Nicholas E. Kurland

Measurement of nanomechanical properties of biomolecules using atomic force microscopy

The capabilities of atomic force microscopy (AFM) have been rapidly expanding beyond topographical imaging to now allow for the analysis of a wide range of properties of diverse materials. The technique of nanoindentation, traditionally performed via dedicated indenters can now be reliably achieved using AFM instrumentation, enabling mechanical property determination at the nanoscale using the high spatial and force resolutions of the AFM. In the study of biological systems, from biomolecules to complexes, this technique provides insight into how mesoscale properties and functions may arise from a myriad of single biomolecules. In vivo and in situ analyses of native structures under physiological conditions as well as the rapid analysis of molecular species under a variety of experimental treatments are made possible with this technique. As a result, AFM nanoindentation has emerged as a critical tool for the study of biological systems in their natural state, further contributing to both biomaterial design and pharmacological research. In this review, we detail the theory and progression of AFM-based nanoindentation, and present several applications of this technique as it has been used to probe biomolecules and biological nanostructures from single proteins to complex assemblies. We further detail the many challenges associated with mechanical models and required assumptions for model validity. AFM nanoindentation capabilities have provided an excellent improvement over conventional nanomechanical tools and by integration of topographical data from imaging, enabled the rapid extraction and presentation of mechanical data for biological samples.

Investigations of Chemical Modifications of Amino-Terminated Organic Films on Silicon Substrates and Controlled Protein Immobilization

Fourier transform infrared spectroscopy by grazing-angle attenuated total reflection (FTIR-GATR), ellipsometry, atomic force microscopy (AFM), UV-visible spectroscopy, and fluorescence microscopy were employed to investigate chemical modifications of amino-terminated organic thin films on silicon substrates, protein immobilization, and the biological activity and hydrolytic stability of immobilized proteins. Amino-terminated organic films were prepared on silicon wafers by self-assembling 3-aminopropyltriethoxysilane (APTES) in anhydrous toluene. Surface amino groups were derivatized into three different linkers: N-hydroxysuccinimide (NHS) ester, hydrazide, and maleimide ester groups. UV-visible absorption measurements and fluorescence microscopy revealed that more than 40% of surface amino groups were chemically modified. Protein immobilization was carried out on modified APTES films containing these linkers via coupling with primary amines (-NH(2)) in intact monoclonal rabbit immunoglobulin G (IgG), the aldehyde (-CHO) of an oxidized carbohydrate residue in IgG, or the sulfhydryl (-SH) of fragmented half-IgG, respectively. FTIR spectra contain vibrational signatures of these functional groups present in modified APTES films and immobilized IgGs. Changes in the APTES film thickness after chemical modifications and protein immobilization were also observed by ellipsometric measurements. The biological activity and long-term hydrolytic stability of immobilized IgGs on modified APTES films were estimated by fluorescence measurements of an adsorbed antigen, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (FITC-Ab). Our results indicate that the FITC-Ab binding capacity of half-IgG immobilized via maleimide groups is greater than that of the oxidized IgG and the intact IgG immobilized via hydrazide and NHS ester groups, respectively. In addition, IgGs immobilized using all coupling chemistries were hydrolytically stable in phosphate-buffered saline (PBS).

Isolation and processing of silk proteins for biomedical applications

Silk proteins of silkworms are chiefly composed of core fibroin protein and glycoprotein sericin that glues fibroin. Unique mechanical properties, cyto-compatibility and controllable biodegradability facilitate the use of fibroin in biomedical applications. Sericin serves as additive in cosmetic and food industries, as mitotic factor in cell culture media, anti-cancerous drug, anticoagulant and as biocompatible coating. For all these uses; aqueous solutions of silk proteins are preferred. Therefore, an accurate understanding of extraction procedure of silk proteins from their sources is critical. A number of protocols exist, amongst which it is required to settle a precise and easy one with desired yield and least down-stream processing. Here, we report extraction of proteins employing methods mentioned in literature using cocoons of mulberry and nonmulberry silks. This study reveals sodium carbonate salt-boiling system is the most efficient sericin extraction procedure for all silk variants. Lithium bromide is observed as the effective fibroin dissolution system for mulberry silk cocoons; whereas heterogeneous species-dependent result is obtained in case of nonmulberry species. We further show the effect of common post processing on nanoscale morphology of mulberry silk fibroin films. This knowledge eases the adoption and fabrication of silk biomaterials in devices and therapeutic delivery systems.

