Adam Kostelnik

Color change of phenol red by integrated smart phone camera as a tool for the determination of neurotoxic compounds

The use of a cell phone as a detection system is easy, simple and does not require trained personnel, which is in contrast to standard laboratory instruments. This paper deals with immobilization of acetylcholinesterase (AChE) in a gelatin matrix, and phenol red, as an indicator of AChE activity, is used in order to establish a method that is easily compatible with a camera device. AChE splits acetylcholine into choline and acetic acid, which changes the pH of a medium, resulting in a phenol red color change. The coloration changed in presence of an AChE inhibitor. Measurements were performed on 3D-printed, tube-shaped holder, and digital photography, with subsequent analysis of red-green-blue (RGB), served for assay purposes. Calibration of AChE inhibitors, tacrine and galantamine, was performed, with limit of detection equal to 1.1 nM and 1.28 µM, respectively. Interferences were also measured, resulting in a proof-of-method stability. The method was further successfully validated for the standard Ellman’s assay, and verified on murine plasma samples spiked with inhibitors.

Electrochemical determination of activity of acetylcholinesterase immobilized on magnetic particles

Acetylcholinesterasa (AChE) je enzym ukoncujici přenos vzruchů v cholinergnim systemu. Aktivita enzymu se běžně vysetřuje Ellmanovou metodou, ktera ma ovsem nedostatky jako je nestabilita použitých reagencii. Dalsi nevýhodou je neschopnost využit enzym znovu. Proto jsme vyvinuli proces využivajici EDC pro imobilizaci AChE na povrch magnetických castic, ktere nabizeji možnost separovat enzym a znovu jej využit. Pro stanoveni enzyove aktivity jsme optimalizovali metodu square wave voltametrie za použiti tistěných elektrod modifikovaných Pruskou modři. Pro testovaný inhibitor AChE, takrin, jsme ziskali detekcni limit 8,1 μM. V porovnani se standardni Ellmanovou metodou, imobilizovaný enzym ma limitovanou citlivost k organickým rozpoustědlům.

Construction of an Acetylcholinesterase Sensor Based on Synthesized Paramagnetic Nanoparticles, a Simple Tool for Neurotoxic Compounds Assay

Magnetic particles (MPs) have been widely used in biological applications in recent years as a carrier for various molecules. Their big advantage is in repeated use of immobilized molecules including enzymes. Acetylcholinesterase (AChE) is an enzyme playing crucial role in neurotransmission and the enzyme is targeted by various molecules like Alzheimer’s drugs, pesticides and warfare agents. In this work, an electrochemical biosensor having AChE immobilized onto MPs and stabilized through glutaraldehyde (GA) molecule was proposed for assay of the neurotoxic compounds. The prepared nanoparticles were modified by pure AChE and they were used for the measurement anti-Alzheimer’s drug galantamine and carbamate pesticide carbofuran with limit of detection 1.5 µM and 20 nM, respectively. All measurements were carried out using screen-printed sensor with carbon working, silver reference, and carbon auxiliary electrode. Standard Ellman’s assay was used for validation measurement of both inhibitors. Part of this work was the elimination of reversible inhibitors represented by galantamine from the active site of AChE. For this purpose, we used a lower pH to get the original activity of AChE after inhibition by galantamine. We also observed decarbamylation of the AChE-carbofuran adduct. Influence of organic solvents to AChE as well as repeatability of measurement with MPs with AChE was also established.

Determination of selected fatty acids in dried sweat spot using gas chromatography with flame ionization detection

A method is described for the determination of fatty acids in dried sweat spot and plasma samples using gas chromatography with flame ionization detection. Plasma and dried sweat spot samples were obtained from a group of blood donors. The sweat was collected from each volunteer during exercise. Sweat was spotted onto collection paper containing butylated hydroxytoluene. Fatty acids were derivatized with acetyl chloride in methanol to form methyl esters of fatty acids. The fatty acids in dried sweat spot samples treated with butylated hydroxytoluene and stored at -20°C were stable for 3 months. Our results indicate that sweat contains, among fatty acids with short chain, also fatty acids with long chain and unsaturated fatty acids. Linear relationships between percentage content of selected fatty acids in dried sweat spot and plasma 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 Ellmans test and verified on murine plasma samples spiked with inhibitors. We consider that the assay is fully suitable for practical performance.

DIAGNOSTIKA AUTOIMUNITNÍCH CHOROB

Jako autoimunitni oznacujeme ty choroby, ktere jsou způsobeny reakci imunitniho systemu proti zdravým, tělu vlastnim strukturam. Diagnostika takovýchto stavů vyžaduje souhru klinicke casti s casti laboratorni. Tento text se zaměřuje na diagnostiku vybraných systemových a organově specifických autoimunitnich chorob. Neklade si ale za cil podrobně popsat jednotlive choroby, spise shrnuje nejvýraznějsi voditka vedouci k jejich spravne diagnoze. Rovněž nastiňuje použivane laboratorni metody a shrnuje jejich výhody a nevýhody.V případě kdy se pacient dostane k lékaři, jako první přichází na řadu fyzikální vyšetření, kdy lékař zjišťuje vnější projevy, např. otok, zarudnutí, popř. prohmatání bolestivého místa atd. Toto vyšetření je následně doprovázeno vyšetřením přístrojovým (např. ultrazvuk, rtg, aj.). Tato vyšetření jsou ovšem nespecifická a musí být doplněna o vyšetření laboratorní, kdy zjišťujeme přítomnost specifických markerů v krvi nebo jiném biologickém materiálu pacienta.

