Abstract: This work describes a technique for the rapid and sensitive electrochemical flow injection monitoring of aflatoxin M-1 (AFM(1)) in cheese samples. Stabilized filter-supported bilayer lipid membranes (BLMs) were used as detectors Single stranded DNA oligomers terminated with alkyl chains (dT(20)-C-16) were incorporated into the membranes to control surface electrostatic properties. The incorporation of dT(20)-C-16 in BLMs lowered the detection limit for the detection of this toxin by one to four orders of magnitude as compared with the detection limit obtained in the absence of DNA. Therefore, it is now possible to continuously monitor this toxin at concentrations that approached those that could be of interest as set by the U.S. Food and Drug Administration and most European countries. The work described herein takes a significant step towards development of a detector of greater practical potential by demonstrating that the incorporation of C-16-ssDNA into lipid membranes results in a combination of properties that provides for a much more sensitive and robust detection system. Injections of AFM(1) were made into flowing streams of a 0.1 M KCI electrolyte solution, and a transient current signal with duration of seconds reproducibly appeared in about 12 s after exposure of the detector element to the toxin. The magnitude of this signal was linearly related to the concentration of AFM(1) with detection limits at subnanomolar level. The effect of interferents such as proteins and lipids was investigated. It was determined that interferences from proteins could be eliminated by adjustment of the flow rate of the carrier electrolyte solution. The technique was applied for the rapid flow injection determination of aflatoxin MI in cheese samples. AFM(1) could be determined in continuous flowing systems with a rate of at least 3 samples min-1. Repetitive cycles of injection of AFM(1) have shown no signal degradation during each cycle for experiments that attempted over 30 cycles of detection.

Abstract: Biosensors were fabricated from filter-supported solventless bilayer lipid membranes (BLM) and used for the determination of substrates of hydrolytic enzymes in a flow-through system. The co-deposition of lipid (dipalmitoylphosphatidic acid) and protein solutions to form a BLM on a microporous glass fiber or polycarbonate ultrafiltration membrane disc is described. Enzymes were immobilized on the membranes by incorporating the protein solution into the lipid matrix at the air-electrolyte interface before BLM formation. BLM containing acetylcholinesterase, urease and penicillinase were used for the determination of acetylcholine, urea and penicillin, respectively, following injection of the substrates into a 0.1 M KCl/10 mM HEPES carrier electrolyte. The enzymatic reaction at the membrane surface caused changes in the electrostatic fields and phase structure of the BLM, resulting in ion current transients; the magnitude of these were linearly related to the substrate concentration down to the µM level. The RSD were ~5%. The effects of pH, amount of enzyme and flow rate are discussed. The response times were ~10 s, the detection limits were 1, 10 and 100 µM for acetylcholine, urea and penicillin, respectively, and the analytical throughput was 220 samples/h. This work describes a technique for the rapid and sensitive determination of acetylcholine, urea, and penicillin in flowing solution streams using stabilized systems of solventless bilayer lipid membranes (BLMs). This method of monitoring substrates of hydrolytic enzyme reactions made use of BLMs which were supported on ultrafiltration membranes such as polycarbonate and glass microfiber; these filter membranes were found to enhance the stability of BLMs for uses in flow injection experiments. The enzymes were immobilized on BLMs by incorporating the protein solution into the lipid matrix at the air/electrolyte interface before the BLM formation, followed by injections of the substrates into flowing streams of a carrier electrolyte solution. Hydronium ions produced by the enzymatic reaction at the BLM surface caused dynamic alterations of the electrostatic fields and phase structure of BLMs, and as a result ion current transients were obtained; the magnitude of these signals was correlated to the substrate concentration, which could be determined at the micromolar level. The response times were ~10 s, and acetylcholine, urea, and penicillin could be determined in continuous flowing systems with a maximum rate of 220 samples/h. It is expected that this analytical utility of stabilized BLMs for flow stream uses will provide new opportunities in this strategy of chemical sensing.

Abstract: Bilayer lipid membranes (BLM) were used as generic transducers to monitor hydrolytic enzyme reactions at the membrane surface. The reactions of urease and penicillase with urea and penicillin were used as test reactions. The BLM could be used as generic transducers of pH; the increase in pH being dependent on the quantity and extent of dipalmitoylphosphatidic acid up to 35%. The results were consistent with an electrostatic mechanism of perturbation of the BLM surface structure. Detection limits were 40 µM-urea and 0.1 mM penicillin. The planar membranes were stable for a few hours and would be best suited to stopped-flow or flow injection systems where solution movement can be controlled.