Supramolecular Peptoid Structure Strengthens Complexation with Polyacrylic Acid Microgels.

We have studied the complexation between cationic antimicrobials and polyanionic microgels to create self-defensive surfaces that responsively resist bacterial colonization. An essential property is the stable sequestration of the loaded (complexed) antimicrobial within the microgel under a physiological ionic strength. Here, we assess the complexation strength between poly(acrylic acid) [PAA] microgels and a series of cationic peptoids that display supramolecular structures ranging from an oligomeric monomer to a tetramer. We follow changes in loaded microgel diameter with increasing [Na+] as a measure of the counterion doping level. Consistent with prior findings on colistin/PAA complexation, we find that a monomeric peptoid is fully released at ionic strengths well below physiological conditions, despite its +5 charge. In contrast, progressively higher degrees of peptoid supramolecular structure display progressively greater resistance to salting out, which we attribute to the greater entropic stability associated with the complexation of multimeric peptoid bundles.

Analytical Cryo-Scanning Electron Microscopy of Hydrated Polymers and Microgels.

Despite the fact that scanning electron microscopes (SEM) coupled with energy-dispersive X-ray microanalysis (EDS) has been commercially available for more than a half-century, SEM/EDS continues to develop and open new opportunities to study the morphology of advanced materials. This is particularly true in applications to hydrated soft matter. Developments in field-emission electron sources that enable low-voltage imaging of uncoated polymers, silicon-drift detectors that enable high-efficiency collection of X-rays characteristic of light elements, and cryogenic methods to effectively cryo-fix hydrated samples have opened new opportunities to apply techniques relatively well established in hard-materials applications to challenging new problems involving synthetic polymers. We have applied cryo-SEM imaging and spatially resolved EDS to collect new information characterizing polyelectrolyte microgels. These are charged gel particles with dimensions in the range of 0.1-100 µm. Perhaps most notable is the fact that the high hydration levels-the samples are mostly water-allow robust calibration curves to be generated using frozen-hydrated buffers with known salt and/or hydrocarbon compositions. Such calibration curves enable quantitative composition measurements in the low-concentration extremes associated with high-swelling hydrogels. We use an experimentally derived carbon calibration curve to determine the microgel swell ratio, Q. The swell ratio, arguably, is the single most important gel characteristic because it is directly related to the mesh size of the networked polymer, which in turn determines many of the gel's mechanical and transport properties. While Q can be experimentally measured in macroscopic gels based on weight measurements in the dry and hydrated states, it is very difficult to measure in a microgel, and the fact that EDS in a cryo-SEM can determine Q from a single X-ray spectrum is significant. Furthermore, because of the electrostatic charge distributed along the polymer chains, the presence and concentration of counter-ions play a critical role in polyelectrolyte systems. While conceptually understood for decades, experimental measurements of counter-ion concentrations have been largely limited to a relatively small set of materials that involve macroscopic samples. By developing calibration curves from frozen-hydrated buffer of known ionic strength, we measure the concentration of Na counter-ions in microgels of poly(acrylic acid) (PAA) with a limit of detection of ∼0.014 M. Such measurements may help resolve some long-standing questions in polyelectrolyte science concerning counter-ion condensation. Even in the absence of a calibration curve, we show that spatially resolved X-ray spectroscopy can map the spatial distribution of a cationic oligopeptide complexed within a hydrated PAA microgel because of the nitrogen fingerprint that, albeit at very low concentration, is unique to the peptide. We look specifically at the case of a microgel with a so-called core-shell structure, where, again, the underlying polyelectrolyte science responsible for core-shell formation remains incompletely understood. These examples highlight how a modern cryo-SEM can be exploited to quantitatively characterize hydrated soft matter. The approach is almost certain to continue its development and impact as the base of experienced practitioners, the accessibility to well-configured microscopes, and the abundance of challenging problems involving hydrated soft matter all continue to grow.

Chemical Orthogonality in Surface-Patterned Poly(ethylene glycol) Microgels.