Biopatterning of Silk Proteins for Soft Micro-optics

Silk proteins from spiders and silkworms have been proposed as outstanding candidates for soft micro-optic and photonic applications because of their optical transparency, unique biological properties, and mechanical robustness. Here, we present a method to form microstructures of the two constituent silk proteins, fibroin and sericin for use as an optical biomaterial. Using photolithography, chemically modified silk protein photoresists are patterned in 2D arrays of periodic patterns and Fresnel zone plates. Angle-dependent iridescent colors are produced in these periodic micropatterns because of the Bragg diffraction. Silk protein photolithography can used to form patterns on different substrates including flexible sheets with features of any shape with high fidelity and resolution over large areas. Finally, we show that these mechanically stable and transparent iridescent architectures are also completely biodegradable. This versatile and scalable technique can therefore be used to develop biocompatible, soft micro-optic devices that can be degraded in a controlled manner.

Self-assembly mechanisms of silk protein nanostructures on two-dimensional surfaces

Self-assembly processes are ubiquitous in natural systems, and their study can provide insight into the harnessing of unique properties for engineering of new materials from the bottom up. Models for diffusion-limited assembly behavior have shown that structures formed have a characteristic fractal dimensionality that is smaller than the embedding space or the lattice. Typically however, these processes have only been studied via theoretical and computational tools, with relatively few natural systems having been reported to approach the limiting conditions assumed. Sericin, a protein critical to silk macrostructure, displays the remarkable ability to self-assemble through different modes of classical and non-universal diffusion-limited aggregation to produce radially-branched dendritic architectures. We report on the characterization of these assemblies by pure proteins from different species of silkworms in the absence of any charge shielding or modulation by salts. It is shown how physical differences between colloidal systems can yield remarkable changes in branching architectures from proteins that are functionally similar. This represents a novel system for fundamental and applied studies of particle aggregation and the development of biomaterials based on self-similarity at multiple length scales.

pH responsive poly amino-acid hydrogels formed via silk sericin templating.

Poly(amino acid) hydrogels have attracted a great deal of attention as biodegradable biomaterials that can limit products of synthetic polymer degradation. Here we report on a stimuli-responsive, porous, composite biomaterial based on the protein templating of the poly(amino acid) hydrogel from poly(aspartic acid) with the silk protein sericin. This low-cost, biocompatible and biodegradable hydrogel demonstrates a greatly increased porosity and improvement in volumetric swelling over networks formed from pure poly(aspartic acid). The swelling capacity measured over a range of pH values surrounding physiological pH 7.0 demonstrates a linear profile, in which hydrogel volume and mass increase to a maximum, with an increase as a function of higher sericin content. In comparison to pure poly(aspartic acid), this demonstrates a nearly 3-fold increase in retention volume at basic pH. The increase in swelling is also demonstrated by the increase in porosity and internal micro-architecture of the hydrogel networks. The biomaterial is then shown to perform well as a scaffold for cells with high mechanical strength and integrity. This protein- and homo poly(amino acid)-based super-swelling hydrogel has applications in drug delivery and tissue engineering as an economical and environmentally friendly biomaterial, in addition to ensuring the species incorporated maintain their biocompatibility during processing.

Silk nanostructures based on natural and engineered self-assembly

Abstract: Self-assembly processes are ubiquitous in natural systems, and can enable the harnessing of unique properties for engineering new materials from the bottom up. Here we present the current understanding on the self-assembly of silks as exhibited by various silk protein solutions, notably those of silkworms and orb-weaving spiders. Such proteins have been employed to form a variety of nanostructures ranging from random fractal assemblies to well-defined nanoparticles. We discuss mechanisms of natural and engineered self-assembly in silk proteins (native fibroin, sericin and recombinant proteins) and focus on self-assembly research undertaking the fabrication of diverse architectures along with their potential applications.