Inhibition of Acetylcholinesterase and Butyrylcholinesterase by a Plant Secondary Metabolite Boldine

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are two enzymes sensitive to various chemical compounds having ability to bind to crucial parts of these enzymes. Boldine is a natural alkaloid and it was mentioned in some older works that it can inhibit some kinds of AChE. We reinvestigated this effect on AChE and also on BChE using acetyl (butyryl) thiocholine and Ellmans reagents as standard substances for spectrophotometric assay. We found out IC50 of AChE equal to 372 μmol/l and a similar level to BChE, 321 μmol/l. We conclude our experiment by a finding that boldine is cholinesterase inhibitor; however we report significantly weaker inhibition than that suggested in literature. Likewise, we tried to investigate the mechanism of inhibition and completed it with in silico study. Potential toxic effect on cholinesterases in real conditions is also discussed.

Superficially Bound Acetylcholinesterase Based on a Chitosan Matrix for Neurotoxic Compound Assay by a Photographic Technique

ABSTRACT Smartphones have become popular in the last decade and they have been used in analytical chemistry as easy detection systems with comparable parameters to standard laboratory equipment. This work focuses on the attachment of enzyme acetylcholinesterase on the previously activated chitosan. Acetylcholine splits the neurotransmitter acetylcholine, as a natural substrate, into choline and acetic acid. However, this compound can cleave alternative substrates, e.g., indoxyl acetate, which was used in this work. The conversion of the substrate is blocked or slowed down by inhibitors from which galantamine and tacrine were tested as model inhibitors with detection limits of 1.1 and 0.18 µM, respectively. The measurement procedure was performed on a three-dimensional printed holder and red–green–blue channels were used for digital photography evaluation. The method was validated using a standard Ellman’s spectrophotometric assay. We successfully attached acetylcholinesterase on the chitosan surface that was used for the inhibitor assay. The long-term stability of the immobilized enzyme as well as the sensitivity to organic solvents were also tested. The proposed method appeared to be suitable for the rapid and inexpensive assay of neurotoxic compounds.

Optical fiber based acetylcholinesterase sensor with activity determination by phenol red

A of tuna fish products is necessary to assure consumers of accurate labeling of food products. The most common methods that can be used to identify tuna species include methods based on detection of specific DNA (PCR and its modification: real-time PCR, digital PCR etc.). The quality of species specific DNA crucially affects the efficiency of amplification during the subsequent PCR. The question in DNA detection in processed products lies in the possibility of the DNA fragmentation during the processing technologies and the use of ingredients that may inhibit the PCR reaction. In this study, three DNA extraction methods were compared: DNeasy Blood and Tissue Kit, DNeasy mericon Food Kit and Chemagic DNA Tissue 10 Kit. The quantity and quality of DNA were estimated by measuring DNA concentration and ratios A260/A280. The amplifiability was tested by using a set of primers designed to detect the fragments of COI gene of different sizes ranges from 100 bp to 500 bp. Several parameters were evaluated: The effect of heat treatment (boiling at 70oC and 90oC), cold and hot smoking, sterilization procedure of whole and mechanically treated muscle used in canned process (high temperature in combination with high pressure), and pate and spread.A peroxidase biosensor is highly demanded in food, healthcare, pharmaceutical and environmental analysis. H2O2 is often a residue component of non-food product. Moreover, H2O2 is used as an antibacterial agent added to milk or it is used for sterilization of equipment related to food and beverage during the both technological manufacturing and packaging. European union countries contain of H2O2 higher than 0.1% which is not allowed in these products. If the level of H2O2 exceeds 6%, health damage or at least skin irritation can be expected. Due to toxicity of H2O2 in higher concentrations sensitive method for its detection is required. In this work, a voltammetric biosensor based on horseradish peroxidase, magnetic particles and Prussian blue entrapped in chitosan membrane onto carbon working electrode was proposed as a suitable tool for the assay purposes. Magnetic particles contained pseudo-peroxidase activity catalyzed reduction of H2O2 and Prussian blue enabled highly sensitive detection at low potential. Square wave voltammetry was used as detection method. Concentration curve replies Michaelis equation with correlation coefficient 0.999 and limit of detection was set to be 0.03% of H2O2. Uric acid, ascorbic acid, trolox, acetaminophen and reduced glutathione were measured as interferents and no significant influence on measured method was observed when presented in equivalent concentrations like the H2O2. No effect of matrix (tooth paste, hand cream and skin tonic) assay was detected. On the basis of gained results, method was considered as highly sensitive, accurate and fast assay for detection of H2O2.

Anti-Parkinson Drug Biperiden Inhibits Enzyme Acetylcholinesterase

Biperiden is a drug used in Parkinson disease treatment and it serves also as an antiseizures compound in organophosphates poisoning. It acts as antagonist of muscarinic receptor activated by acetylcholine while the enzyme acetylcholinesterase (AChE) cleaves acetylcholine in synaptic junction into choline and acetic acid. This enzyme is inhibited by various compounds; however there has not been proposed evidence about interaction with biperiden molecule. We investigated this interaction using standard Ellmans assay and experimental findings were critically completed with an in silico prediction by SwissDock docking software. Uncompetitive mechanism of action was revealed from Dixon plot and inhibition constant (Ki) was calculated to be 1.11 mmol/l. The lowest predicted binding energy was −7.84 kcal/mol corresponding to H-bond between biperiden molecule and Tyr 341 residuum in protein structure of AChE. This interaction seems to be further stabilized by π-π interaction with Tyr 72, Trp 286, and Tyr 341. In conclusion, biperiden appears as a very weak inhibitor but it can serve as a lead structure in a pharmacological research.