In vitro synthesis of native, fibrous long spacing and segmental long spacing collagen.

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

Combinatorial polyelectrolyte multilayer film fabrication.

A combinatorial strategy for the fabrication of a library of polyelectrolyte multilayer films is presented in this paper. This innovative approach involves the parallel formation of polyelectrolyte multilayer films in the individual wells of polystyrene microtitre plates under various deposition conditions. The progress of film formation was monitored via the intensity of the UV-vis absorbance of one of the depositing polyelectrolytes using a conventional microplate reader. We demonstrate the utility of this technique by building a library of 120 distinct polyelectrolyte multilayer films. Both the primer layer composition and salt content of the polyeletrolyte solutions were systematically varied in the preparation of films of nine bilayers. Film growth did not follow a linear adsorption regime for the first three bilayers regardless of the composition of the primer layer. We observed that increasing the sodium chloride concentration in the polyelectrolyte solutions resulted in increased polyelectrolyte absorption for all the conditions studied. The approach presented here is a convenient method of producing and characterizing multiple films rapidly and reproducibly, making it a valuable tool for optimizing film fabrication.

Potassium ion mediated collagen microfibril assembly on mica.

Potassium ion can critically effect the interaction between collagen microfibrils and mica leading to different ordered structures that vary dramatically with changing ion concentration. AFM images of the structures formed at different ion concentrations appear to be intermediate stages in the progression from disordered to ordered film. At 200 mM potassium ion concentration, a nanometer-thick array of aligned and bundled microfibrils covering large areas can be created easily and reproducibly on mica.

An enzyme-amplified diffraction-based immunoassay.

We have previously shown that a gold-conjugated secondary label can be used to reduce the limit of detection in a diffraction-based assay by more than 40-fold. We now show that by using a combination of a peroxidase-conjugated secondary label and a precipitating substrate the limit of detection in a diffraction-based assay can be reduced by more than 1000-fold. The response to secondary enhancement was linear for concentrations from 50 to 2000 pg/mL of antidigoxin.

A quantitative diffraction-based sandwich immunoassay.

It is shown that diffraction-based sensing can be enhanced for diagnostic purposes through the use of a secondary label. The limit of detection for anti-rabbit IgG was reduced more than 40-fold by using a gold-conjugated secondary antibody. The response to secondary antibody binding was linear for concentrations from 25 to 500 ng/ml of anti-rabbit IgG, suggesting that quantitative determinations can be readily done. Moreover, the binding of the secondary antibody was observed as soon as 1 min after its introduction to the surface-bound primary complex.

Diffraction-based assay for detecting multiple analytes.

The principles of diffraction are utilized to enable the simultaneous detection of multiple analytes in solution, forming the basis of a multi-analyte sensor. Probe molecules are immobilized on a substrate such that each type of molecule defines a specific pattern within the same region of substrate. The binding of a target molecule to its complementary probe is heralded by a characteristic diffraction image. This principle is demonstrated using antibody conjugates.