Cholinesterase surfaces sensors

Experiment 2. Cholinesterase as a sensor on the cell surface A target of the allelochemical may also be a surface sensor-cholinesterase (Fig. 10). If after the staining with Red analogue of Ellman reagent the blue colour is absent in the allelochemical treated microspore, possible target is the enzyme (Roshchina, 2001a,b) as for alkaloid berberine tested. If after the treatment by the test allelochemical, the colour is absent or light, the compound inhibits the enzyme (also see biochemical assay in Chapter 11). 

The aim of our investigation was the development of the amperometric enzyme immunosensor for the determination of Klebsiella pneumoniae bacterial antigen (Ag), causes the different inflammatory diseases. The biosensing pail of the sensors consisted of the enzyme (cholinesterase) and antibodies (Ab) immobilized on the working surface of the screen-printed electrode. Bovine seiaim albumin was used as a matrix component. 

I.G. Mourzina, T. Yoshinobu, Y.E. Ermolenko, Y.G. Vlasov, M.J. Schoning and H. Iwasaki, Immobilization of urease and cholinesterase on the surface of semiconductor transducer for the development of light-addressable potentiometric sensors, Microchim. Acta, 144(1-3) (2004) 41-50. 

Piezoelectric sensors have become a versatile tool in biosensorics to study protein-protein and protein-small molecule interactions. Here we present theoretical background on piezoelectric sensors and instructions, how to modify their surface with various recognition elements for cholinesterases. These recognition elements comprise an organophosphate (paraoxon), a cocaine derivative (BZE-DADOO), and a tricyclic, aromatic compound (propidium). Additionally, a guide to the kinetic evaluation of the obtained binding curves is given in this chapter. 

Here we show how to modify the piezoelectric surface to develop affinity sensors for detection of small molecular compounds using antibodies and (acetyl)cholinesterases (AChE), respectively. The chemical structures of the described surface modifications are shown in Fig. 

Sensor systems based on the reversible, competitive inhibition of AChE and BChE by monosulfonate tetraphenyl porphyrin (TPPSi) were developed. TPPSi (350 iM in 50 mM pH 7 NaPi with 25% edianol) was applied to surfaces of immobilized enzymes and allowed to interact for 20 min before rinsing the excess solution off with excess buffer (50 mM NaPi pH 7). TPPSi was found to be a competitive inhibitor of cholinesterase activity for both AChE and BChE 12, 14, 15). The absorbance spectrum was unique for the AChE-TPPS complex (TAC) as compared to that of the BChE-TPPSi complex (TBC) with characteristic peaks at 446 nm for TAC and 412 nm for TBC. When TPPSi was applied to a siuface bearing both AChE and BChE (TABC), the characteristic peaks for both TAC and TBC were observed in the absorbance spectrum. 

A piezoelectric biosensor, for the detection of several organophosphorus pesticides was developed (Halamek et al., 2005). The sensor was based on the immobilization of a reversible inhibitor of cholinesterase on the surface of the sensor. The binding of AChE to this inhibitor was monitored with a mass-sensitive piezoelectric quartz crystal. In the presence of an inhibiting substance in the sample, the binding of the enzyme to the immobilized compound was reduced, and the decrease of mass change was proportional to the concentration of the analyte in the sample. This sensor was applied to the determination of pesticides in river water samples. 

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