Because of its widely known antifouling properties, a variety of lithographic approaches has been used to pattern surfaces with poly(ethylene glycol) (PEG) to control surface interactions with biomolecules and cells over micro- and nanolength scales. Often, however, particular applications need additional functions within PEG-patterned surfaces. Monofunctional films can be generated using PEG modified to include a chemically functional group. We show that patterning with focused electron beams, in addition to cross-linking a monofunctional PEG homopolymer thin-film precursor and grafting the resulting patterned microgels to an underlying substrate, induces additional chemical functionality by radiation chemistry along the polymer main chain and that this second functionality can be orthogonal to the initial one. Specifically, we explore the reactivity of biotin-terminated PEG (PEG-B) as a function of electron dose using 2 keV electrons. At low doses (∼4-10 µC/cm2), the patterned PEG-B microgels are reactive with streptavidin (SA). As dose increases, the SA reactivity decays as biotin is damaged by the incident electrons. Independently, amine reactivity appears at higher doses (∼150-500 µC/cm2). At both extremes, the patterned PEG microgels retain their ability to resist fibronectin adsorption. We confirm that the amine reactivity derives from the PEG main chain by demonstrating similar dose response in hydroxy-terminated PEG (PEG-OH), and we attribute this behavior to the formation of ketones, aldehydes, and/or carboxylic acids during and after electron-beam (e-beam) patterning. Based on relative fluorescent intensities, we estimate that the functional contrast between the differentially patterned areas is about a factor of six or more. This approach provides the ability to easily pattern biospecific functionality while preserving the ability to resist nonspecific adsorption at length scales relevant to controlling protein and cell interactions.

Integrating nucleic acid sequence-based amplification and microlensing for high-sensitivity self-reporting detection.

We use electron-beam patterned functional microgels to integrate self-reporting molecular beacons, dielectric microlenses, and solid-phase and/or solution-phase nucleic acid amplification in a viral-detection microarray model. The detection limits for different combinations of these elements range from 10-10 M for direct target-beacon hybridization alone to 10-18 M when all elements are integrated simultaneously.

Counterion Exchange in Peptide-Complexed Core-Shell Microgels.

The complexation of polyvalent macroions with oppositely charged polyelectrolyte microgels can lead to core-shell structures. The shell is believed to be highly deswollen with a high concentration of counter-macroions. The core is believed to be relatively free of macroions but under a uniform compressive stress due to the deswollen shell. We use cryo-scanning electron microscopy (SEM) with X-ray microanalysis to confirm this understanding. We study poly(acrylic acid) (PAA) microgels which form a core-shell structure when complexed with a small cationic antimicrobial peptide (L5). We follow the spatial distribution of polymer, water, Na counterions, and peptide based on the characteristic X-ray intensities of C, O, Na, and N, respectively. Frozen-hydrated microgel suspensions include buffers of known composition from which calibration curves can be generated and used to quantify both the microgel water and sodium concentrations, the latter with a minimum quantifiable concentration less than 0.048 M. We find that as-synthesized PAA microgels are enriched in Na relative to the surrounding buffer as anticipated from established ideas of counterion shielding of electrostatic charge. The shell in L5-complexed microgels is depleted in Na and enriched in peptide and contains relatively little water. Our measurements furthermore show that shell/core interface is diffuse over a length scale of a few micrometers. Within the limits of detection, the core Na concentration is the same as that in as-synthesized microgels, and the core is free of peptide. The core has a slightly lower water concentration than as-synthesized controls, consistent with the hypothesis that the core is under compression from the shell.

Solid-Phase Nucleic Acid Sequence-Based Amplification and Length-Scale Effects during RNA Amplification.

Solid-phase oligonucleotide amplification is of interest because of possible applications to next-generation sequencing, multiplexed microarray-based detection, and cell-free synthetic biology. Its efficiency is, however, less than that of traditional liquid-phase amplification involving unconstrained primers and enzymes, and understanding how to optimize the solid-phase amplification process remains challenging. Here, we demonstrate the concept of solid-phase nucleic acid sequence-based amplification (SP-NASBA) and use it to study the effect of tethering density on amplification efficiency. SP-NASBA involves two enzymes, avian myeloblastosis virus reverse transcriptase (AMV-RT) and RNase H, to convert tethered forward and reverse primers into tethered double-stranded DNA (ds-DNA) bridges from which RNA- amplicons can be generated by a third enzyme, T7 RNA polymerase. We create microgels on silicon surfaces using electron-beam patterning of thin-film blends of hydroxyl-terminated and biotin-terminated poly(ethylene glycol) (PEG-OH, PEG-B). The tethering density is linearly related to the PEG-B concentration, and biotinylated primers and molecular beacon detection probes are tethered to streptavidin-activated microgels. While SP-NASBA is very efficient at low tethering densities, the efficiency decreases dramatically with increasing tethering density due to three effects: (a) a reduced hybridization efficiency of tethered molecular beacon detection probes; (b) a decrease in T7 RNA polymerase efficiency;

Microlens Enhancement of Surface-Tethered Molecular Beacons.

