Nanoassemblies of acetylcholinesterase and β-lactamase immobilized on magnetic nanoparticles as biosensors to detect pollutants in water.

Abstract

The use of enzymes immobilized on magnetic nanoparticles to detect contaminants in aqueous samples has gained interest, since it allows the magnetic control, concentration and reuse of the enzymes. In this work, the detection of trace amounts of organophosphate pesticides (chlorpyrifos) and antibiotics (penicillin G) in water was attained by developing a nanoassembly formed by either inorganic or biomimetic magnetic nanoparticles used as substrates to immobilize acetylcholinesterase (AChE) and β-lactamase (BL). Other than the substrate, the optimization of the nanoassembly was done by testing enzyme immobilization both through electrostatic interaction (also reinforced with glutaraldehyde) and covalent bonds (by carbodiimide chemistry). Temperature (25°C), ionic strength (150mM NaCl) and pH (7) were set to ensure enzymatic stability and to allow both the nanoparticles and the enzymes to present ionic charges that would allow electrostatic interaction. Under these conditions, the enzyme load on the nanoparticles was ⁓0.1mg enzyme per mg nanoparticles, and the preserved activity after immobilization was 50-60% of the specific activity of the free enzyme, being covalent bonding the one which yielded better results. Covalent nanoassemblies could detect trace concentrations of pollutants down to 1.43nM chlorpyrifos and 0.28nM penicillin G. They even permitted the quantification of 14.3μM chlorpyrifos and 2.8μM penicillin G. Also, immobilization conferred higher stability to AChE (⁓94% activity after 20 days storage at 4°C) and allowed to reuse the BL up to 12 cycles.