Amperometrijski biosenzor za određivanje D-laktata

A carbon paste electrode biosensor for D-lactic acid analysis in biological samples was developed. D-lactate cytochrome c oxidoreductase from baker’s yeast (EC 1.1.2.4) was used as biorecognition element of the electrode in combination with phenazine methosulphate (PMS)–Reinecke’s salt precipitate as mediator. The enzyme was bound on paste due to Enzacryl using glutardialdehyde. The electrode response to D-lactate addition was linear in the concentration range of 0.015–100 mM. Limit of detection was equal to 56 μM when signal vs. noise criterium (S/N=3) is considered. Specificity for only D-lactate was proven. Relative standard deviation of 6.6 % for 12 mM D-lactate was found. The biosensor exerts long-term stability and also good stability during repeated measuring cycles. The electrode has a good potential for use in practical samples.Za određivanje D-laktata u biološkim uzorcima razvijen je biosenzor s elektrodom od ugljikove paste. Kao element biološkog prepoznavanja na elektrodi korišten je D-laktat citokrom c oksidoreduktaza iz pekarskog kvasca (EC 1.12.4) u kombinaciji s fenazin metosulfatom istaloženim s Reinecke soli. Enzim je vezan na pastu na polimer Enzacryl te učvršćen glutaraldehidom. Odziv elektrode na dodatak D-laktata bio je linearan u rasponu koncentracije od 0,015 do 100 mM. Limit detekcije pri omjeru S/N=3 signala i šuma iznosio je 56 μM. Dokazana je specifičnost elektrode za D-laktat, te je utvrđena relativna standardna devijacija od 6,6 % za 12 mM D-laktata. Biosenzor je pokazao dugoročnu stabilnost, te dobru stabilnost pri ponavljanju ciklusa mjerenja. Elektroda se može primijeniti u praksi za analizu uzoraka

Variation of Cholinesterase-Based Biosensor Sensitivity to Inhibition by Organophosphate Due To Ionizing Radiation

A cholinesterase based biosensor was constructed in order to assess the effects of ionizing radiation on exposed AChE. Although the primary objective of the experiment was to investigate the effect of ionizing radiation on the activity of the biosensor, no changes in cholinesterase activity were observed. Current provided by oxidation of thiocholine previously created from acetylthiocholine by enzyme catalyzed reaction was in a range 395–455 nA. No significant influence of radiation on AChE activity was found, despite the current variation. However, a surprising phenomenon was observed when a model organophosphate paraoxon was assayed. Irradiated biosensors seem to be more susceptible to the inhibitory effects of paraoxon. Control biosensors provided a 94 ± 5 nA current after exposure to 1 ppm paraoxon. The biosensors irradiated by a 5 kGy radiation dose and exposed to paraoxon provided a current of 49 ± 6 nA. Irradiation by doses ranging from 5 mGy to 100 kGy were investigated and the mentioned effect was confirmed at doses above 50 Gy. After the first promising experiments, biosensors irradiated by 5 kGy were used for calibration on paraoxon and compared with the control biosensors. Limits of detection 2.5 and 3.8 ppb were achieved for irradiated and non-irradiated biosensors respectively. The overall impact of this effect is discussed

ELISA Detection of Francisella tularensis using Polyclonaland Monoclonal Antibodies

The mouse monoclonal and polyclonal antibodies were produced for the detection of intracellular pathogenand potential warfare agent Francisella tularensis. Antibody titers obtained were 1:640 for polyclonal antibodiesand 1:320 for monoclonal antibodies. Both antibodies were used in the indirect enzyme-linked immunosorbentassay (ELISA) found to detect F. tularensis whole cells. The limit of detection was 5.4×106 CFU/ml for polyclonalantibodies and 6.9×106 CFU/ml for monoclonal antibodies. The value sample could  be distinguished from anyconcentration of another gram-negative bacterium: Escherichia coli.Defence Science Journal, 2008, 58(5), pp.698-702, DOI:http://dx.doi.org/10.14429/dsj.58.169

Sarin Assay using Acetylcholinesterases and Electrochemical Sensor Strip

An electrochemical sensor strip was used for sarin assay. Three different acetylcholinesterases (AChEs) were chosen as promising recognition elements. viz., human recombinant, electric eel, and bovine erythrocytes origin. Human recombinant AChE seems to be the most sensitive to inhibition by sarin when the achieved limit of detection (0.45×10-8 mol/l) and IC50 [(9.77± 8.08)×10-6 mol/l] are considered. On the contrary, AChE from bovine erythrocytes proved to reach highest IC50 (5.37± 4.52)×10-7 mol/l and the one from electric eel reached the highest limit of detection 0.93×10-8 mol/l. From the AChEs tested as biorecognition element, human recombinant seems to be the best for construction of new ChE detectors.Defence Science Journal, 2009, 59(3), pp.300-304, DOI:http://dx.doi.org/10.14429/dsj.59.152

