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Low Melting Point Agarose Gel as a Protection Layer in the Preparation of Aligned Binary Protein Patterns by Photolithography

Lap Man Lee1, Ronald L. Heimark2, Roberto Guzman3, Yitshak Zohar1, and James C. Baygents3. (1) Aerospace & Mechanical Engineering, The University of Arizona, Tucson, AZ 85721, (2) Surgery, Cell Biology & Anatomy, The University of Arizona, Tucson, AZ 85721, (3) Chemical & Environmental Engineering, The University of Arizona, Tucson, AZ 85721

Over the last decade, Bio-Microelectromechanical Systems (Bio-MEMS) and Lab-on-a-Chip devices have been developed from synergetic applications of microsystem technology and biotechnology [1, 2]. The design and fabrication of biologically functional surfaces are important steps in the production of such microdevices and this provides a substantial driving force to develop novel surface nanotechnologies, including protein patterning methods [3]. Selectively modified surfaces, with biologically functional components, are essential working elements in biosensors [4,5] and devices for cell manipulation and tissue engineering [68].

Two prominent methods for producing patterned surface modifications are photolithography and soft lithography. Soft lithography, also known as micro-contact printing, employs a pre-formed stamp or mold, often made from poly(dimethlysiloxane) (PDMS), to transfer patterns onto a substrate [9]. While this technology has many promising uses [9,10], soft lithography is not especially suited to applications where precise alignment is required to position a sequence of surface modifications and the stamping process can result in a loss of biological function in the deposited layer [10].

Standard photolithography, on the other hand, is a well-established technique for the bulk manufacture of integrated circuits. In photolithography, a pattern defined by a photomask is transferred to an overlying layer of photoresist, and from the photoresist to underlying layers. The method provides for high resolution and precise feature alignment, but the conventional solutions used to develop and strip the photoresist are largely incompatible with biological molecules and adversely affect their function [11].

Here we present a novel photolithography method to build binary, self-assembled patterns of two different proteins. Chessboard patterns of 125micron by 125micron squares are constructed on a silicon dioxide substrate, using standard photoresist chemistries in combination with low-temperature oxygen plasma etching. Low-melting-point agarose (LPMA) is used to protect underlying protein layers and, at the appropriate stage, the digestive enzyme GELase is used to selectively remove the prophylactic LMPA layers. Two antibodies, mouse-IgG and human-IgG, were immobilized and patterned by this procedure. The patterned antibodies maintained the specificity of their antigen-antibody binding, as demonstrated by fluorescence microscopy. In addition, normalized fluorescence intensity profiles illustrate that the patterned proteins layers are uniform (standard deviations below 0.05). Finally, a trypsin activity test was conducted to probe the effect of the patterning protocol on immobilized enzymes; results imply that LMPA protection preserves 70% of immobilized enzyme activity.

References

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