INTEGRATED BIOSENSOR-BASED PROCESSES FOR MULTIPATHOGENIC ANALYTE DETECTION
Location: Molecular Characterization of Foodborne Pathogens
Title: Protein-Based Microarray for the Detection of Pathogenic Bacteria
Submitted to: Journal of Rapid Methods and Automation in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 30, 2007
Publication Date: March 4, 2007
Citation: Gehring, A.G., Albin, D.M. 2007. Protein-Based Microarray for the Detection of Pathogenic Bacteria. Journal of Rapid Methods and Automation in Microbiology. 15:49-66
Interpretive Summary: There is a desire to check food samples for the presence of different, harmful bacteria in a single test. A detection technique called microarrays, has the ability to address this need by using a test surface containing up to thousands of different reactive sites with a single sample. These reactive sites contain biological recognition elements (typically antibodies or nucleic acid) that may selectively react with and specifically adhere to bacteria or bacterial components (e.g., cell walls, cell fragments, toxins). As a prototype system, we have developed an antibody-based microarray and demonstrated that it could detect in tact Escherichia coli (E. coli) O157:H7 cells. E. coli O157:H7 is a harmful bacterium that is responsible for numerous outbreaks of foodborne illness, and even death, in the United States every year. Specifically, the test first captured E. coli O157:H7 with an antibody that was attached to a charged glass slide. Then, a second antibody with a fluorescent marker that could be easily detected and measured with a laser scanner was used to determine if any E. coli O157:H7 were captured. In this research, the microarray approach was optimized in several ways to facilitate rapid detection of intact bacterial cells. This microarray-based test may be used by regulatory agencies as well as food producers to rapidly assess for the presence of multiple, harmful bacteria in numerous food samples.
Microarrays have been used for gene expression and protein interaction studies, but recently, multianalyte diagnostic assays have employed the microarray platform. We developed a microarray immunoassay for bacteria, with biotinylated capture antibodies on streptavidin slides. To complete the fluorescent sandwich immunoassay following capture of bacteria (Escherichia coli O157:H7), a fluorescein-labeled antibody was used to label antibody-bound bacteria. The assay time was less than 4 h. As this method was developed, it became apparent that several methodological factors markedly affected the results. Therefore, a series of experiments was conducted to investigate methodological factors that affected the assay including analysis of variation in normal printing, use of a coverslip to contain sample exposure to the microarrayed antibodies, surface chemistries of capture antibody immobilization/attachment, and assay reaction time. The use of a coverslip during immunological reactions reduced the fluorescent signal by approximately 50% compared to the use of an uncovered, hydrophobic barrier that was also used to contain sample solution during exposure to the microarray. Also, when protein G was used for capture antibody attachment, a lower signal was generated than when biotinylated capture antibody was used. At high bacterial concentrations (108 or 109 cells/mL), the assay could be shortened to less than 20 min. Antibody microarrays were effectively used to detect bacteria, but assay parameters markedly affected the results and required careful design.