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Micromechanical bending experiments using atomic force microscopy were performed to study the mechanical properties of native and carbodiimide-cross-linked single collagen fibrils. Fibrils obtained from a suspension of insoluble collagen type I isolated from bovine Achilles tendon were deposited on a glass substrate containing microchannels. Force-displacement curves recorded at multiple positions along the collagen fibril were used to assess the bending modulus. By fitting the slope of the force-displacement curves recorded at ambient conditions to a model describing the bending of a rod, bending moduli ranging from 1.0 GPa to 3.9 GPa were determined. From a model for anisotropic materials, the shear modulus of the fibril is calculated to be 33 +/- 2 MPa at ambient conditions. When fibrils are immersed in phosphate-buffered saline, their bending and shear modulus decrease to 0.07-0.17 GPa and 2.9 +/- 0.3 MPa, respectively. The two orders of magnitude lower shear modulus compared with the Young's modulus confirms the mechanical anisotropy of the collagen single fibrils. Cross-linking the collagen fibrils with a water-soluble carbodiimide did not significantly affect the bending modulus. The shear modulus of these fibrils, however, changed to 74 +/- 7 MPa at ambient conditions and to 3.4 +/- 0.2 MPa in phosphate-buffered saline.

A new micromechanical technique was developed to study the mechanical properties of single collagen fibrils. Single collagen fibrils, the basic components of the collagen fiber, have a characteristic highly organized structure. Fibrils were isolated from collagenous materials and their mechanical properties were studied with atomic force microscopy (AFM). In this study, we determined the Young's modulus of single collagen fibrils at ambient conditions from bending tests after depositing the fibrils on a poly(dimethyl siloxane) (PDMS) substrate containing micro-channels. Force-indentation relationships of freely suspended collagen fibrils were determined by loading them with a tip-less cantilever. From the deflection-piezo displacement curve, force-indentation curves could be deduced. With the assumption that the behavior of collagen fibrils can be described by the linear elastic theory of isotropic materials and that the fibrils are freely supported at the rims, a Young's modulus of 5.4 +/- 1.2 GPa was determined. After cross-linking with glutaraldehyde, the Young's modulus of a single fibril increases to 14.7 +/- 2.7 GPa. When it is assumed that the fibril would be fixed at the ends of the channel the Young's moduli of native and cross-linked collagen fibrils are calculated to be 1.4 +/- 0.3 GPa and 3.8 +/- 0.8 GPa, respectively. The minimum and maximum values determined for native and glutaraldehyde cross-linked collagen fibrils represent the boundaries of the Young's modulus.

Adopting supramolecular chemistry for immobilization of proteins is an attractive strategy that entails reversibility and responsiveness to stimuli. The reversible and oriented immobilization and micropatterning of ferrocene-tagged yellow fluorescent proteins (Fc-YFPs) onto β-cyclodextrin (βCD) molecular printboards was characterized using surface plasmon resonance (SPR) spectroscopy and fluorescence microscopy in combination with electrochemistry. The proteins were assembled on the surface through the specific supramolecular host-guest interaction between βCD and ferrocene. Application of a dynamic covalent disulfide lock between two YFP proteins resulted in a switch from monovalent to divalent ferrocene interactions with the βCD surface, yielding a more stable protein immobilization. The SPR titration data for the protein immobilization were fitted to a 1:1 Langmuir-type model, yielding K(LM) = 2.5 × 10(5) M(-1) and K(i,s) = 1.2 × 10(3) M(-1), which compares favorably to the intrinsic binding constant presented in the literature for the monovalent interaction of ferrocene with βCD self-assembled monolayers. In addition, the SPR binding experiments were qualitatively simulated, confirming the binding of Fc-YFP in both divalent and monovalent fashion to the βCD monolayers. The Fc-YFPs could be patterned on βCD surfaces in uniform monolayers, as revealed using fluorescence microscopy and atomic force microscopy measurements. Both fluorescence microscopy imaging and SPR measurements were carried out with the in situ capability to perform cyclic voltammetry and chronoamperometry. These studies emphasize the repetitive desorption and adsorption of the ferrocene-tagged proteins from the βCD surface upon electrochemical oxidation and reduction, respectively.

Biological molecules such as proteins and oligonucleotides are the greatest source of inspiration for supramolecular chemists.[1] With the increased knowledge about supramolecular architectures in water, [2] applications of synthetic supramolecular architectures to investigate the biological molecules that initially inspired their design has become possible. The combination of synthetic supramolecular systems with proteins, for example, allows the control and study of these proteins.[3] The combination of designed supramolecular interactions with proteins allows for a powerful application of recognition motifs that are bio-orthogonal and thus provides an independent opportunity to modulate proteins. An important target in the field of protein nanotechnology, for example, is control over protein immobilization onto surfaces for diverse biochemical and biomedical applications.[4] Immobilization approaches based on biological supramolecular recognition motifs have been reported in recent years, such as DNA-protein interactions, [5] NiNTA-His 6 -tagged protein systems, [6] and the biotin and avidin interaction.[7] Supramolecular protein immobilization through synthetic supramolecular elements such as adamantane [8] and ferrocene [9] has also been reported; it relies on multivalent interactions of randomly and multiply attached supramolecules. However, a simple, strong, reversible, and monovalent supramolecular protein immobilization strategy is still required. A bio-orthogonal and monovalent supramolecularprotein system would allow for a high level of control over the homogeneity of the protein monolayers and site specificity of immobilization, whilst also displaying controllable binding kinetics.Herein we demonstrate how a single, synthetic supramolecular host-guest interaction allows for stable, but reversible, site-selective, monovalent protein immobilization on a self-assembled monolayer. This work utilizes the site-selective incorporation of ferrocene into a protein followed by immobilization onto a cucurbit [7]uril (CB[7]) monolayer. The technique allows printing of stable protein monolayers in well-defined formats to be achieved with controlled protein orientation and with subsequent replacement of the protein monolayer by a small synthetic ligand. The scope of this new immobilization strategy was investigated by using X-ray photoelectron (XPS) and Fourier transform IR reflection absorption (IR-RAS) spectroscopy, surface plasmon resonance (SPR), cyclic voltammetry (CV), atomic force (AFM) and fluorescence microscopy (FM).We used expressed protein ligation as a site-selective protein modification technique to insert a single supramolecular guest molecule.[10] This allows for the generation of a homogenously labeled protein and avoids random and multiple tagging of proteins, with concomitant problems in protein immobilization. The supramolecular guest (ferrocenylmethyl)trimethyl-ammonium iodide features a strong binding affinity of K % 10 11 m À1 to CB [7] in solution.[11] Consequently, we selected the protonata...