Abstract
Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 105 cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.
Highlights
According to the U.S Centers for Disease Control and Prevention, approximately 48 million illnesses; 128,000 hospitalizations; and 3000 deaths per year in the United States alone are attributed to ingestion of contaminated foods [1]
Sickness associated with foodborne E. coli O157:H7 is an important problem in the United States where past multistate outbreaks have been associated with meat [6] and produce [7]
A major part of this investigation compared the efficacy of binding for bacteria (E. coli O157:H7) versus the biomolecule, Shiga toxin 1 (Stx1; a protein synthesis inhibitor that is produced by Shigatoxigenic strains of E. coli), at the capture surface of a microarray substrate as influenced by various incubation conditions
Summary
According to the U.S Centers for Disease Control and Prevention, approximately 48 million illnesses; 128,000 hospitalizations; and 3000 deaths per year in the United States alone are attributed to ingestion of contaminated foods [1]. Traditional microbial culture methods can detect and identify a single, specific bacterium contaminant in foods, but the approach may require days or weeks to complete and typically, quantitative data is not generated Such specific detection of very small numbers (e.g., 1 cell/mL) of pathogenic bacteria in complex food matrices necessitates methods with extremely high sensitivity. Dielectrophoresis [26] and direct radiation force combined with ultrasound acoustic streaming [27] have been employed as means to improve immobilization via active partitioning of bacteria from liquid phase to static, antibody-coated, solid substrates Other groups, such as Ball et al [28], have employed centrifugation to mechanically force bacteria to a capture surface. A major part of this investigation compared the efficacy (i.e., increased fluorescence responses associated with bacterial capture) of binding for bacteria (E. coli O157:H7) versus the biomolecule, Shiga toxin 1 (Stx; a protein synthesis inhibitor that is produced by Shigatoxigenic strains of E. coli), at the capture surface of a microarray substrate as influenced by various incubation conditions (static, mixing, and centrifugation)
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