Vamsi K. Yadavalli

Controlled Molecular Release Using Nanoporous Alumina Capsules

The use of mechanically robust nanoporous alumina capsules, with highly uniform pores of 25 nm to 55 nm, for controled drug delivery is demonstrated. The nanoporous alumina capsules were fabricated by anodization of an aluminum tube, resulting in a highly uniform, large surface area, relatively inexpensive device suitable for biofiltration applications. Characterization of diffusion from the nanoporous capsules using fluorescein isothiocyanate and dextran conjugates of varying molecular weight, showed that molecular transport could be readily controlled by selection of capsule pore size. A branched membrane structure, with a stepwise change in pore size from large to small, is used to provide small pore-sized membranes with sufficient mechanical strength for handling.

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).

Effect of substrate stiffness on early human embryonic stem cell differentiation

BackgroundThe pluripotency and self renewing properties of human embryonic stem cells (hESC) make them a valuable tool in the fields of developmental biology, pharmacology and regenerative medicine. Therefore, there exists immense interest in devising strategies for hESC propagation and differentiation. Methods involving simulation of the native stem cell microenvironment, both chemical and physical, have received a lot of attention in recent years. Equally important is evidence that cells can also sense the mechanical properties of their microenvironment. In this study, we test the hypothesis that hESCs accept mechanical cues for differentiation from the substrate by culturing them on flexible polydimethylsiloxane (PDMS) of varying stiffness.ResultsPDMS substrates were prepared using available commercial formulations and characterized for stiffness, surface properties and efficiency of cell attachment and proliferation. Across different substrate stiffness, cell numbers, cell attachment and cell surface area were found to be similar. Expression of pluripotency markers decreased with increased time in culture across all PDMS substrates of varying stiffness. Analysis of gene expression of differentiation markers indicates that the differentiation process becomes less stochastic with longer culture times.ConclusionsWe evaluated the utility of PDMS substrates for stem cell propagation and substrate mediated differentiation. The stiffness affected gene expression of pluripotent and differentiation markers with results indicating that these substrate systems could potentially be used to direct hESC fate towards early mesodermal lineages. This study suggests that coupled with soluble factors, PDMS substrates could potentially be useful in generating defined populations of differentiated cells.

Conducting polymer-silk biocomposites for flexible and biodegradable electrochemical sensors.

UNLABELLED Approaches to form flexible biosensors require strategies to tune materials for various biomedical applications. We report a facile approach using photolithography to fabricate poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) ( PEDOT PSS) sensors on a fully biodegradable and flexible silk protein fibroin support. A benchtop photolithographic setup is used to fabricate high fidelity and high resolution PEDOT PSS microstructures over a large (cm) area using only water as the solvent. Using the conductive micropatterns as working electrodes, we demonstrate biosensors with excellent electrochemical activity and stability over a number of days. The fabricated biosensors display excellent nonspecific detection of dopamine and ascorbic acid with high sensitivity. These devices are mechanically flexible, optically transparent, electroactive, cytocompatible and biodegradable. The benign fabrication protocol allows the conducting ink to function as a matrix for enzymes as shown by a highly sensitive detection of glucose. These sensors can retain their properties under repeated mechanical deformations, but are completely degradable under enzymatic action. The reported technique is scalable and can be used to develop sensitive, robust, and inexpensive biosensors with controllable biodegradability, leading to applications in transient or implantable bioelectronics and optoelectronics.

Surface immobilization of DNA aptamers for biosensing and protein interaction analysis.

