Covalent immobilization of beta-galactosidase onto a gold-coated magnetoelastic transducer via a self-assembled monolayer: toward a magnetoelastic biosensor.
The enzyme beta-galactosidase has been covalently immobilized onto a gold-coated magnetoelastic film via a self-assembled monolayer (SAM) of omega-carboxylic acid alkylthiol. Use of magnetoelastic transduction allows for the wireless monitoring of enzymatic activity through the associated change in the frequency and amplitude of magnetic fields. The formations of SAMs of 3-mercaptopropanoic acid and thioctic acid were monitored by magnetoelastic transduction. After coupling of beta-galactosidase to the SAMs, the enzyme activity was monitored by using a substrate that forms an insoluble product upon action of the enzyme. Specifically, an indolyl galactopyranoside substrate was employed in conjunction with an azo dye as the precipitating system. The immobilized enzyme was evaluated and found to have an apparent Michaelis-Menten constant (KM) of 1.2 mM for the indolyl galactopyranoside. Calibration plots for both substrates and inhibitors were generated to establish the versatility of this sensing system. Kinetic parameters for nonprecipitating substrates were determined in conjunction with a precipitating enzymatic substrate by way of a competitive inhibition study using beta-galactosidase attached to magnetoelastic strips. The methods developed within this work allow for the fabrication of wireless enzyme sensing systems, which can also be used as another means of screening for enzyme inhibitors.
Ball
J Christopher
JC
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055, USA.
Puckett
Libby G
LG
Bachas
Leonidas G
LG
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004800:Enzymes, Immobilized; D006046:Gold / Q000737:chemistry*; D008280:Magnetics / Q000295:instrumentation*; D008567:Membranes, Artificial; D015394:Molecular Structure; D013997:Time Factors; D014159:Transducers; D001616:beta-Galactosidase / Q000737:chemistry*
D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D006046:Gold; D001616:beta-Galactosidase