Colorimetric Detection of Organophosphate Pesticides Based on Acetylcholinesterase and Cysteamine Capped Gold Nanoparticles as Nanozyme

Organophosphates (OPs) are neurotoxic agents also used as pesticides that can permanently block the active site of the acetylcholinesterase (AChE). A robust and sensitive detection system of OPs utilising the enzyme mimic potential of the cysteamine capped gold nanoparticles (C-AuNPs) was developed. The detection assay was performed by stepwise addition of AChE, parathion ethyl (PE)-a candidate OP, acetylcholine chloride (ACh), C-AuNPs, and 3, 3′, 5, 5′-tetramethylbenzidine (TMB) in the buffer solution. The whole sensing protocol completes in 30–40 min, including both incubations. The Transmission Electron Microscopy (TEM) results indicated that the NPs are spherical and have an average size of 13.24 nm. The monomers of C-AuNPs exhibited intense catalytic activity (nanozyme) for the oxidization of TMB, revealed by the production of instant blue colour and confirmed by a sharp peak at 652 nm. The proposed biosensor’s detection limit and linear ranges were 5.8 ng·mL−1 and 11.6–92.8 ng·mL−1, respectively, for PE. The results strongly advocate that the suggested facile colorimetric biosensor may provide an excellent platform for on-site monitoring of OPs.

A novel luminol-based chemiluminescence method for detecting acetylcholine

Aim. To develop а new simple non-enzymatic method for the determination of acetylcholine (ACh) by the chemiluminescent reaction of luminol under conditions of the enzymatic hydrolysis of acetylcholine (pH 8.5).Experimental part. The method proposed is based on the perhydrolysis reaction of ACh by the excess ofhydrogen peroxide with the formation of peracetic acid. The latter was further determined by the activation effect of the luminol chemiluminescent oxidation reaction in the presence of hydrogen peroxide. The analytical signal was the summary luminescence (Σ) registered within certain time.Results and discussion. The pH range of the analytically applicable system was from 8.2 to 8.5. The effect of ACh + H2O2 incubation period on the reaction progress was also studied. The increase of the incubation period enhanced the sensitivity of the method (the limit of detection (LOD)), but because of practical reasons (especially the detection speed) and practical experience the incubation period was set to 30 min. The linear dependence was observed in the acetylcholine chloride concentration range of (0.8 – 2.8) × 10-4 mol/L. While determining acetylcholine chloride in the concentration range of (1.1 – 2.2) × 10-4 mol/L the relative standard deviation (RSD) did notexceed 3 % ((X – μ) × 100 %/μ = –0.5…+0.5 %). The Limit of Quantitation (LOQ, 10S) was 7.7 × 10-5 mol/L.Conclusions. A new non-enzymatic kinetic method for the chemiluminescent determination of ACh in aqueous solutions and the pharmaceutical formulation Acetylcholinchlorid Injeel® has been proposed. This method is simple, fast, inexpensive, and thus appropriate for the routine ACh quality control in the laboratories of hospitals, pharmaceutical industries and research institutions.Key words: acetylcholine; chemiluminescence method

IN VITRO ANTICONVULSANT EFFECT OF ETHYL ACETATE FRACTION OF TITANUS LEAF (LEEA AEQUATA L.) ON ISOLATED COLON

Objective: The excessive activity of the parasympathetic nervous system in the intestinal smooth muscle is important role in the increased intestinal motility, so antispasmodic medications are needed that can decrease intestinal motility such as atropine. Treatment may also use medicinal plants that are cheaper and easy to obtain, and also believed to have smaller side effects than modern antispasmodic drugs. This study aimed to determine the anticonvulsant or relaxation effects of the ethyl acetate fraction of titanus leaf (EAFTL) against contracted guinea pig colon.Methods: The parameters measured are smooth muscle relaxation. Before testing, guinea pig colon was equilibrated for 45 min to obtain a stable condition in Tyrode’s solution with a temperature of 37°C aerated with carbogen gas (O2:CO2) with a ratio of 95%:5%. The relaxing effect of the colon was tested after inducing by acetylcholine chloride; then, each colon was given cumulative concentration of EAFTL and atropine sulfate. The concentration of acetylcholine chloride required to increase the contraction of the guinea pig colon was 1.76×10−4 Mol. The cumulative concentration of EAFTL given was 0.5–4 mg/Ml and cumulative concentration of atropine sulfate given was 6.95×10−6–2.08×10−2 mg/Ml.Results: The EAFTL has a relaxing effect. Statistical analysis of EAFTL at a concentration of 4 mg/Ml (100.000±1.7417) in reducing the smooth muscle of colon contraction induced by acetylcholine chloride 1.76×10−4 Mol (p>0.005), has not statistically significant compared to that of atropine sulfate 6.95×10−3mg/Ml (105.7292±0.8161).Conclusion: EAFTL has relaxing effect on the smooth muscle of the colon.

Generation and Role of Oscillatory Contractions in Mouse Airway Smooth Muscle

Background/Aims: Tetraethylammonium chloride (TEA) induces oscillatory contractions in mouse airway smooth muscle (ASM); however, the generation and maintenance of oscillatory contractions and their role in ASM are unclear. Methods: In this study, oscillations of ASM contraction and intracellular Ca2+ were measured using force measuring and Ca2+ imaging technique, respectively. TEA, nifedipine, niflumic acid, acetylcholine chloride, lithium chloride, KB-R7943, ouabain, 2-Aminoethoxydiphenyl borate, thapsigargin, tetrodotoxin, and ryanodine were used to assess the mechanism of oscillatory contractions. Results: TEA induced depolarization, resulting in activation of L-type voltage-dependent Ca2+ channels (LVDCCs) and voltage-dependent Na+ (VNa) channels. The former mediated Ca2+ influx to trigger a contraction and the latter mediated Na+ entry to enhance the contraction via activating LVDCCs. Meanwhile, increased Ca2+-activated Cl- channels, inducing depolarization that resulted in contraction through LVDCCs. In addition, the contraction was enhanced by intracellular Ca2+ release from Ca2+ stores mediated by inositol (1,4,5)-trisphosphate receptors (IP3Rs). These pathways together produce the contractile phase of the oscillatory contractions. Furthermore, the increased Ca2+ activated the Na+-Ca2+ exchanger (NCX), which transferred Ca2+ out of and Na+ into the cells. The former induced relaxation and the latter activated Na+/K+-ATPase that induced hypopolarization to inactivate LVDCCs causing further relaxation. This can also explain the relaxant phase of the oscillatory contractions. Moreover, the depolarization induced by VNa channels and NCX might be greater than the hypopolarization caused by Na+/K+-ATPase alone, inducing LVDCC activation and resulting in further contraction. Conclusions: These data indicate that the TEA-induced oscillatory contractions were cooperatively produced by LVDCCs, VNa channels, Ca2+-activated Cl- channels, NCX, Na+/K+ ATPase, IP3Rs-mediated Ca2+ release, and extracellular Ca2+.