Piezo dispensed microarray of multivalent chelating thiols for dissecting complex protein-protein interactions.
The fabrication of a novel biochip, designed for dissection of multiprotein complex formation, is reported. An array of metal chelators has been produced by piezo dispensing of a bis-nitrilotriacetic acid (bis-NTA) thiol on evaporated gold thin films, prestructured with a microcontact printed grid of eicosanethiols. The bis-NTA thiol is mixed in various proportions with an inert, tri(ethylene glycol) hexadecane thiol, and the thickness and morphological homogeneity of the dispensed layers are characterized by imaging ellipsometry before and after back-filling with the same inert thiol and subsequent rinsing. It is found that the dispensed areas display a monotonic increase in thickness with increasing molar fraction of bis-NTA in the dispensing solution, and they are consistently a few Angströms thicker than those prepared at the same molar fraction by solution self-assembly under equilibrium-like conditions. The bulkiness of the bis-NTA tail group and the short period of time available for chemisorption and in-plane organization of the dispensed thiols are most likely responsible for the observed difference in thickness. Moreover, the functional properties of this biochip are demonstrated by studying multiple protein-protein interactions using imaging surface plasmon resonance. The subunits of the type I interferon receptor are immobilized as a composition array determined by the surface concentration of bis-NTA in the array elements. Ligand dissociation kinetics depends on the receptor surface concentration, which is ascribed to the formation of a ternary complex by simultaneous interaction of the ligand with the two receptor subunits. Thus, multiplexed monitoring of binding phenomena at various compositions (receptor densities) offers a powerful tool to dissect protein-protein interactions.
Klenkar
Goran
G
Division of Molecular and Applied Physics, Department of Physics, Chemistry, and Biology, Linköping University, S-581 83 Linköping, Sweden.
Valiokas
Ramûnas
R
Lundström
Ingemar
I
Tinazli
Ali
A
Tampé
Robert
R
Piehler
Jacob
J
Liedberg
Bo
B
D002614:Chelating Agents / Q000737:chemistry*; D015394:Molecular Structure; D009571:Nitrilotriacetic Acid; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D020349:Surface Plasmon Resonance
D002614:Chelating Agents; D011506:Proteins; D013438:Sulfhydryl Compounds; D009571:Nitrilotriacetic Acid