3.1.Enzymeimmobilization,biosensorstabilityandcalibration
Two biosensorconfigurations withtheAChEenzymeim-
mobilized inchitosanweretested:achemicallydepositedchitosan
layerwithphysicallyentrappedAChEandalayer-by–layerap-
proach withtheenzymeentrappedinalternatelayersofchitosan
and alginate.PhysicaladsorptionofAChEwasalsotestedbutthe
enzyme leachedfromtheelectrodesurfaceandthebiosensorwas
not stable.Thebiosensorwastestedside-by-sidewithabiosensor
with covalentlyimmobilizedAChEusingglutaraldehyde.This
comparison allowedustodeterminetheeffectofchitosanonthe
enzyme activityandevaluatethesolvent’s effect. Fig. 1 showsthe
calibration curveofthethreetypesofbiosensors.Theoptimal
configurationwasdeterminedbasedonthestabilityandresponse
times. Table1 summarizes theaveragecurrentintensityandre-
sponse times.Thebiosensorwiththechitosan–alginate assembly
showedsignificantly highercurrentintensityduetohigheren-
zyme loading,demonstratinggoodretentionoftheenzymatic
activity.Theaveragecurrentintensityshowedhighstandardde-
viation andahigherresponsetime(4min)duetodiffusional
constraints ofthemultiplelayers.Whenassayedmultipletimes
the biosensorshowedacontinuousdecreaseinthecurrentre-
sponse indicatingthattheenzymelayerwasnotstable.Bycom-
parison, thechemicallydepositedsinglelayerchitosan film was
consistent andstronglyattached.Theresponsetimeforthissensor
was1min.Thebiosensorwithglutaraldehydebindingwasalso
stable witharesponsetimeof1min.Bothofthesesensorshad
good reproducibility(relativestandarddeviationsforatleast
7 sensorspreparedinidenticalconditionsareshownin Table1).
The biosensorwiththephysicallyentrappedenzymehadahigher
sensitivityascomparedtotheonebasedoncovalentcross-linking
with glutaraldehyde.Bothofthesebiosensorswereusedforfur-
ther characterizationtoestablishtheeffectoforganicsolventsand
for thedetectionofparaoxon.