Amperometric TNT biosensor based on the oriented immobilization of a nitroreductase maltose binding protein fusion.
The preparation and characterization of an amperometric 2,4,6-trinitrotoluene (TNT) biosensor based on the surface immobilization of a maltose binding protein (MBP) nitroreductase (NR) fusion (MBP-NR) onto an electrode modified with an electropolymerized film of N-(3-pyrrol-1-ylpropyl)-4,4'-bipyridine (PPB) are described. The MBP domain of MBP-NR exhibits a high and specific affinity toward electropolymerized films of PPB with the immobilized enzyme retaining virtually all of its enzymatic activity. Under similar conditions, the wild-type NR enzyme (i.e., without the MBP domain) loses most of its enzymatic activity. The kinetics of the catalytic reaction between the biosensor and TNT and 2,4-dinitrotoluene (DNT) were characterized using rotated disk electrode and cyclic voltammetry techniques, and values of 1.4 x 10(4) and 7.1 x 10(4) M(-1) s(-1) were obtained for TNT and DNT, respectively. The apparent Michaelis-Menten constants (KM) for MBP-NR in solution and on the surface, using TNT as substrate, were determined to be 27 and 95 microM, respectively. The corresponding value for "wild-type" NR in solution containing TNT was 78 microM, which is very close to the value obtained for MBP-NR on the surface. The limits of detection for both TNT and DNT were estimated to be 2 microM, and the sensitivities were determined to be 205 and 222 nA/microM, respectively.
Naal
Z
Z
Department of Chemistry & Chemical Biology, Cornell University, Ithaca, New York 14853, USA.
Park
J H
JH
Bernhard
S
S
Shapleigh
J P
JP
Batt
C A
CA
Abruña
H D
HD
D015374:Biosensing Techniques / Q000379:methods* / Q000592:standards; D002352:Carrier Proteins; D004785:Environmental Pollutants / Q000032:analysis; D004800:Enzymes, Immobilized; D058619:Maltose-Binding Proteins; D008839:Microelectrodes / Q000592:standards; D009601:Nitroreductases; D011993:Recombinant Fusion Proteins; D014303:Trinitrotoluene / Q000032:analysis*
D002352:Carrier Proteins; D004785:Environmental Pollutants; D004800:Enzymes, Immobilized; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D014303:Trinitrotoluene; D009601:Nitroreductases