Development of an Electrochemical Insulin Sensor Based on a High Affinity DNA Sequence Found in the Insulin-linked Polymorphic Region

The detection of pertinent biomarkers has the potential provide an early indication of disease progression before considerable damage has been incurred. A decrease in an individual’s sensitivity to insulin, which may be quantified as the ratio of insulin to glucose in the blood after a glucose pulse, has recently been reported as an early predictor of insulin-dependent diabetes mellitus. Routine measurement of insulin levels is therefore desirable in the care of diabetes-prone individuals. A rapid, simple, and reagentless method for insulin detection would allow for wide-spread screenings that provide earlier signs of diabetes onset. The aim of this thesis is to develop a folding-base electrochemical sensor for the detection of insulin. The sensor described herein consists of a DNA probe immobilized on a gold disc electrode via an alkanethiol linker and embedded in an alkanethiol self-assembled monolayer. The probe is labeled with a redox reporter, which readily transfers electrons to the gold electrode in the absence of insulin. In the presence of insulin, electron transfer is inhibited, presumably due to a binding-induced conformational or dynamic change in the DNA probe that significantly alters the electron-tunneling pathway. A 28-base segment of the insulin-linked polymorphic region that has been reported to bind insulin with high affinity serves as the capture element of the DNA probe. Three probe constructs that vary in their secondary structure and position of the redox label are evaluated for their utility as insulin-sensing elements on the electrochemical platform. The effects of probe modification on secondary structure are also evaluated using circular dichroism spectroscopy.

Detection Methods for the Genus Lysobacter and the Species Lysobacter enzymogenes

Strains of Lysobacter enzymogenes, a bacterial species with biocontrol activity, have been detected via 16S rDNA sequences in soil in different parts of the world. In most instances, however, their occurrence could not be confirmed by isolation, presumably because the species occurred in low numbers relative to faster-growing species of Bacillus or Pseudomonas. In this study, we developed DNA-based detection and enrichment culturing methods for Lysobacter spp. and L. enzymogenes specifically. In the DNA-based method, a region of 16S rDNA conserved among Lysobacter spp. (L4: GAG CCG ACG TCG GAT TAG CTA GTT), was used as the forward primer in PCR amplification. When L4 and universal bacterial primer 1525R were used to amplify DNA from various bacterial species, an 1100-bp product was found in Lysobacter spp. exclusively. The enrichment culturing method involved culturing soils for 3 days in a chitin-containing broth amended with antibiotics. Bacterial strains in the enrichment culture were isolated on yeast-cell agar and then identified by 16S rDNA sequence analysis. A strain of L. enzymogenes added to soils was detected at populations as low as 102 and 104 CFU/g soil by PCR amplification and enrichment culturing, respectively. In a survey of 58 soil samples, Lysobacter was detected in 41 samples by PCR and enrichment culture, out of which 6 yielded strains of Lysobacter spp. by enrichment culture. Among isolated strains, all were identified to be L. enzymogenes, with the exception of a strain of L. antibioticus. Although neither method alone is completely effective at detecting L. enzymogenes, they are complementary when used together and may provide new information on the spatial distribution of the species in soil.