A sensitive enzymatic method for paraoxon detection based on enzyme inhibition and fluorescence quenching

A sensitive and selective method for the paraoxon detection based on enzyme inhibition and fluorescence quenching was presented in this study. Under the catalytic effect of acetylcholinesterase (AChE), acetylthiocholine (ATCh) hydrolysis released thiocholine (TCh) which could react with N-(7-dimethylamino-4-methylcoumarin-3-yl) maleimide (DACM) to produce a blue fluorescence compound. Subsequently, AChE catalytic activity was inhibited with the addition of paraoxon, which caused TCh decreased, leading to a significant decrease of the blue fluorescent compound. Meanwhile, p-nitrophenol, the hydrolysis product of paraoxon, would lead to a quenching of the fluorescence. Therefore, fluorescence intensity of the system would decrease dramatically by a combined effect of enzyme inhibition and fluorescence quenching. Under optimal experimental conditions, an excellent linear relationship between the decrease of fluorescence intensity and paraoxon concentration over the range from 5.5 × 10⁻¹² to 1.8 × 10⁻¹⁰ mol L⁻¹ was obtained. Fluorescence background caused by nonenzymatic hydrolysis of ATCh or other matters was relatively low, the proposed approach offered adequate sensitivity for the detection of paraoxon at 3.5 × 10⁻¹² mol L⁻¹.     

Photolithographically patterned enzyme membranes for the detection of pesticides and copper(II) based on enzyme inhibition

Summary A non-aqueous and an aqueous photopolymer system with an enzyme are used to prepare photolithographically patterned enzyme membranes for amperometric (thinfilm platinum electrode) and potentiometric (ISFET) sensors based on enzyme inhibition. Flow methods for enzyme inhibition tests are described. The decrease in enzyme (AChE) activity after incubation in a solution of dichlorvos as inhibitor is detected amperometrically. The enzyme urease is immobilized onto the pH-sensitive gate area of an ISFET. Such a biosensor is able to detect copper-(II) in water in the ppm-range without preconcentration.Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthday     

Multibiosensor based on enzyme inhibition analysis for determination of different toxic substances

An original concept of an enzyme multibiosensor for determination of toxic substances based on enzyme inhibition analysis has been proposed and its main performances have been analysed. For the development of this multibiosensor, two types of transducers such as potentiometric pH-sensitive field-effect transistors and conductometric thin-films interdigitated electrodes, and three enzymes, namely urease, acetylcholinesterase and butyrylcholinesterase have been used. The experimental data have been treated by multivariate correspondence analysis. A complete procedure for a simultaneous determination of some heavy metal ions and pesticides has been proposed and its advantages have been discussed.     

Use of a peroxidase reactor in flow injection analysis for the determination of chloramine and the inhibition kinetics

A method for the determination of chloramine using a flow injection peroxidase reactor based on the inhibition reaction of the enzyme is developed. The immobilisation of the horseradish peroxidase is performed on the commercial polymer carrier VA Epoxy Biosynth. The peroxidase activity is detected photometrically based on the dehydration of the dye 2,2′-azino-di-[3-ethylenebenzthiazoline-6-sulphonate] (ABTS). The calibration of the method gives a linear concentration range from 0.026 to 1.04 mmol l⁻¹ (SD_n=3=below 5%). The detection limit was calculated to 26 μmol l⁻¹. A mixture out of competitive and non-competitive inhibition was analysed based on the Lineweaver–Burk plots.     

An inhibition type amperometric biosensor based on tyrosinase enzyme for fluoride determination

In this study, a new biosensor based on the inhibition of tyrosinase for the determination of fluoride is described. To construct the biosensor tyrosinase was immobilized by using gelatine and cross-linking agent glutaraldehyde on a Clark type dissolved oxygen (DO) probe covered with a teflon membrane which is sensitive for oxygen. The phosphate buffer (50 mM, pH 7.0) at 30 °C were established as providing the optimum working conditions. The method is based on the measurement of the decreasing of dissolved oxygen level of the interval surface that related to fluoride concentration added into reaction medium in the presence of catechol. Inhibitor effect of fluoride results in decrease in dissolved oxygen concentration. The biosensor response depends linearly on fluoride concentration between 1.0 and 20 μM with a response time of 3 min.In the characterization studies of the biosensor some parameters such as reproducibility, substrate specificity and storage stability were carried out. From the experiments, the average value (x), Standard deviation (S.D) and coefficient of variation (C.V %) were found as 10.5 μM, ± 0.57 μM, 5.43%, respectively for 10 μM fluoride standard.     