The tethering of molecular beacon oligonucleotide detection probes to surface-patterned poly(ethylene glycol) (PEG) microgels has enabled the integration of molecular beacons into a microarray format. The microgels not only localize the probes to specific surface positions but also maintain them in a waterlike environment. Here we extend the concept of microgel tethering to include dielectric microlenses. We show that streptavidin-functionalized polystyrene microspheres (3 µm diameter) can be colocalized with molecular beacons using biotinylated PEG gels in patterns ranging from pseudocontinuous microgel pads with lateral dimensions on the order of tens of micrometers to individual microgels with lateral dimensions on the order of 400-500 nm. We use a simplex assay based on Influenza A detection to study the lensing behavior. The microspheres increase the effective numerical aperture of the collection optics, and we find that a tethered microsphere increases the peak intensity collected from hybridized beacons between 1.5 and 10 times depending on the specific pattern size and areal density of microgels. The highest signal increase occurs when a single microsphere is tethered to a single isolated microgel. The tethering is highly self-directed and occurs in the individual-microgel case only when the microgel is close to the optic axis of the microsphere. This alignment minimizes spherical aberration and maximizes coupling of emitted fluorescent intensity into the collection optics.

Substrate effects on cell-envelope deformation during early-stage Staphylococcus aureus biofilm formation.

Bacterial adhesion to a surface is the first step in biofilm formation, and adhesive forces between the surface and a bacterium are believed to give rise to planktonic-to-biofilm phenotypic changes. Here we use Focused-Ion-Beam (FIB) tomography with backscattered scanning electron microscopy (SEM) to image Staphyolococcus aureus (S. aureus) biofilms grown on Au-coated polystyrene (PS) and Au-coated PS modified by mixed thiols of triethylene glycol mono-11-mercaptoundecyl ether (EG3) and 1-dodecanethiol (CH3). The FIB-SEM technique enables a direct measurement of the contact area between individual bacteria and the substrate. The area of adhesion is effectively zero on the EG3 substrate. It is nonzero on all of the other substrates and increases with increasing hydrophobicity. The fact that the contact area is highest on the unmodified gold, however, indicates that other forces beyond hydrophobicity are significant. The magnitude of bacterial deformation suggests that the adhesive forces are on the order of a few nN, consistent with AFM force measurements reported in the literature. The resolution afforded by electron microscopy furthermore enables us to probe changes in the cell-envelope thickness, which decreases within and near the contact area relative to other parts of the same bacterium. This finding supports the idea that mechanosensing due to stress-induced membrane thinning plays a role in the planktonic-to-biofilm transition associated with bacterial adhesion.

Molecular Crowding Effects on Microgel-Tethered Oligonucleotide Probes.

Microgel tethering is a nontraditional method with which to bind oligonucleotide hybridization probes to a solid surface. Microgel-tethering physically positions the probes away from the underlying hard substrate and maintains them in a highly waterlike environment. This paper addresses the question of whether molecular crowding affects the performance of microgel-tethered molecular beacon probes. The density of probe-tethering sites is controlled experimentally using thin-film blends of biotin-terminated [PEG-B] and hydroxyl-terminated [PEG-OH] poly(ethylene glycol) from which microgels are synthesized and patterned by electron beam lithography. Fluorescence measurements indicate that the number of streptavidins, linear DNA probes, hairpin probes, and molecular beacon probes bound to the microgels increases linearly with increasing PEG-B/PEG-OH ratio. For a given tethering-site concentration, more linear probes can bind than structured probes. Crowding effects emerge during the hybridization of microgel-tethered molecular beacons but not during the hybridization of linear probes, as the tethering density increases. Crowding during hybridization is associated with conformational constraints imposed by the close proximity of closed and hybridized structured probes. The signal-to-background ratio (SBR) of hybridized beacons is highest and roughly constant for low tethering densities and decreases at the highest tethering densities. Despite differences between microgel tethering and traditional oligonucleotide surface-immobilization approaches, these results show that crowding defines an optimum tethering density for molecular beacon probes that is less than the maximum possible, which is consistent with previous studies involving various linear and structured oligonucleotide probes.

Dip-pen microarraying of molecular beacon probes on microgel thin-film substrates.

