AbstractsBiology & Animal Science

Since the development of the immunoassay, biomolecule detection via binding assays has become vitally important in many fields. High sensitivity in such assays usually requires low molecular weight labeling species (i.e., tracers) that typically must be detected using complex instrumentation (fluorimeter, etc.). In this thesis, the use of a specific enzyme cofactor reconstitution reaction employing pyrroloquinoline quinone (PQQ) with apo-glucose dehydrogenase (apo-GDH) is explored as a simple and high sensitivity tracer system for detecting DNA and antimicrobial peptides, either visually or using conventional spectrophotometry. First, PQQ is encapsulated in liposomes, which are tagged with DNA and used in a sandwich-type heterogeneous assay for the detection of single-stranded DNA. After the binding reaction, the bound liposomes are lysed to release the PQQ. This assay has a detection limit of 62 fmol of single-stranded DNA, and thus rivals more conventional fluorescence-based DNA detection systems, even though only a visual read is required to detect the presence of target DNA. PQQ-loaded liposomes are also utilized in the homogeneous detection of the membrane bilayer permeabilization induced by antimicrobial peptides. Detection of such peptides at nM levels is possible using this assay scheme. Further, PQQ is doped into polymeric nanospheres, which are subsequently tagged with DNA, and also employed in a sandwich-type assay for single-stranded DNA detection. The nanoparticles show enhanced PQQ-loading capacity compared to the liposomes, and could eventually exhibit even lower limits of detection than the liposome-based assay. Lastly, initial results with a PQQ-linked oligonucleotide probe for homogeneous endpoint PCR detection of target DNA at levels of 15 molecules with only 40 PCR cycles are demonstrated, along with initial work to optimize the synthesis of the required probe. With further optimization, the PQQ-based reconstitution assay could be adapted as a tracer system to devise a variety of high-sensitivity field test devices to monitor important biomolecules.