Amperometrijski biosenzor za određivanje D-laktata

A carbon paste electrode biosensor for D-lactic acid analysis in biological samples was developed. D-lactate cytochrome c oxidoreductase from baker’s yeast (EC 1.1.2.4) was used as biorecognition element of the electrode in combination with phenazine methosulphate (PMS)–Reinecke’s salt precipitate as mediator. The enzyme was bound on paste due to Enzacryl using glutardialdehyde. The electrode response to D-lactate addition was linear in the concentration range of 0.015–100 mM. Limit of detection was equal to 56 μM when signal vs. noise criterium (S/N=3) is considered. Specificity for only D-lactate was proven. Relative standard deviation of 6.6 % for 12 mM D-lactate was found. The biosensor exerts long-term stability and also good stability during repeated measuring cycles. The electrode has a good potential for use in practical samples.Za određivanje D-laktata u biološkim uzorcima razvijen je biosenzor s elektrodom od ugljikove paste. Kao element biološkog prepoznavanja na elektrodi korišten je D-laktat citokrom c oksidoreduktaza iz pekarskog kvasca (EC 1.12.4) u kombinaciji s fenazin metosulfatom istaloženim s Reinecke soli. Enzim je vezan na pastu na polimer Enzacryl te učvršćen glutaraldehidom. Odziv elektrode na dodatak D-laktata bio je linearan u rasponu koncentracije od 0,015 do 100 mM. Limit detekcije pri omjeru S/N=3 signala i šuma iznosio je 56 μM. Dokazana je specifičnost elektrode za D-laktat, te je utvrđena relativna standardna devijacija od 6,6 % za 12 mM D-laktata. Biosenzor je pokazao dugoročnu stabilnost, te dobru stabilnost pri ponavljanju ciklusa mjerenja. Elektroda se može primijeniti u praksi za analizu uzoraka

Evaluation of Cholinesterase Activities During in Vivo Intoxication Using an Electrochemical Sensor Strip – Correlation With Intoxication Symptoms

Cholinesterase activity in blood of laboratory rats was monitored. Rats were intoxicated with paraoxon at dosis of 0 – 65 – 125 – 170 – 250 – 500 nmol. The 250 nmol dose was found to be the LD50. An electrochemical sensor was found useful to provide information about cholinesterase activity. The decrease of cholinesterase activity was correlated to intoxication symptoms and mortality level. It was found that the symptoms of intoxication are not observed while at least 50% of cholinesterase activity in blood remains. The minimal cholinesterase activity essential to survival is around 10%, when compared with the initial state. No changes in levels of low moleculary weight antioxidants were observed

Acetylcholinesterase Inhibitors Assay Using Colorimetric pH Sensitive Strips and Image Analysis by a Smartphone

Smartphones are widely spread and their usage does not require any trained personnel. Recently, smartphones were successfully used in analytical chemistry as a simple detection tool in some applications. This paper focuses on immobilization of acetylcholinesterase (AChE) onto commercially available pH strips with stabilization in the gelatin membrane. AChE degrades acetylcholine into choline and acetic acid which causes color change of acid-base indicator. Smartphone served as a tool for measurement of indicator color change from red to orange while inhibitors blocked this process. AChE inhibitors were measured with limits of detection, 149 nM and 22.3 nM for galanthamine and donepezil, respectively. Organic solvents were measured for method interferences. Measurement procedure was performed on 3D printed holder and digital photography was evaluated using red-green-blue (RGB) channels. The invented assay was validated to the standard Ellman's test and verified on murine plasma samples spiked with inhibitors. We consider that the assay is fully suitable for practical performance

Ferric Reducing Antioxidant Power and Square Wave Voltammetry for Assay of Low Molecular Weight Antioxidants in Blood Plasma: Performance and Comparison of Methods

The purpose of the present study was to employ two methods—square wave voltammetry (SWV) performed on screen printed sensors and ferric reducing antioxidant power (FRAP)—as suitable tools for the assay of low-molecular-weight antioxidants (LMWAs). LMWAs were assayed by both methods and the resulting data were statistically compared. Plasma samples from five Cinereous vultures accidentally intoxicated with lead were used to represent real biological matrices with different levels of LMWAs. Blood was collected from the birds prior to and one month after treatment with Ca-EDTA. SWV resulted in two peaks. The first peak, with the potential value of 466 ± 15 mV, was recognized as ascorbic and uric acids, while the second one (743 ± 30 mV) represented glutathione, tocopherol, ascorbic acid and in a minor effect by uric acid, too. Contribution of individual antioxidants was recognized by separate assays of LMWA standards. Correlation between peaks 1 and 2 as well as the sum of the two peaks and FRAP was analysed. While peak 1 and the sum of peaks were in close correlation to FRAP results (correlation coefficient of 0.97), the relation between peak 2 and FRAP may be expressed using a correlation coefficient of 0.64. The determination of thiols by the Ellman assay confirmed the accuracy of SWV. Levels of glutathione and other similar structures were stable in the chosen model and it may be concluded that SWV is appropriate for assay of LMWAs in plasma samples. The methods employed in the study were advantageous in minimal sample volume consumption and fast acquisition of results