Cholinesterases immobilization

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. 

In subsequent studies (64,65), various cholinesterases were tested as coatings, and were immobilized using different agents glutaraldehyde, bovine serum albumin, diazo coupling, and carbodiimide. Eel Cholinesterase, immobilized with glutaraldehyde exhibited the highest sensitivity for DIMP. 

In another report biosensors with cholinesterases immobilized on membranes were used in a FIA system. Paraoxon and carbaryl were determined with a detection limit of 0.1 M with 4-aminophenyl acetate as the substrate. A commercial Metrohm flow-through cell with a glassy carbon electrode was used. In such a system determinations in extracts from kiwi fruits treated with carbaryl were carried out. For a concentration of 3 ppm of the analyte in the extract a good agreement with the UV determinations was obtained. 

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. 

Durand P, Nicaud JM, Mallevialle J. 1984. Detection of organophosphorus pesticides with an immobilized cholinesterase electrode. J Anal Toxicol 8 112-117. 

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. 

Andreou V., Clonis Y., A portable fiber-optic pesticide biosensor based on immobilized cholinesterase and sol-gel entrapped bromocresol purple for in-field use, Biosens. Bioelectr. 2002 17 61-69. 

Procedure Cholinesterase activity in analyzed tissue or the matrix (biotest with immobilized AChE) is determined in the incubation media [consisting of substrate ATCh - 34 mmol maleate buffer 0.1 M, pH = 6.0- 6.5 ml sodium citrate 0.1 M - 0.5 ml CuS045H20 0.03M -1.0 ml distilled H20 (or inhibitor in variant with toxin analyzed) -1.0 ml potassium ferricyanide 0.005 M -1 ml.] Volume of incubation media in one test - 400 mcl. As a blank (control sample), a treatment of the exposure without the substrate is used. If inhibitory effects of allelochemical (or any toxin) are analyzed, before the substrate addition the sample was preliminary exposed to allelochemical inhibitor. Two methods for the AChE-biotests may be recommended (i) in microcells ( stationary conditions ) and (ii) in flowing columns-reactors ( dynamic conditions ). 

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). 

M.N. Hendji, N. Jaffrezic-Renault, C. Martelet, P. Clechet, A.A. Shulga, V.I. Strikha, L.I. Netchipruk, A.P. Soldatkin, and W.B. Wlodarski, Sensitive detection of pesticides using a differential ISFET-based system with immobilized cholinesterases. Anal. Chim. Acta 281, 3—11 (1993). 

K.C. Gulla, M.D. Gouda, M.S. Thakur, and N.G. Karanth, Reactivation of immobilized acetyl cholinesterase in an amperometric biosensor for organophosphorus pesticide. Biochim. Biophys. Acta... 

Mechanism of Action A benzimidazole carbamate anthelmintic that degrades parasite cytoplasmic microtubules, irreversibly blocks cholinesterase secretion, glucose uptake in helminth and larvae (depletes glycogen, decreases ATP production, depletes energy). Vermicidal. Therapeutic Effect Immobilizes and kills worms. Pharmacokinetics Poorly and variably absorbed from GI tract. Widely distributed, cyst fluid and including cerebrospinal fluid (CSF). Protein binding 70%. Extensively metabolized in liver. Primarily excreted in urine and bile. Not removed by hemodialysis. Half-life 8-12 hr. 

Brown WE, Shamoo AY, Hill BL, et al. 1984. Immobilized cholinesterase to detect airbome concentrations of hexamethylene diisocyanate (HDl). Toxicol Appl Pharmacol 73(1) 105-109. 

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. 

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

W.E. Brown, A.Y. Shamoo, B.L. Hill and M.H. Karol, Immobilized cholinesterase to detect airborne concentrations of hexamethylene diisocyanate (HDI), Toxicol. Appl. Pharmacol., 73 (1984) 105-109. 

Immobilization of cholinesterase, like that of other enzymes, has a number of potentially valuable applications, including active-site studies, economical use of the enzyme as an analytical reagent, and enhancement of the storage life of the enzyme. 

Enzyme electrodes are essentially immobilized enzyme systems. Crochet and Montalvo (C15) developed a technique for cholinesterase based on coupling a small pH electrode to a thin polymer membrane. At the electrode surface, cholinesterase interacted with acetylcholine to produce acetic acid, which was detected by the pH electrode. Excellent sensitivity was achieved by the use of a very thin film of enzyme solution, with extremely low strength of buffer containing the enzyme and almost complete suppression of spontaneous hydrolysis of the substrate. 

Aldrich, F. L., Usdin, V., and Vasta, B, Method of preparing immobilized serum cholinesterase and product thereof. U.S, Patent 3,223,593 (1965). 

Degani, Y., and Miron, T., Immobilization of cholinesterase in cross-linked polyacrylamide. Biochim. Biophys. Acta 212, 362-364 (1970). 

G29. Goutier, R., Etude 61ectrophor6tique des esterases s6riques et de la fixation du DF P dans le s6rum chez le lapin et la cobage. Biochim, Biophys. Acta 19, 524-534 (1956). Guilbault, G. G., and Das, J., Immobilization of cholinesterase and urease. Anal. Biochem. 33, 341-355 (1970). 

This reaction system is being utilized commercially in the Bioalarm instruments of Thom EMI (England) and Midwest Research Instruments (USA) incorporating immobilized cholinesterase. 

Fig. 1. Structures of the immobilized ligands used for cholinesterase recognition BZE-DADOO, paraoxon-aminohexanol, paraoxon-hexahistidine-Ni2+-NTA-lysine, propidium (from top), each bound to MUAon gold.

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. 

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