Real-time femtomolar detection of cancer biomarkers from photoconjugated antibody-phage constructs.

Here we describe novel covalent conjugates of antibody-phage for the detection of multiple cancer biomarkers using real time immuno-polymerase chain reaction (immuno-PCR). While the conventional process of immuno-PCR utilizes DNA-conjugated antibodies, chemical modification of antibodies not only reduces antibody affinity but also creates a heterogeneous population of products. However, phage naturally encapsulate genomic DNA, which can be used as a PCR template. To produce covalently conjugated antibody-phage constructs without recombinant antibody expression or chemical modification of antibodies, we incorporated a photocrosslinkable non-canonical amino acid within an antibody-binding domain displayed on one of the phage coat proteins. To correlate antigen presence to a specific DNA sequence, the phage genomes were modified with domains that recognized specific sets of primers. The crosslinked antibody-phage conjugates were then tested in a sandwich-type immunoassay using real-time PCR where low pg ml-1 concentrations of antigen could be detected and identified from a single solution containing a mixture of three different types of cancer biomarkers.

Measuring cellular forces using bis-aliphatic hydrazone crosslinked stress-relaxing hydrogels.

Studies focused on understanding the role of matrix biophysical signals on cells, especially those when cells are encapsulated in hydrogels that are locally remodelled, are often complicated by appropriate methods to measure differences between the bulk and local material properties. From this perspective, stress-relaxing materials that allow long-term culture of embedded cells provide an opportunity to elucidate aspects of this biophysical signalling. In particular, rheological characterization of the stress relaxation properties allows one to link a bulk material measurement to local aspects of cellular functions by quantifying the corresponding cellular forces that must be applied locally. Here, embryonic stem cell-derived motor neurons were encapsulated in a well-characterized covalently adaptable bis-aliphatic hydrazone crosslinked PEG hydrogel, and neurite outgrowth was observed over time. Using fundamental physical relationships describing classical mechanics and viscoelastic materials, we calculated the forces and energies involved in neurite extension, the results of which provide insight to the role of biophysical cues on this process.

Biomimetic synthesis of ordered silica structures mediated by block copolypeptides.

In biological systems such as diatoms and sponges, the formation of solid silica structures with precisely controlled morphologies is directed by proteins and polysaccharides and occurs in water at neutral pH and ambient temperature. Laboratory methods, in contrast, have to rely on extreme pH conditions and/or surfactants to induce the condensation of silica precursors into specific morphologies or patterned structures. This contrast in processing conditions and the growing demand for benign synthesis methods that minimize adverse environmental effects have spurred much interest in biomimetic approaches in materials science. The recent demonstration that silicatein-a protein found in the silica spicules of the sponge Tethya aurantia--can hydrolyse and condense the precursor molecule tetraethoxysilane to form silica structures with controlled shapes at ambient conditions seems particularly promising in this context. Here we describe synthetic cysteine-lysine block copolypeptides that mimic the properties of silicatein: the copolypeptides self-assemble into structured aggregates that hydrolyse tetraethoxysilane while simultaneously directing the formation of ordered silica morphologies. We find that oxidation of the cysteine sulphydryl groups, which is known to affect the assembly of the block copolypeptide, allows us to produce different structures: hard silica spheres and well-defined columns of amorphous silica are produced using the fully reduced and the oxidized forms of the copolymer, respectively.

Silicatein filaments and subunits from a marine sponge direct the polymerization of silica and silicones in vitro.

Nanoscale control of the polymerization of silicon and oxygen determines the structures and properties of a wide range of siloxane-based materials, including glasses, ceramics, mesoporous molecular sieves and catalysts, elastomers, resins, insulators, optical coatings, and photoluminescent polymers. In contrast to anthropogenic and geological syntheses of these materials that require extremes of temperature, pressure, or pH, living systems produce a remarkable diversity of nanostructured silicates at ambient temperatures and pressures and at near-neutral pH. We show here that the protein filaments and their constituent subunits comprising the axial cores of silica spicules in a marine sponge chemically and spatially direct the polymerization of silica and silicone polymer networks from the corresponding alkoxide substrates in vitro, under conditions in which such syntheses otherwise require either an acid or base catalyst. Homology of the principal protein to the well known enzyme cathepsin L points to a possible reaction mechanism that is supported by recent site-directed mutagenesis experiments. The catalytic activity of the "silicatein" (silica protein) molecule suggests new routes to the synthesis of silicon-based materials.