The integration of microarray-based nucleic acid detection technologies and microfluidics is attractive, because the combination of small sample volumes, relatively short diffusion distances, and solid-phase detection enhances the development of multiplexed assays with improved sensitivity and minimal sample size. However, traditional microarray spotting methods typically create probe spot sizes of ∼50-100 µm diameter, comparable to the dimensions of many microfluidic channels. In addition, detection of hybridization events typically requires a post-hybridization labeling step. We address both issues by exploring the use of dip-pen nanolithography (DPN) to pattern linear oligonucleotides and self-reporting molecular beacon (MB) probes on streptavidin-functionalized poly(ethylene glycol) microgel thin-film substrates. In contrast to many systems involving DPN deposition, the fluorescence of the labeled probes enables their amount and spatial distribution to be characterized by optical microscopy. Their deposition rate decreases with increasing DPN dwell time, consistent with a Langmuir adsorption model, but the linear relationship between spot diameter and time(1/2) indicates that spot size is diffusion controlled. We then use DPN to pattern MB probes for the mecA and spa genes in Staphylococcus aureus as a 2-column array with 1 µm spot sizes and 5 µm spot spacings, and we use this array to differentiate targets characteristic of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus. This duplexed self-reporting gel-tethered MB microarray not only shows high specificity but also a high signal-to-background ratio.

Extracellular matrix reorganization during cryo preparation for scanning electron microscope imaging of Staphylococcus aureus biofilms.

Biofilms are three-dimensional communities of bacteria distributed in a highly hydrated extracellular matrix (ECM). They can be visualized by scanning electron microscopy (SEM), but the requisite SEM sample preparation can modify the biofilm morphology. Here, four different approaches to prepare biofilms of hydrated Staphylococcus aureus for SEM imaging are compared. In order of increasing cooling effectiveness these are: (1) drying in air; (2) plunging in liquid nitrogen; (3) plunging in liquid ethane; and (4) high pressure freezing with liquid nitrogen. These different methods give rise to markedly different biofilm morphologies, which are revealed by cryo-SEM imaging. Significantly, high-pressure frozen biofilms exhibit a rich network of nanoscale ECM fibers surrounding individual bacteria throughout the biofilm thickness. This structure is entirely lost when similar biofilms are dried in air, and it is substantially modified when these biofilms are plunged into liquid nitrogen or liquid ethane.

Elasto-plastic effects on shape-shifting electron-beam-patterned gel-based micro-helices.

Shape-shifting helical gels have been created by various routes, notably by photolithography. We explore electron-beam lithography as an alternative to prescribe microhelix formation in tethered patterns of pure poly(acrylic acid). Simulations indicate the nanoscale spatial distribution of deposited energy that drives the loss of acid groups and crosslinking. Upon exposure to buffer, a patterned line converts to a 3D helix whose cross section comprises a crosslinked and hydrophobic core surrounded by a high-swelling pH-responsive corona. Through-thickness asymmetries generate out-of-plane bending to drive helix formation. The relative core and corona fractions are determined by the electron dose which in turn controls the helical radius and pitch. Increasing pH substantially raises the swelling stress and the rod elongates plastically. The pitch concurrently changes from minimal to non-minimal. The in-plane asymmetry driving this change can be attributed to shear-band formation in the hydrophobic core. Subsequent pH cycling drives elastic cycling of the helical properties. These findings illustrate the effects of elastoplastic deformation on helical properties and elaborate unique attributes of electron lithography as an alternate means to create shape-shifting structures.

The 2023 Orthopedic Research Society's international consensus meeting on musculoskeletal infection: Summary from the in vitro section.

Antimicrobial strategies for musculoskeletal infections are typically first developed with in vitro models. The In Vitro Section of the 2023 Orthopedic Research Society Musculoskeletal Infection international consensus meeting (ICM) probed our state of knowledge of in vitro systems with respect to bacteria and biofilm phenotype, standards, in vitro activity, and the ability to predict in vivo efficacy. A subset of ICM delegates performed systematic reviews on 15 questions and made recommendations and assessment of the level of evidence that were then voted on by 72 ICM delegates. Here, we report recommendations and rationale from the reviews and the results of the internet vote. Only two questions received a ≥90% consensus vote, emphasizing the disparate approaches and lack of established consensus for in vitro modeling and interpretation of results. Comments on knowledge gaps and the need for further research on these critical MSKI questions are included.

An in vitro model of microbial contamination in the operating room.

