionally the very fast UV cross-linking was used to immobilize antibodies. The optimized system was used to produce antibody microarrays and with it microarray ELISA experiments were performed successfully.
authors.affiliation: IMTEK-University of Freiburg, Laboratory for MEMS Applications, Georges-Koehler-Allee 103, D-79110 Freiburg, Germany. ogutmann@imtek.de
authors.forename: Oliver
authors.initials: O
authors.lastname: Gutmann
authors.forename: Ruben
authors.initials: R
authors.lastname: Kuehlewein
authors.forename: Stefanie
authors.initials: S
authors.lastname: Reinbold
authors.forename: Remigius
authors.initials: R
authors.lastname: Niekrawietz
authors.forename: Chris P
authors.initials: CP
authors.lastname: Steinert
authors.forename: Bas
authors.initials: B
authors.lastname: de Heij
authors.forename: Roland
authors.initials: R
authors.lastname: Zengerle
authors.forename: Martina
authors.initials: M
authors.lastname: Daub
mesh_terms: D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000528:radiation effects; D012680:Sensitivity and Specificity; D013499:Surface Properties; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D011506:Proteins