To utilize aptamers as molecular recognition agents in biosensors and biodiagnostics, it is important to develop strategies for reliable immobilization of aptamers so that they retain their biophysical characteristics and binding abilities. Here we report on quartz crystal microbalance (QCM) measurements and atomic force microscope (AFM)-based force spectroscopy studies to evaluate aptasensors fabricated by different modification strategies. Gold surfaces were modified with mixed self assembled monolayers (SAMs) of aptamer and oligoethylene glycol (OEG) thiols (HS-C(11)-(EG)(n)OH, n=3 or 6) to impart resistance to nonspecific protein adsorption. By affinity analysis, we show that short OEG thiols have less impact on aptamer accessibility than longer chain thiols. Backfilling with OEG as a step subsequent to aptamer immobilization provides greater surface coverage than using aptamer and OEG thiol to form a mixed SAM in one-step. Immunoglobulin E and vascular endothelial growth factor (VEGF) were studied as target proteins in these experiments. Binding forces obtained by these strategies are similar, demonstrating that the biophysical properties of the aptamer on the sensors are independent from the immobilization strategy. The results present mixed SAMs with aptamers and co-adsorbents as a versatile strategy for aptamer sensor platforms including ultrasensitive biosensor design.

The fractal self-assembly of the silk protein sericin

Silk consists primarily of two proteins—a fibrous core protein, fibroin, and a glue protein, sericin, which envelops the fibroin fibers with sticky layers thus helping in the formation of cocoon, achieved by cementing the silk fibers together. Sericin, a water soluble protein, has traditionally been discarded in silk processing, despite great potential for use as a biomaterial. Here we show that this glue protein, sericin, has the ability to form self-assembled nano and microstructures with hierarchical self-similarity across length scales in the form of a diffusion-limited, fractal assembly. Sericin obtained from two silkworms, a domesticated mulberry Bombyx mori and a wild non-mulberry Antheraea mylitta, were studied, with particular insight into its structure and morphology as it dried on a surface. High-resolution atomic force microscopy was used to image the self-assembled protein, with investigations on the various factors that influenced this process. We describe the self-assembly patterns formed by the sericin protein and analyze the images based on the theory of diffusion limited aggregation to explain the fractal nature of these architectures observed. The unique physical behavior and fractal nature of this glue protein may represent a key step in understanding its biological relevance as well as the role it plays in the assembly and formation of silks.

Nanomechanical measurements of polyethylene glycol hydrogels using atomic force microscopy.

Poly(ethylene glycol) (PEG)-based hydrogels are among the most widely used synthetic polymers for biomedical applications. Critical parameters of importance for PEG hydrogels are their mechanical properties which can be highly tuned. While properties such as elastic moduli have been measured at the bulk scale, it is often important to measure them at the micro and nanoscales. Further, non-destructive measurements of material properties can enable in situ and high-throughput monitoring for applications including modulating cellular interactions. In this research, the elastic modulus and the stiffness of polyethylene glycol diacrylate (PEG-DA) hydrogel matrices at the nanoscale are determined via nanoindentation using an atomic force microscope (AFM). The effect of varying parameters including monomer molecular weight, initiator concentration and rates of hydration on the mechanical strength of photopolymerized hydrogels were investigated. We present the effects of indentation parameters including loads and indent depths on such measurements. Mechanical characteristics of versatile PEG hydrogels can be adjusted based on polymer chain length and crosslinking, while completely hydrated hydrogels have mechanical properties similar to articular cartilage. A better understanding of these properties can enable tailoring hydrogel based biomaterials for various applications in scaffolds and tissue engineering.

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.

Nanoscale measurements of the assembly of collagen to fibrils

Observing the self-assembly of collagen from single collagen monomers to higher order fibrils and fibers provides a bottom-up approach to engineering its ultrastructure in comparison to structural studies of already formed collagen fibers. This approach can be used for the fabrication of controlled collagen-based biomaterials with varying mechanical properties. Here, we investigate the time-dependent self-assembly of collagen into single fibrils in vitro through high resolution imaging of collagen type 1 prior to fibrillogenesis. This was confirmed by comparing persistence length and diameter in controlled experiments and studying the morphology and mechanical properties of nanoscale collagen fibrils through AFM nanoindentation measurements. The Youngs modulus of these collagen fibrils was estimated to be around 1GPa in the dehydrated state. The stability and mechanical characteristics of collagen obtained in these experiments indicate the hierarchical assembly occurs at both a structural and mechanical level.