We constructed surface-patterned hydrogels using low-energy focused electron beams to locally crosslink poly(ethylene glycol) (PEG) thin films on silanized glass substrates. Derived from electron-beam lithography, this technique was used to create patterned hydrogels with well-defined spatial positions and degrees of swelling. We found that cells of the bacterium Staphylococcus epidermidis adhered to and grew on the silanized glass substrates. These cells did not, however, adhere to surfaces covered by high-swelling lightly crosslinked PEG hydrogels. This finding is consistent with the cell-repulsiveness generally attributed to PEGylated surfaces. In contrast, S. epidermidis cells did adhere to surfaces covered by low-swelling highly crosslinked hydrogels. By creating precise patterns of repulsive hydrogels combined with adhesive hydrogels or with exposed glass substrate, we were able to spatially control the adhesion of S. epidermidis. Significantly, adhesive areas small enough to trap single bacterial cells could be fabricated. The results suggest that the lateral confinement imposed by cell-repulsive hydrogels hindered the cell proliferation and development into larger bacterial colonies.

We engineered surfaces that permit the adhesion and directed growth of neuronal cell processes but that prevent the adhesion of astrocytes. This effect was achieved based on the spatial distribution of sub-micron-sized cell-repulsive poly(ethylene glycol) [PEG] hydrogels patterned on an otherwise cell-adhesive substrate. Patterns were identified that promoted cellular responses ranging from complete non-attachment, selective attachment, and directed growth at both cellular and subcellular length scales. At the highest patterning density where the individual hydrogels almost overlapped, there was no cellular adhesion. As the spacing between individual hydrogels was increased, patterns were identified where neurites could grow on the adhesive surface between hydrogels while astrocytes were unable to adhere. Patterns such as lines or arrays were identified that could direct the growth of these subcellular neuronal processes. At higher hydrogel spacings, both neurons and astrocytes adhered and grew in a manner approaching that of unpatterned control surfaces. Patterned lines could once again direct growth at cellular length scales. Significantly, we have demonstrated that the patterning of sub-micron/nano scale cell-repulsive features at microscale lengths on an otherwise cell-adhesive surface can differently control the adhesion and growth of cells and cell processes based on the difference in their characteristic sizes. This concept could potentially be applied to an implantable nerve-guidance device that would selectively enable regrowing axons to bridge a spinal-cord injury without interference from the glial scar.

We are interested in controlling the spatial distribution of proteins on surfaces at cellular and subcellular length scales. To do this, we use a variation of e-beam lithography in a field-emission scanning electron microscope (SEM) to radiation crosslink thin films of water- soluble polymers such as poly(ethylene glycol) [PEG] and poly (carboxylic acids). We can simultaneously pattern the resulting hydrogels on silicon or glass surfaces with nanoscale and microscale feature sizes. Using hydroxy-terminated PEG 6800 we create gels with swell ratios between unity and fifteen depending on the degree of radiation crosslinking, and the swelling properties can be interpreted in terms of the Flory-Rehner formulation modified for one-dimensional swelling. While lightly-crosslinked PEG gels resist protein adsorption and cell adhesion as expected, highly crosslinked PEG gels adsorb such proteins as fibronectin and laminin and consequently become adhesive to fibroblasts, macrophages, and neurons. By spatially modulating the degree of crosslinking, we can localize these cells on surfaces and, for example, direct neurite outgrowth. If instead of using hydroxy-terminated PEG we use amine- terminated PEG, we introduce the additional flexibility of creating high-swelling PEG gels that resist nonspecific protein adsorption but to which specific proteins can be covalently bound. These can be surface patterned at submicron spacings, and we can pattern 7500 nanohydrogels in a 100 micron diameter arrays in 10 seconds. This is an areal density ~104 times greater than a modern DNA/protein chip, and the required bioreagents for chip fabrication and processing are proportionately less. We can bind fibronectin and laminin to different arrays, and we show that these proteins maintain their biospecificity after binding to the nanohydrogels with high fidelity. Looking to applications in next-generation protein-chip technology, our most recent experiments compare the performance of nanohydrogel arrays to that of standard protein microarrays using oligonucleotides which specifically bind nucleic acid-binding proteins.

We used a focused electron beam to crosslink hydroxyl- and amine- terminated poly(ethylene glycol) to create nanosized hydrogels and thus present a new method to bring the attractive biocompatibility associated with macroscopic hydrogels into the submicron length-scale regime. Using poly(ethylene glycol) thin films on silicon and glass substrates, we generate nanohydrogels with lateral dimensions of order 200 nm which can swell by a factor of at least five, and high-density arrays of such nanohydrogels can be flexibly patterned onto silicon or glass surfaces. Significantly, the amine groups remain functional after e-beam exposure, and we show that they can be used to covalently bind proteins and other molecules. We use bovine serum albumin to amplify the number of amine groups, and we further demonstrate that different proteins can be covalently bound to different hydrogel pads on the same substrate to create multifunctional surfaces. This approach opens a new means for patterning of proteins and other biological molecules on surfaces and may be useful for controlling surface bioactivity in emerging bio/proteomic and sensor technologies.