Infection associated with tissue-contacting biomedical devices is a compelling clinical problem initiated by the microbial colonization of the device surface. Among the possible sources of contaminating bacteria is the operating room (OR) itself, where viable bacteria in the atmosphere can sediment onto a device surface intraoperatively. We have developed an aerosolizing system that can reproducibly spray small quantities of aerosolized bacteria onto a surface to mimic OR contamination. This paper describes the design of the system and characterizes key aspects associated with its operation. The area density of sprayed bacteria is on the order of 102 /cm2 . Using titanium (Ti) alloy coupons as test substrates contaminated by staphylococci, we quantify the fraction of bacteria that are well adhered to the substrate, those that can be removed by sonication, and those that are not recovered after spraying. Despite the relatively low levels of surface contamination, we furthermore show that such a model is able to demonstrate a statistically significant reduction in colonization of Ti coupons modified by antimicrobial quaternary ammonium compounds relative to unmodified controls.

Self-Defensive Antimicrobial Surfaces Using Polymyxin-Loaded Poly(styrene sulfonate) Microgels.

Self-defensive antimicrobial surfaces are of interest because they can inhibit bacterial colonization while minimizing unnecessary antimicrobial release in the absence of a bacterial challenge. One self-defensive approach uses self-assembly to first deposit a submonolayer coating of polyelectrolyte microgels and subsequently load those microgels by complexation with small-molecule antimicrobials. The microgel/antimicrobial complexation strength is a key parameter that controls the ability of the antimicrobial both to remain sequestered within the microgels when exposed to medium and to release in response to a bacterial challenge. Here we study the relative complexation strengths of two FDA-approved cationic antibiotics─colistin (polymyxin E) and polymyxin B─with microgels of poly(styrene sulfonate) (PSS). These polymyxins are similar cyclic polypeptides with +5 charge at pH 7.4. However, polymyxin B substitutes an aromatic ring for a dimethyl moiety in colistin, and this aromaticity can influence complexation via π and hydrophobic interactions. Coarse-grained molecular dynamics shows that the free-energy change associated with polymyxin B/PSS complexation is more negative than that of colistin/PSS complexation. Experimentally, in situ optical microscopy of microgel deswelling shows that both antibiotics load quickly from low-ionic-strength phosphate buffer. The enhanced polymyxin B/PSS complexation strength is then manifested by subsequent exposure to flowing antibiotic-free buffer with varying NaCl concentration. Microgels loaded with polymyxin B remain stably deswollen to higher salt concentrations than do colistin/PSS microgels. Importantly, exposing loaded microgels to E. coli in nutrient-free-flowing phosphate buffer shows that bacteria are killed by physical contact with the loaded microgels consistent with the contact-transfer mechanism of self-defensiveness. In vitro culture experiments show that these same surfaces, nevertheless, support the adhesion, spreading and proliferation of human fetal osteoblasts. These findings suggest a pathway to create a self-defensive antimicrobial surface effective under physiological conditions based on the nonmetabolic bacteria-triggered release of FDA-approved antibiotics.

Polymer multilayers with pH-triggered release of antibacterial agents.

We report on the layer-by-layer design principles of poly(methacrylic acid) (PMAA) ultrathin hydrogel coatings that release antimicrobial agents (AmAs) in response to pH variations. The studied AmAs include gentamicin and an antibacterial cationic peptide L5. Adipic acid dihydrazide (AADH) is a cross-linker which, relative to ethylenediamine (EDA), increases the hydrogel hydrophobicity and introduces centers for hydrogen bonding to AmAs. AmA retention in AADH-cross-linked hydrogels in high-salt solutions was enhanced while AmA release at low pH was suppressed. L5 retains its antibacterial activity toward planktonic Staphylococcus epidermidis after release from PMAA hydrogels in response to pH decreases in the surrounding medium due to bacterial growth. Staphylococcus epidermidis adhesion and colonization was almost completely inhibited by L5 loading of hydrogels. The AmA-releasing and AmA-retaining properties of these hydrogel coatings provide new opportunities to study the fundamental mechanisms of AmA-coating-bacteria interactions and develop a new class of clinically relevant antibacterial coatings for medical devices.

Self-defensive antimicrobial biomaterial surfaces.

