Optical Biosensing of Nitrate Ions Using a Sol–Gel Immobilized Nitrate Reductase

Abstract

The coupling of enzymes with sol–gel technology creates exciting possibilities for biosensing. Enzymes can be highly selective and will only respond to specific analytes. Sol–gels provide a unique matrix in which various biomaterials can be immobilized without any loss of enzyme activity. These two components have been combined for the optical biosensing of nitrate ions. The periplasmic nitrate reductase (Nap) extracted from the denitrifying bacterium Thiosphaera pantotropha reacts specifically with the nitrate (NO ₃ ^- ) anion. The encapsulation of this enzyme in a sol–gel structure for the optical biosensing of nitrate ions is reported. The reduction of nitrate by periplasmic nitrate reductase results in a characteristic change in the UV/VIS absorption spectrum of the nitrate reductase. This spectroscopic change has been quantitatively calibrated against nitrate concentration. The nitrate biosensing system is fully reversible and is highly sensitive and selective to nitrate ions. The results obtained show that the activity of the enzyme is not affected by the sol–gel matrix, even after a storage period of up to six months. As no leaching of the Nap from the sol–gel matrix was observed, it is clear that the encapsulation of this nitrate sensitive enzyme in a sol–gel medium represents an ideal anionic recognition element of an optical biosensor for the detection of nitrate ions in the µmol l ^{- 1} range.