Immunomagnetic separation with mediated flow injection analysis amperometric detection of viable Escherichia coli O157.
The coupling of an immunological separation (using immunomagnetic beads) with amperometric flow injection analysis detection of viable bacteria is presented. Using a solution containing Escherichia coli O157, the electrochemical response with two different mediators [potassium hexacyanoferrate(III) and 2,6-dichlorophenolindophenol] was evaluated in the FIA system. Antibody-derivatized Dynabeads were used to selectively separate E. coli O157 from a matrix. The kinetics and the capacity parameters regarding the attachment of bacteria to the immunobeads were studied. The immunomagnetic separation was then used in conjunction with electrochemical detection to measure the concentration of viable bacteria. A calibration curve of colony-forming units (cfu) against electrochemical response was obtained. The detection limit for this rapid microbiological method was 10(5) cfu mL-1, and the complete assay was performed in 2 h. Some advantages over ELISA methods are the direct detection of viable cells (and not total bacterial load) and the need for only one antibody (not enzyme-labeled), thus making the assay faster (only one washing step is necessary) and less expensive.
Pérez
F G
FG
Dipartimento di Sanità Pubblica, Università degli Studi di Firenze, Italy.
Mascini
M
M
Tothill
I E
IE
Turner
A P
AP
D015086:2,6-Dichloroindophenol / Q000737:chemistry; D015169:Colony Count, Microbial; D004563:Electrochemistry; D004797:Enzyme-Linked Immunosorbent Assay; D019453:Escherichia coli O157 / Q000276:immunology / Q000302:isolation & purification*; D005292:Ferricyanides / Q000737:chemistry; D017022:Flow Injection Analysis / Q000379:methods; D018189:Immunomagnetic Separation / Q000379:methods*
D005292:Ferricyanides; D015086:2,6-Dichloroindophenol; C028033:potassium ferricyanide