Sensors cholinesterase

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). 

Biosensors ai e widely used to the detection of hazardous contaminants in foodstuffs, soil and fresh waters. Due to high sensitivity, simple design, low cost and real-time measurement mode biosensors ai e considered as an alternative to conventional analytical techniques, e.g. GC or HPLC. Although the sensitivity and selectivity of contaminant detection is mainly determined by a biological component, i.e. enzyme or antibodies, the biosensor performance can be efficiently controlled by the optimization of its assembly and working conditions. In this report, the prospects to the improvement of pesticide detection with cholinesterase sensors based on modified screen-printed electrodes are summarized. The following opportunities for the controlled improvement of analytical characteristics of anticholinesterase pesticides ai e discussed ... 

The optimization of the biorecognition layer by the modification of a transducer used. Nanostmctured poly aniline composite comprising Prussian Blue or poly-ionic polymers has been synthesized and successfully used in the assembly of cholinesterase sensors. In comparison with non-modified sensors, this improved signal selectivity toward electrochemically active species and decreased the detection limits of Chloropyrifos-Methyl and Methyl-Pai athion down to 10 and 3 ppb, respectively. 

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. 

The working conditions of the immunosensor (enzyme and antigen concentrations, dilutions of the antibodies, pH of the buffer solution) were found. The cholinesterase immobilized demonstrated the maximum catalytic activity in phosphate buffer solution with pH 8.0. The analytical chai acteristics of the sensor - the interval of the working concentrations and detection limit - have been obtained. The proposed approach of immunoassay made possible to detect 5T0 mg/ml of the bacterial antigen. 

The design and implementation of a portable fiber-optic cholinesterase biosensor for the detection and determination of pesticides carbaryl and dichlorvos was presented by Andreou81. The sensing bioactive material was a three-layer sandwich. The enzyme cholinesterase was immobilized on the outer layer, consisting of hydrophilic modified polyvinylidenefluoride membrane. The membrane was in contact with an intermediate sol-gel layer that incorporated bromocresol purple, deposited on an inner disk. The sensor operated in a static mode at room temperature and the rate of the inhibited reaction served as an analytical signal. This method was successfully applied to the direct analysis of natural water samples (detection and determination of these pesticides), without sample pretreatment, and since the biosensor setup is fully portable (in a small case), it is suitable for in-field use. 

In AChE-based biosensors acetylthiocholine is commonly used as a substrate. The thiocholine produced during the catalytic reaction can be monitored using spectromet-ric, amperometric [44] (Fig. 2.2) or potentiometric methods. The enzyme activity is indirectly proportional to the pesticide concentration. La Rosa et al. [45] used 4-ami-nophenyl acetate as the enzyme substrate for a cholinesterase sensor for pesticide determination. This system allowed the determination of esterase activities via oxidation of the enzymatic product 4-aminophenol rather than the typical thiocholine. Sulfonylureas are reversible inhibitors of acetolactate synthase (ALS). By taking advantage of this inhibition mechanism ALS has been entrapped in photo cured polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ) to prepare an amperometric biosensor for... 

S. Andreescu, V. Magearu, A. Lougarre, D. Fournier, and J.L. Marty, Immobilization of enzymes on screen-printed sensors via a histidine tail. Application to the detection of pesticides using modified cholinesterase. Anal. Lett. 34, 529-540 (2001). 

A. Ivanov, G. Evtugyn, L.V. Lukachova, E.E. Karyakina, HC. Budnikov, S.G. Kiseleva, A.V. Orlov, G.P. Karpacheva, and A.A. Karyakin, Cholinesterase potentiometric sensor based on graphite screen-printed electrode modified with processed polyaniline. IEEE Sensors J. 3, 333-340 (2003). 

A. Ivanov, G. Evtugyn, H. Budnikov, F. Ricci, D. Moscone, and G. Palleschi, Cholinesterase sensors based on screen-printed electrodes for detection of organophosphorus and carbamic pesticides. Anal. Bioanal. Chem. 377, 624-631 (2003). 

E.V. Gogol, G.A. Evtyugin, E.V. Suprun, G.K. Budnikov, and V.G. Vinter, Determination of residual pesticide in plant materials using planar cholinesterase sensors modified with nation. J. Anal. Chem. 56, 963-970 (2001). 

J. Halamek, A. Makower, K. Knosche, P. Skladal and F.W. Scheller, Piezoelectric affinity sensors for cocaine and cholinesterase inhibitors, Talanta, 65 (2005) 337-342. 

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. 

G.A. Evtyugin, I.I. Stoikov, C.K. Budnikov and E.E. Stoikova, A cholinesterase sensor based on a graphite electrode modified with 1,3-disub-stituted calixarenes, J. Anal. Chem., 58 (2003) 1151-1156. 

Ultrasensitive determination of pesticides via cholinesterase-based sensors for environmental analysis... 

P. Skladal and M. Mascini, Sensitive detection of pesticides using am-perometric sensors based on cobalt phthalocyanine-modified composite electrodes and immobilised cholinesterase, Biosens. Bioelectron, 7 (1992) 335-343. 

With regard to assaying the inhibitory activity of extracts electro-chemically, one of the problems of assays using sensors based on cholinesterase was that considerable time, e.g. 30-45 min [46,47], could be needed for the activity of the enzyme electrode to fall below control levels. The time increased as the level of inhibition decreased. Such lengthy assays make any number of serial assays impractical. In previous work [48,49], it had been noted that if sensors were exposed to solution containing inhibitors and then allowed to dry, they could be... 

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. 

For regeneration, 500mM formic acid solution was used to dissociate the BZE-DADOO-cholinesterase complex. The sensor was used for more than 40 regeneration steps. Figure 3 shows the binding records for various concentrations of butyryl-cholinesterase. The binding of the enzyme to the immobilized BZE-DADOO could be detected at protein concentration down to 5 pg/mL. For the experiments with DFP, a certain BChE concentration was chosen (25 pg/mL) and incubated with different concentrations of DFP for 30 min. The results are shown in Fig. 4. The organophosphate could be detected at concentrations down to 0.1 nM. 

Teller, C., Halamek, J., Makower,A., Fournier, D., Schulze, H., and Scheller, F. W. (2006) A piezoelectric sensor with propidium as a recognition element for cholinesterases. Sensors and. Actuators B-Chemical 113, 214—221... 

Tran-Minh C., Pandey P.C., Kumaran S., (1990) Studies on acetylcholine sensor and its analytical application based on the inhibition of cholinesterase. Bios. Bioelectron., 5, 461-471... 

Cholinesterase based bioreactor for determination of pesticides Sensors Actuators B 18-19 689-93... 

Optimisation of the assembly of cholinesterase sensors is commonly directed toward the improvement of substrate detection, i.e. to achieve maximum signal, prolonged shelf-life, stable and reliable response. 

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