AFM imaging was performed on a scan size of 25.0 m, with a scan rate of 0.7 Hz, and 512 samples/line. == Film thickness measurements == Film thicknesses from spin coating solutions of 5 L HRP (60 M in PBS) and 25 L poly(SET) (0.3, 0.5 or 1 wt%) were measured. patterns at the micrometer and nanometer scale with the advantage compared to Vorasidenib indirect strategies that there is no limit in the number of different proteins that can be patterned. Yet despite these advantages, only a few examples have been reported to directly pattern biomolecules on surfaces at the submicron scale.15The majority have employed physical deployment onto surfaces using an atomic force microscopy tip such as dip pen lithography or polymer pen lithography; these techniques involve pressing or flowing the material onto a surface and do not require a resist. This is because approaches in which radiation is utilized, such as electron-beam lithography (EBL) require the development of resists that are not only water soluble, but also protect the biomolecules from denaturation during irradiation along with other control methods. These requirements have significantly hindered the investigation of direct-write biomolecule patterns by techniques such as EBL. EBL is a maskless patterning technique that produces user designed complex patterns at high resolution. Although a serial technique, EBL gives nanometer level alignment ability, which enables inter-feature spacings that are so small that different protein features may be touching or arrayed one on top of the other allowing for complex, multiplexed patterns.6The majority of protein patterning by EBL has been accomplished by indirect approaches,611because the high vacuum and high energy radiation inactivates proteins. Indeed, the harsh conditions required have been exploited to pattern by selective ablation of proteins upon electron beam exposure.7,8Thus far, there have only been two resists that have been employed for direct protein pattering by EBL; proteins bacteriorhodopsin and green fluorescent protein were patterned using poly(acrylic acid) and silk as resists.1,3In both studies, the authors noted the proteins used had exceptionally stable structures, which Vorasidenib enabled them to be stable under harsh conditions of EBL. Herein, we describe a resist material, a Vorasidenib trehalose glycopolymer that stabilizes a variety of proteins including antibodies and sensitive growth factors to repeated exposure to vacuum and to electron beams allowing for direct create by EBL, as well as multiplexing. The second option is critical for full realization of the technology in applications such as sensors. == Results == == Trehalose glycopolymer patterning by EBL == The trehalose glycopolymer explored for EBL has a polystyrene backbone and trehalose part chains (polystyrenyl ether trehalose or poly(Collection),Number Vorasidenib 1). This particular backbone was chosen because polystyrene is known as a negative-tone EBL resist.11,12Thus, it was anticipated the polymer would cross-link less than e-beam irradiation. Indeed, upon exposure to e-beams, this polymer was cross-linked and covalently bound to the silicon/silicon dioxide surfaces. Overexposure was Chuk observed at doses above 70 C/cm2while the patterns looked sharp at an area dose of 40 C/cm2(Supplementary Number 1). These doses are much lower than that required for patterning polystyrene enabling much faster patterning which makes poly(Collection) a cost effective resist. The polymer likely cross-links to the surface and to additional polymer chains by a radical cross-linking mechanism similar to that observed for additional polymers such as poly(ethylene glycol) (PEG).6,9 == Number 1. Electron beam lithography process for multiple protein patterns. == I, Spin-coating with poly(Collection)-protein-1 remedy and writing of the 1st coating.IWe, Rinsing of the unexposed poly(Collection)-protein-1 followed by spin-coating poly(Collection)-protein-2, alignment to the first coating, and writing of the second coating.IIIIV, Multiple protein Vorasidenib patterns are obtained by repeated spin-coating, alignment,.