Self-defensive biomaterial surfaces are being developed in order to mitigate infection associated with tissue-contacting biomedical devices. Such infection occurs when microbes colonize the surface of a device and proliferate into a recalcitrant biofilm. A key intervention point centers on preventing the initial colonization. Incorporating antimicrobials within a surface coating can be very effective, but the traditional means of antimicrobial delivery by continuous elution can often be counterproductive. If there is no infection, continuous elution creates conditions that promote the development of resistant microbes throughout the patient. In contrast, a self-defensive coating releases antimicrobial only when and only where there is a microbial challenge to the surface. Otherwise, the antimicrobial remains sequestered within the coating and does not contribute to the development of resistance. A self-defensive surface requires a local trigger that signals the microbial challenge. Three such triggers have been identified as: (1) local pH lowering; (2) local enzyme release; and (3) direct microbial-surface contact. This short review highlights the need for self-defensive surfaces in the general context of the device-infection problem and then reviews key biomaterials developments associated with each of these three triggering mechanisms.

Poly-N-acetylglucosamine matrix polysaccharide impedes fluid convection and transport of the cationic surfactant cetylpyridinium chloride through bacterial biofilms.

Biofilms are composed of bacterial cells encased in a self-synthesized, extracellular polymeric matrix. Poly-beta(1,6)-N-acetyl-d-glucosamine (PNAG) is a major biofilm matrix component in phylogenetically diverse bacteria. In this study we investigated the physical and chemical properties of the PNAG matrix in biofilms produced in vitro by the gram-negative porcine respiratory pathogen Actinobacillus pleuropneumoniae and the gram-positive device-associated pathogen Staphylococcus epidermidis. The effect of PNAG on bulk fluid flow was determined by measuring the rate of fluid convection through biofilms cultured in centrifugal filter devices. The rate of fluid convection was significantly higher in biofilms cultured in the presence of the PNAG-degrading enzyme dispersin B than in biofilms cultured without the enzyme, indicating that PNAG decreases bulk fluid flow. PNAG also blocked transport of the quaternary ammonium compound cetylpyridinium chloride (CPC) through the biofilms. Binding of CPC to biofilms further impeded fluid convection and blocked transport of the azo dye Allura red. Bioactive CPC was efficiently eluted from biofilms by treatment with 1 M sodium chloride. Taken together, these findings suggest that CPC reacts directly with the PNAG matrix and alters its physical and chemical properties. Our results indicate that PNAG plays an important role in controlling the physiological state of biofilms and may contribute to additional biofilm-associated processes such as biocide resistance.

Biomaterial surfaces self-defensive against bacteria by contact transfer of antimicrobials.

Despite extensive engineering of tissue-contacting biomedical devices to control healing, these devices remain susceptible to bacterial colonization, biofilm formation, and chronic infection. The threat of selecting for resistance genes largely precludes sustained antimicrobial elution as a wide-spread clinical solution. In response, self-defensive surfaces have been developed where antimicrobial is released only when and where there is a bacterial challenge. We explore a new self-defensive approach using anionic microgels into which small-molecule cationic antimicrobials are loaded by complexation. We identify conditions where antimicrobial remains sequestered within the microgels for periods as long as weeks. However, bacterial contact triggers release and leads to local bacterial killing. We speculate that the close proximity of bacteria alters the local thermodynamic environment and interferes with the microgel-antimicrobial complexation. The contact-transfer approach does not require bacterial metabolism but instead appears to be driven by differences between the microgels and the bacterial cell envelope where there is a high concentration of negative charge and hydrophobicity. Contact with metabolizing macrophages or osteoblasts is, however, insufficient to trigger antimicrobial release, indicating that contact transfer can be specific to bacteria and suggesting an avenue to biomedical device surfaces that can simultaneously promote healing and resist infection.

Functional Changes during Electron-Beam Lithography of Biotinylated Poly(ethylene glycol) Thin Films.

In contrast to photolithography where particular wavelengths of light can couple to specific photochemistries, electron-beam lithography can drive competing chemistries. To separate surface-grafting, cross-linking, and chemical functionality, we studied the effects of 2 keV electrons on thin films of poly(ethylene glycol) end-functionalized with hydroxyls (PEG-OH) or biotins (PEG-B). Similarities in the dose-dependent thickness changes of the patterned PEGs indicate that surface grafting and cross-linking primarily involve the ethylene oxide main chain. While higher doses create thicker patterns with more biotin, the concurrent increase in thiol reactivity indicates that cross-linking competes with biotin degradation. The dose window for optimal e-beam patterning of biotinylated PEG is very narrow. Biotin is entirely consumed at higher doses. Its modified functionality is reactive with 5-((2-(and-3)-S-(acetylmercapto) succinoyl) amino) (SAMSA). This effect creates a dose-dependent orthogonal functionality that can be patterned from a single precursor thin film.