This contribution summarizes some strategies that we followed for the optimization of supramolecular architectures aiming at controlling the binding and organization of biopolymers at solid/solution-interfaces in a membrane-mimetic approach. The examples given concern mostly self-assembly processes and the biorecognition between biotin and streptavidin.

Coatings of genetically engineered protein polymers based on the crystalline segment of B. Mori silk fibroinand cell binding domains from extracellular matrix proteins (ProNectin™ by Protein Polymer Technologies, Inc.) were applied to bare silicon wafers used to mimic microcircuit devices. These silicon devices have applications in the stimulation and monitoring of central nervous system activity but need bioactive coatings for integration into nervous tissue. Gel point coating technology was developed by preparing solutions at the onset of phase separation in protein polymer/formic acid/ ethanol systems. Dipping silicon wafer substrates into such solutions produced homogeneous thin protein polymer coatings. Quench coating techniques that create rough surfaces in a controlled manner were explored by driving protein polymer solutions through different regions of the protein polymer/formic acid/methanol system before drying. Atomic force microscopy was used to characterize the protein films.

Self-assembled monolayer (SAM) films of organosilanes have been characterized as surfaces for the spatially-controlled, selective attachment of single-stranded synthetic DNA. Geometric patterns of DNA on silica substrates have been created by photochemical modification of the SAMs to produce regions which promote or resist DNA attachment. The attachment of DNA to the modified surfaces was followed using UV spectroscopy, radioassay, ellipsometry, and enzyme-linked colorimetric assay. We have also demonstrated that immobilized DNA is capable of hybridization with a complementary strand under stringent conditions.

The coupling of a photodynamic protein to an electroactive polymer may be employed in opto-electronic signal transduction for optical displays, color mimicking and biosensor applications. We have synthesized a novel biomaterial, biotin-LC-hydrazide substituted poly(3-undecylthiophene-co-3-thienylcarboxaldehyde) [B-PUAT], which is capable of incorporating any biotinylated molecule through a bridging streptavidin protein. A long spacer group between the conjugated polymer backbone and the biotin side group improves monolayer formation at the airwater interface and enhances the material's ability to bind protein. The LB technique has been employed to couple this biomaterial and streptavidin through the well known biotin-streptavidin complexation. Biotinylated phycoerythrin [B-PE] is incorporated into the above monolayer by binding the unoccupied biotin binding sites on the bound streptavidin.

Crystalline cell surface layers (S-layers) represent the outermost cell envelope component in many bacteria. The oblique, square or hexagonal lattices are formed of assemblies of a single protein or glycoprotein species. Isolated S-layer subunits are endowed with the ability to assemble into monomolecular arrays in suspension, on solid surfaces (e.g. metals, polymers, glass, carbon, silicon), at the air/water interface, or on lipid films generated by the Langmuir Blodgett technique. S-layer lattices are isoporous structures with functional groups located on the surface and in the pores in an identical position and orientation. These characteristic features have led to applications of S-layers as (i) ultrafiltration membranes with pores of identical size and morphology and a broad chemical modification potential, (ii) matrices for the controlled and reproducible immobilization of functional macromolecules, as required for affinity and enzyme membranes, affinity microcarriers and biosensors, (iii) carriers for Langmuir-Blodgett films and reconstituted biological membranes, (iv) immobilization matrices and adjuvants for weakly immunogenic antigens and haptens and (v) patterning elements in molecular nanotechnology.

The formation of two-dimensional (2-D) crystals of biological macromolecules is of interest for nanotechnological applications. Protein 2-D crystals may be used as molecular sieves and/or support devices as components of biosensors etc. [1]. Functionally specialized 2-D crystals, containing transport or catalytic proteins, provide a certain function in a highly efficient and vectorial manner. Future developments may allow the design of more complex structures such as multilayers made from different proteins or arrays of functionally linked oligo- or multimeric complexes consisting of multiple protein species [2]. Regular 2-D arrays, either truely crystalline or densely packed molecules, are one of the basic structures taht might be used to construct more sophisticated protein-based devices. 2-D crystals have some interesting features:

Protease-activated triggers have been introduced Into a pore-forming protein, staphylococcal a-hemolysin (αHL). The hemolysin was remodeled by genetic engineering to form two-chain constructs with redundant polypeptide sequences at the central loop, the Integrity of which Is crucial for efficient pore formation. The new hemolysins are activated when the polypeptide extensions are removed by proteases. By alterating the protease recognition sequence in the loop, selective activation by specified proteases can be obtained. Protease-triggered pore-forming proteins might be used for the selective destruction of cancer cells that bear tumor-associated proteases. When certain two-chain constructs are treated with proteases, a full-length polypeptide chain forms as the result of a protease-mediated transpeptidation reaction. This reaction might be used to produce chimeric hemolysins that are Inaccessible by conventional routes.

