In this paper, we describe a novel nonlabeled biosensor with high diagnostic potential for rapid and sensitive detection of antigens in complex biological samples. The biosensor comprises a piezoimmunosensor (PZ) displaying a specially constructed recombinant antibody on its surface. The recombinant single-chain fragment variable (scFv) antibody contained a cysteine within the linker amino acid sequence used to join the scFv variable heavy and light chains. The presence of cysteine induced the scFv construct to self-assemble as a densely packed rigid monolayer on the gold surface of a quartz crystal microbalance. scFv molecules in this self-assembled mono-layer (SAM) exhibited a defined orientation and high areal densities, with scFv-modified microbalance surfaces displaying 35 times as many variable antigen-binding sites per square centimeter as surfaces modified with whole antibody. Experimental data show that the scFv SAM PZ is superior to Fab fragment, Fab fragment containing a free sulfhydryl group (i.e., Fab-SH), and whole antibody PZs regarding sensitivity and specificity. Because of their small uniform size (MW ≈ 27000) and the ease with which they can be modified using genetic engineering, scFv's have significant advantages over whole antibodies in microbalance biosensor systems. We demonstrate here that the use of scFv containing a cysteine within the scFv linker sequence (i.e., scFv-cys) for preparation of biosensor surfaces markedly increases the density of available antigen-binding sites, yielding a system that is highly selective, rapid, and capable of detecting low concentrations of antigens in complex samples.Biosensor systems that detect biological and chemical agents have important medical, environmental, public safety, and defense applications. An ideal biosensor would be sensitive, rapid, reliable, robust, and inexpensive. Piezoimmunosensors (PZs) are a type of biosensor utilizing antibodies and a quartz crystal microbalance (QCM) to detect minute changes in mass as antigens bind to the antibodies on the QCM surface. 1,2 Although their diagnostic potential is theoretically quite high, in practice, the usefulness of PZs has been limited by the fact that typical IgG antibodies can trap or nonspecifically bind irrelevant molecules, thus yielding false positive signals in assays. Additionally, there remains some skepticism concerning their applicability as biosensors due to the complexity of the physical properties of biofilms in a liquid that make it difficult to establish an explicit relationship between the added mass and a change in the resonant frequency. The QCM gives a direct response signal that characterizes a binding event between an antibody layer, immobilized on the surface of the QCM or other transducers, and the antigen to be detected. The mass change on the QCM surface is estimated using the Sauerbrey equation, 3 Δf = −2Δmnf 0 2 /[A(µ q ρ q ) 1/2 ], where n is the overtone number, µ q is the shear modulus of the quartz [2.947 × 10 11 g/(cm·s 2 )], ρ q is the density of the ...
