A simple sensor method was developed for aflatoxin M1 analysis to be applied directly with milk by using antibody modified screen-printed carbon working electrode with carbon counter and silver–silver chloride pseudo-reference electrode. A competitive ELISA assay format was constructed on the surface of the working electrode using 3,3,5′,5′-tetramethylbenzidine dihyrochloride (TMB)/ H2O2 electrochemical detection scheme with horseradish peroxidase (HRP) as the enzyme label. The performance of the assay and the sensor was optimised and characterised in pure buffer conditions before applying to milk samples. Extensive interference to the electroanalytical signal was observed upon the analysis of milk. Through a series of chemical fractionations of the milk, and testing the electrochemical properties of the fractions, the interference was attributed to whey proteins with focus towards α-lactalbumin. A simple pre- treatment technique of incorporating 18 mM calcium chloride, in the form of Dulbucco's PBS, in a 1:1 ratio to the milk sample or standards and also to the washing buffer stabilised the whey proteins in solution and eliminate the interfering signal. The resulting immunosensor was interference free and achieved a limit of detection of 39 ng l−1 with a linear dynamic detection range up to 1000 ng l−1. The developed immunosensor method was compared to a commercial ELISA kit and an in-house HPLC method. The immunsensor was comparable, in term of sensitivity, but vastly superior in term of portability and cost therefore a key instrument for the detection of aflatoxin M1 at the source of the co
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