Alternating polyelectrolyte films constructed by the sequential adsorption of poly(allylamine hydrochloride) (PAH) and poly(styrenesulfonate) (PSS) have been used as substrates for the immobilization of immunoglobulin G (IgG) and anti-IgG. Anti-IgG has also been immobilized in multilayer films by the alternate deposition of PSS and anti-IgG. The assembly process of the multilayer films was monitored using a quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). Film growth was achieved up to at least nine (5 anti-IgG and 4 PSS) layers. The utility of these films for immunosensing has been investigated via their subsequent interaction with IgG. The alternating polyelectrolyte/protein layers were constructed in order to increase the binding layer capacity (i.e. sensitivity) of the thin film with respect to IgG detection. The sensitivity, determined using IgG mass uptake data from quartz crystal microgravimetry, was found to be linearly dependent on the number of anti-IgG layers (and hence the amount of IgG incorporated) in the polyelectrolyte film when the anti-IgG layers are separated by one PSS layer. In contrast, for films where anti-IgG layers are separated by five polyelectrolyte (PSS(PAH/PSS) 2) layers, only the outer anti-IgG layer is immunologically active. This is attributed to the formation of a dense polyelectrolyte film through which antibody permeation is restricted. The films evaluated have promise in that the sensitivity can be tuned by fabricating the desired number of protein layers, whilst the selectivity can be modified by selecting the desired biospecific biomolecule.

A novel method for preparation of biomacromolecular imprinted nanoparticles is described. Combinations of functional monomers were polymerized in the presence of the imprinting peptide melittin in aqueous solution at room temperature to produce a small library of polymer nanoparticles. The template peptide and unreacted monomers are subsequently removed by dialysis. Nanoparticles (NPs) from the library were evaluated for their binding to melittin by 27 MHz QCM analysis. NPs prepared with optimized functional monomer combinations bind strongly to the target molecule. Nanoparticles that were polymerized in the absence of template peptide were found to have little affinity to the peptide. Binding affinity and the size of imprinted particles are comparable to those of natural antibodies. They interact specifically with the target peptide and show little affinity for other proteins. These NPs are of interest as inert and stable substitutes for antibodies. Extension of this approach to other targets of biological importance and the applications of these materials are currently being evaluated.

A highly sensitive 27-MHz quartz-crystal microbalance, on which a 10-30-mer oligonucleotide was immobilized as a probe molecule, was employed to detect hybridization of complementary oligonucleotides in aqueous solution. From frequency decreases (mass increases due to the hybridization) with passage of time, kinetic parameters such as association constants (K(a)) and binding and dissociation rate constants (k(1) and k(-1)) could be obtained, as well as binding (hybridization) amount at the nanogram level (delta m). Kinetic studies were carried out by changing various parameters: (i) the immobilization method of a probe oligonucleotide on Au electrode, (ii) number of mismatching bases in sequences of target oligonucleotides, (iii) length of both probe and target oligonucleotides, (iv) hybridization temperature, and (v) ionic strength in solution. The obtained results were compared with those obtained by a surface plasmon resonance method using a BIAcore system.