Detection of Multiple Pathogens by Surface Plasmon Resonance Imaging

Open Access
Author:
Walker, Stephen Paul
Graduate Program:
Agricultural and Biological Engineering
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
May 04, 2010
Committee Members:
  • Paul Heinz Heinemann, Dissertation Advisor
  • Paul Heinz Heinemann, Committee Chair
  • Jeffrey M Catchmark, Committee Member
  • Roy Edward Young, Committee Member
  • Chitrita Debroy, Committee Member
Keywords:
  • Biosensors
  • Food Safety
  • Surface Plasmon
  • SPR Imaging
Abstract:
The goal of this research was to determine if a surface plasmon resonance imaging device could be used to detect multiple pathogenic microorganisms that are commonly connected to food related illness. First, the dynamic range of the SPR imaging device was improved by the conversion of the device from a fixed-angle configuration to a scanning angle configuration by the addition of a computer-controlled stepper motor on the prism assembly. The enhanced dynamic range was challenged by studying the application of multiple, alternating thin polymer films of Poly(L-lysine) hydrobromide and poly(L-glutamate). The original fixed angle configuration was theoretically limited to differentiating less than 5 pairs of alternating films while the scanning angle configuration was tested to 7 pairs of alternating films without reaching the limit of dynamic range. The scanning imaging SPR device was applied to the detection and differentiation of: E. coli O157 and two non-O157 STEC strains, E. coli O128 and E. coli O101. To functionalize the gold surface of the SPR slide with an array of antibodies, self-assembled layers of 11-mercaptoundecanoic acid, poly(ethylenimine) and ExtrAvidin were applied. A commercial biotin-conjugated anti-rabbit IgG was mated to the ExtrAvidin surface and then antibody probes were acquired directly from the three types of rabbit antiserum. The results for this methodology were disappointing, with no differences between the antibody-functionalized array spots and non-functionalized control spots for any of the three E. coli strains. The methodology and goals were simplified and the SPR imaging device, restored to a fixed-angle configuration, was applied to the detection of E. coli O157, Salmonella enterica, and Listeria monocytogenes in pure cultures and in apple juice. Again using a self-assembled multilayer approach, the surface was functionalized with layers of 11-Amino-undecanethiol hydrochloride, N-succinimidyl-S-acetylthiopropionate, and streptavidin-maleimide resulting in a streptavidin surface layer. Then, purified, biotin-conjugated antibodies for each of the three target bacteria were coupled to the surface in an array configuration. This methodology was partially successful, with E. coli O157 and Salmonella enterica detected at the 108 cfu/ml level in pure culture. One of the three experimental trials resulted in evidence that Listeria monocytogenes was detected at the 108 cfu/ml level but across the three experimental trials the differences with the controls were not significant at any reasonable confidence interval. None of the target bacteria were successfully detected in apple juice.