We studied the protein adsorption and subsequent macrophage adhesion on poly(ethylene glycol) [PEG] hydrogels crosslinked using a focused electron beam. Thin-film gels were patterned on silicon substrates and could be formed with swell ratios (hydrated height/ dry height) anywhere between fifteen and unity. We have shown that laminin does not adsorb onto highly swelling gels but that it does adsorb on heavily-crosslinked low-swelling gels. As part of ongoing research on patterning surfaces to control neurite growth in the context of the inflammatory environment of a spinal cord injury, we are interested in how these gel surfaces interact with macrophages. We show that the high-swelling PEG gels resist macrophage adhesion, but the macrophages adhere to low-swelling gels pre-exposed to laminin. By spatially patterning combinations of low and high swelling gels, we show that macrophage adhesion can be confined to specific locations on a surface.

PEG is a remarkable material. It is nontoxic, biocompatible, and has been approved by the US Food and Drug Administration for both oral and topical applications. It can be found in a variety of cosmetics, soaps, biomedical devices, and drug formulations. Also, despite the fact that it has been produced commercially for many decades(1) and reviewed extensively(2,3), PEG continues to be investigated in the context of biomaterials science because of its ability to control the bioactivity of surfaces and bulk materials.

We have used focused electron-beam cross-linking to create nanosized hydrogels and thus present a new method with which to bring the attractive biocompatibility associated with macroscopic hydrogels into the submicron length-scale regime. Using amine-terminated poly(ethylene glycol) thin films on silicon substrates, we generate nanohydrogels with lateral dimensions of order 200 nm which can swell by a factor of at least five, depending on the radiative dose. With the focused electron beam, high-density arrays of such nanohydrogels can be flexibly patterned onto silicon surfaces. Significantly, the amine groups remain functional after e-beam exposure, and we show that they can be used to covalently bind proteins and other molecules. We use bovine serum albumin to amplify the number of amine groups, and we further demonstrate that different proteins can be covalently bound to different hydrogel pads on the same substrate to create multifunctional surfaces useful in emerging bio/proteomic and sensor technologies.

This paper describes research addressing the question of whether microscopic hydrogels can be created from poly(ethylene glycol) [PEG 6800] and poly(ethylene oxide) [PEO 200K] using spatially resolved radiation from a scanning electron microscope with an approach similar to that used in the electron-beam patterning of polymeric photoresists. We demonstrate that, indeed, PEG hydrogels with micrometer and submicrometer feature sizes can be created by this approach, and we call these microhydrogels. Using solvent-free PEG 6800 and PEO 200K films similar to50-100-nm thick, we have identified sets of irradiation conditions where sufficient cross-linking occurs so that the exposed patterned polymer remains while the unexposed polymer dissolves during a post-irradiation solvent rinse. Arbitrary spatial patterns can be made. We have generated patterned dots with diameters below 200 rim. Using atomic force microscopy, in air and water, to study similar to5 x 5 mum PEG and PEO pads on silicon, we show that the patterned features generated by electron-beam cross-linking swell when exposed to water. The extent of swelling depends on the incident electron dose. Maximum swelling ratios of 14-16 have been observed. The swelling ratio decreases with increasing dose toward a limit of unity at the highest doses studied. Because of the significance of PEG in biomaterials applications, we examined the adsorption of fibronectin fragments onto the PEG microhydrogels using immunofluoresence optical microscopy. Undetectable Fn levels are observed on microhydrogels subjected to the lowest radiative exposure conditions where maximum swelling occurs. Fn adsorption increases with increasing dose and reaches a maximum at the highest doses where swelling ratios of unity are observed. This approach opens a new means for arbitrarily patterning the spatial distribution of proteins on surfaces and may be useful for controlling surface bioactivity.

The fact that cell expression and differentiation is affected by the nature of its adhesion to a synthetic substrate has been demonstrated using polymeric microspheres [1] and soft lithography [2]. These approaches have been used to present to cells surfaces with adhesion-promoting proteins whose spatial distribution is controlled at length scales comparable to cell sizes. We are developing analogous methods based on electron-beam lithography to control cell adhesion behavior. Thusfar we have developed protocols for the e-beam patterning of biocompatible polymers and studied the adsorption of fibronectin on these.