FORMATION OF MICROSCALE GRADIENTS OF PROTEIN USING HETEROBIFUNCTIONALPHOTOLINKERS

Gradients of biological molecules on a microscale have been postulated to elicit cellular responses, such as migration. However, it has been difficult to prepare such gradients for experimental testing. A means for producing such gradients has been developed using a heterobifunct ional photolinking agent with laser light activation. The photolinking agent synthesized includes an N-hydroxysuccinimide group and a photor eactive benzophenone (BP) separated by a tetraethylene glycol (TEG) sp acer. The presence of the tetraethylene glycol spacer renders the phot olinker hydrophilic, a desirable trait for conjugation in aqueous solu tions. The linker was then conjugated to R-phycoerythrin (R-PE), a flu orescent protein. The resulting photolinker-R-phycoerythrin conjugate (BP-TEG-PE) was then immobilized onto a polystyrene surface by laser i rradiation on a motorized stage. By varying exposure time of the sampl e to the beam, the amount of BP-TEG-PE immobilized on the surface was changed over an order of magnitude over a distance of 250 mu m. This m ethod can be applied to prepare gradients of proteins that elicit biol ogical responses, such as extracellular matrix proteins or growth fact ors, and to study the biological effects of such gradients.