We are adapting proteins that form pores in lipid bilayers for use as components of biosensors. Specifically, we have produced genetically engineered variants of the α hemolysin (αHL) from Staphylococcus aureus with properties that are sensitive to low concentrations of divalent cations. For example, the pore-forming activity of one mutant (αHL-H5: residues 130–134 inclusive replaced with histidine) is inhibited by Zn2+ at concentrations as low as 1 μM, as judged by the reduction in its ability to lyse rabbit red blood cells and to increase the conductance of planar lipid bilayer membranes. When αHL-H5 is added to the aqueous phase bathing one side of a planar membrane, the subsequent addition of 100 μM Zn2+ to either side blocks the pores that form. This result suggests that at least part of the mutated region lines the channel lumen. Ca2+ and Mg2+ do not block the channel and therefore the H5 mutation confers a degree of analyte specificity to the αHL pore. The results suggest that genetically engineered pores have great promise for the rapid and sensitive detection of metal cations and we discuss the merits and potential limitations for their use in this application. Specifically, we examine the issues of selectivity, sensitivity, response time, dynamic range and longevity. Some of these properties are interdependent. For example, the goals of high sensitivity and rapid response time can be in conflict.

We have prepared sugar-based hydrogels using a chemoenzymatic synthetic approach. Specifically, the regioselectivity of enzymes was used to prepare sugar acrylate monomers. These monomers were polymerized with a crosslinker in a standard free radical polymerization. The polymers consist of a polyacrylate backbone with sucrose, (α-methyl glucoside, or β-methyl galactoside pendant groups. These materials are highly water absorbent swelling to more than 100 times their initial weight when placed in water. The degree of swelling for poly(β-methyl galactoside 6-acrylate) hydrogel was relatively insensitive to pH (from pH 4.5-9) and ionic strength (0.15-3 M NaCl). Potential applications of these materials include waterabsorbents and drug delivery systems.

A material having the characteristics of very low density and high mechanical stability has been developed. This material is prepared by crosslinking the biopolymer alginic acid. Bulk densities as low as 0.15 g/cc have been obtained, while still possessing useful strength, 10% deformation at 347 psi. The physical properties can be varied by controlling the processing conditions, which affect pore volume, pore diameter, bulk density and surface area. Preparation of this material involves solvent exchange and subsequent crosslinking. The resin's minimum compressibility and physical strength allow this material to be considered for a number of applications including adsorbents, insulation and as a polymeric carrier for time release.

The failure processes of the bessbeetle cuticle were studied through the use of scanning electron microscopy, light microscopy, and image analysis techniques. Specimens of elytra and pronotum were failed in tension under the light microscope. The failure process was documented in real time through the use of image analysis equipment and a video camera. Failure occurs primarily in a mixed-mode fashion due to the inherent curvature of the cuticle specimens and their complex hierarchical microstructure. A variety of energy-absorbing mechanisms were observed including remote microcracking, fiber pullout, and crack bridging. Damage accumulation around the failed region is extensive consisting of a significant amount of delamination, fiber-matrix disbonds, microcracking, and intra-fiber failure. The experimental methods and results will be discussed.

Lactose and heparin were covalently coupled to poly(chloromethyl styrene) and the modified polymer was used as a substrate for rat hepatocyte culture. Lactose and heparin are recognized by rat hepatocytes and can be used to mediate cell attachment to the substrate. Rat hepatocytes cultured in serum-free media on these substrates were able to maintain enzymes and peptides important in the detoxification functions of hepatocytes, without the addition of hormones such as dexamethasone, media additives such as DMSO, or complex biological extracellular factors.

The growing interest in large-scale cell culture and in tissue engineering requires substrates of different geometries. Therefore, we have fabricated the derivatized polymers into microcarriers and, most recently, foams. These three-dimensional structures, combined with the chemistries of the polymers, provided the hepatocytes with more cell-cell interactions and in vivo-like geometries than conventional flat-dish culture.

