Researchers have developed a procedure for using the multiphoton polymerization effect to harden biocompatible polymers into three-dimensional structures that may help create biosensors with greater surface area. The group — from the Foundation for Research and Technology in Iraklion, Greece — recently tested the method by binding biotin to the surface of a structure and studying the result with fluorescence microscopy and a surface acoustic wave technique. According to Maria Farsari, the principal investigator, most micropatterned biological sensors — many of which draw on techniques developed in the semiconductor industry — are strictly two-dimensional. “By going from 2-D to 3-D, one can greatly increase the active surface area and, thus, the sensitivity of the system without sacrifices to the size of the sensor,” she explained. Using the multiphoton effect to harden the biocompatible polymer Ormocer, researchers created 3-D structures that could be useful in developing lab-on-a-chip-type analytical devices because they significantly increase the active surface area. Images courtesy of Maria Farsari. The group starts with a UV-photocurable organic-inorganic polymer called Ormocer, from Micro Resist Technology of Berlin, that is transparent in the visible and near-infrared. The polymer also can be cross-linked and is mechanically and thermally stable. It takes both UV radiation and the presence of a photoinitiator to cause the polymer to set. Farsari and her colleagues first used the three-photon effect to create a 3-D structure from the polymer in 2005. As described in the Oct. 2 issue of Applied Physics Letters, to activate the polymer, they first used Amplitude Systemes’ laser femtosecond oscillator operating at 1028 nm with an average power of 1 W, a pulse duration The researchers built several microscopic components that consist of a latticework of step-in squares, looking something like a grate. Biocompatibility After creating the structures, they attached biotin to them using UV-activated cross-linking of photobiotin. They chose biotin because, once it has been immobilized, it readily reacts with avidin, which reacts again with biotin or biotinylated molecules. A wide range of macromolecules, including proteins, polysaccha-rides and nucleic acids, can be linked readily to biotin without serious effect on their biological, chemical or physical characteristics. “The strength of the avidin-biotin bond and the generic nature of the biotinylation process make avidin-biotin technology highly amenable to selective patterning of various properties,” Farsari said. To adhere the biotin to the component surface, the researchers covered the components with photobiotin, dried the samples in the dark and exposed them to UV radiation from a KrF laser made by Tui-Laser AG of Munich, Germany. Then they covered them for at least an hour with Atto 565 streptavidin, a fluorophore that binds strongly to biotin. After rinsing the excess fluorophore off the components, the scientists watched the structures’ fluorescence under a Nikon microscope equipped with a camera and green filter. They observed very strong fluorescence from each part of the component. Neither Ormocer nor photobiotin fluoresces when excited by green light. The group saw no fluorescence from components that had been treated only with biotin. This helped confirm that the fluorescences came from the bound biotin and not from biotin somehow trapped in the pores of the material. To determine how much biotin was cross-linked to the component, they employed a Love wave device. This instrument, which uses a shear horizontal-surface acoustic wave device and a thin polymer layer as a waveguide, can monitor the adsorption process because mass adsorbed to the polymer surface modifies the phase change of the surface acoustic wave. This was the most challenging part of the experiment, Farsari said. “The materials system is very complex, and the fluorescence could originate from other sources,” she said. “The acoustic wave sensor proved very useful.” The investigators spin-coated the Ormocer onto a specially prepared piezoelectric crystal and tested several concentrations of photobiotin. Each concentration was then cross-linked. Once completed in this way, the crystals were exposed to an avidin solution, and the binding was monitored in real time using the device. The results demonstrated more dramatic phase changes for higher concentrations of photobiotin. They also showed that the photobiotin in concentrations exceeding 100 mg/ml achieves saturation of the Ormocer base. Farsari said that he and his colleaques are planning to combine the technique with other methods, with an eye toward creating patterns of biomolecules by binding them to the immobilized biotin.