High order self-diffraction is observed from a 5.2 OD570 thin film containing chemically enhanced bacteriorhodopsin. Up to fourteen diffracted orders (±7) could be discerned by eye at a laser intensity of 23 mW/cm2. The results for the first and second order diffracted beams are analyzed in terms of a modified form of the recent self-diffraction model proposed by Fragnito et al.[1].

This article shows the effect of point mutations on the photoelectric behavior of membranes reconstituted from mutant bacteriorhodopsins.

A novel three-dimensional optical memory system based on a light transducing protein, bacteriorhodopsin, is investigated. The system uses the nonlinear optical properties of bacteriorhodopsin to accomplish reading and writing operations. A nondestructive method of reading information in three-dimensional optical memory that uses second harmonic generation is demonstrated. This method has the advantage of fast speed, is nondestructive, and has the potential for parallel access.

Controlled modification of bacteriorhodopsin (BR) by means of genetic engineering is a powerful tool to create BR-variants with photophysical properties significantly different from the wildtype molecule and, in addition, adapted to the physical demands of various optical information processing techniques. The application of BR-films containing the variant D96N in real-time interferometry is described.

Bilayer lipid membranes (BLMs), especially self-assembled bilayer lipid membranes on solid supports (s-BLMs), have been used in the last three years as lipid bilayer-based biosensors. In this paper, we will describe as well as review briefly our work on s-BLMs and closely related systems including the preparation and characterization of s-BLMs, hydrogen peroxide-sensitive electrodes based on s-BLMs, alkaline-pretreated aluminum electrodes as pH sensors, immobilization of ferrocene on a s-BLM as anamperometric sensor of Fe(CN)63-/4- ions, cyclic voltanmmetry studies of s-BLMs modified with electron mediators, TCPBQ/TCOBQ modified s-BLMs for pH measurements, TCNQ-modified s-BLMs for glucose detection, and s-BLMs on interdigital electrodes prepared by microelectronic techniques.

A biomimetic approach utilizing biomolecular self-assembly was used to tailor quantum-dot composites for use as nonlinear optical media. Yeast tRNA was utilized as an ion-exchange/nucleation site within a polymeric matrix (polyacrylamide). Cadmium ion-exchange and subsequent sulfide precipitation resulted in quantum-dot formation. Illumination of samples with an Argon laser (514 nm) utilizing the Z-scan measurement method resulted in χ3 values of +3.7 ×10−6 esu.

Hemoglobin (Hb) and myoglobin (Mb) were encapsulated in transparent silica glasses prepared by the sol-gel method. The preparation of the silica glasses was tailored so that when proteins were entrapped in the pores of the inorganic matrix, they retained their biochemical activity, i.e. they could bind ligands reversibly. Using optical spectroscopy to monitor ligand binding, we studied the binding of O2, CO, and NO with these two heme proteins encapsulated in silica glasses and compared them to heme proteins in aqueous buffer. Both Hb and Mb in the sol-gel glass bound O2, CO, and NO, producing the same spectroscopic properties as those in aqueous buffer. In addition, silica encapsulated Mb was used to evaluate the rate of ligand (O2) transport through the pores of the glass. When varying oxygen concentration and measuring the time required for full conversion of deoxyMb to MbO2 in the silica gel, the time vs. concentration data followed an exponential trend, as expected for diffusion controlled processes.

Screening and detecting virus by receptor-ligand interactions presents an important challenge in medical and environmental diagnostics, and in drug development. We have developed a direct colorimetric detection method based on a polymeric bilayer assembly. The bilayer is composed of a self-assembled monolayer of octadecyl siloxane and a Langmuir-Blodgett layer of polydiacetylene. The polydiacetylene layer is functionalized with receptor-specific ligands such as analogs of sialic acid. The ligand serves as a molecular recognition element, while the conjugated polymer backbone signals binding at the surface by a chromatic transition. The color transition is readily visible to the naked eye as a blue to red color change and can be quantified by visible absorption spectroscopy. The color transition can be inhibited by the presence of soluble inhibitors. Raman spectroscopic analysis shows that the color transition may arise from binding induced strain on the material resulting in bond elongation and conjugation length reduction.