A new X-ray method for the study of crystallization kinetics

Summary A new method for the study of crystallization kinetics has been developed which is based on time dependent intensity measurements of the diffuse X-ray small-angle scattering. This scattering is predominantly due to the electron density fluctuations within the crystalline and amorphous domains and has been found to vary linearly with the crystallinity.The method has been applied to the study of the crystallization kinetics of unstretched natural rubber at 248 K. The precision of the data obtained permit a quantitative check of the validity of the Avrami equation for the interpretation of the crystallization isotherm up to relatively high crystallinities. It is shown that this check allows an accurate determination of the final crystallinity related to the primary crystallization process together with the parameters of the Avrami equation.

---

Zusammenfassung Es wurde ein neues Verfahren zur Untersuchung der Kristallisationskinetik entwickelt, das auf zeitabhängigen Intensitätsmessungen der diffusen Röntgenkleinwinkelstreuung beruht. Das Verfahren benutzt die Tatsache, daß die diffuse Röntgenkleinwinkel-streuung überwiegend durch die Elektronendichtefluktuation innerhalb der kristallinen und amorphen Bereiche bedingt ist und sich linear mit der Kristallinität ändert.Zur experimentellen Überprüfung wurde das Verfahren für die Untersuchung der Kristallisationskinetik von unverstrecktem Naturkautschuk bei 248 K benutzt. Die Genauigkeit der hierbei erhaltenen Meßdaten gestattet eine quantitative Überprüfung der Gültigkeit der Avrami-Gleichung für die Auswertung der Kristallisationsisothermen bis zu relativ hohen Kristallinitäten. Es zeigt sich, daß dieses Verfahren für eine genaue Bestimmung der zum Prozeß der Primärkristallisation gehörenden Endkristallinität zusammen mit der Ermittlung der Parameter der Avrami-Gleichung geeignet ist.

     

Graphic rule for non-steady-state enzyme kinetics and protein folding kinetics

When going more deeply into the principles of enzyme action as well as protein folding, one is often confronted with transient process systems. Based on the recent progress in graphic methods of enzyme kinetics, in this article a graphic rule is described, which can be used to deal with transient processes occurring in both enzyme-catalyzed reaction systems and protein folding systems. Introducing the graphic method to nonsteady-state systems can raise the efficiency of the calculations and provide an intuitive picture, helping the analysis of the mechanisms concerned. For instance, using the current graphic rule, one can immediately write out the phase concentrations of enzyme species or protein folding states. Calculation work such as setting up differential equations, making Laplace transformations, and expanding determinants, which is both tedious and liable to error, is completely avoided. The mathematical proof of the non-steady-state graphic rule is given in the appendix.Presented at the Symposium on Applied Graph Theory and Discrete Mathematics in Chemistry held at the University of Saskatchewan, Saskatoon, Canada, September 12-14, 1991, in honor of Professor Frank Harary on the occasion of his 70th birthday.     

Automated portable analyzer for lead(II) based on sequential flow injection and nanostructured electrochemical sensors

A fully automated portable analyzer for toxic metal ion detection based on a combination of a nanostructured electrochemical sensor and a sequential flow injection system has been developed in this work. The sensor was fabricated from a carbon paste electrode modified with acetamide phosphonic acid self-assembled monolayer on mesoporous silica (Ac-Phos SAMMS) which was embedded in a very small wall-jet (flow-onto) electrochemical cell. The electrode is solid-state and mercury-free. Samples and reagents were injected into the system and flowed through the electrochemical cell by a user programmable sequential flow technique which required minimal volume of samples and reagents and allowed the automation of the analyzer operation. The portable analyzer was evaluated for lead (Pb) detection due to the excellent binding affinity between Pb and the functional groups of Ac-Phos SAMMS as well as the great concern for Pb toxicity. Linear calibration curve was obtained in a low concentration range (1-25 ppb of Pb(II)). The reproducibility was excellent; the percent relative standard deviation was 2.5 for seven consecutive measurements of 10 ppb of Pb(II) solution. Excess concentrations of Ca, Ni, Co, Zn, and Mn ions in the solutions did not interfere with detection of Pb, due to the specificity and the large number of the functional groups on the electrode surface. The electrode was reliable for at least 90 measurements over 5 days. This work is an important milestone in the development of the next-generation metal ion analyzers that are portable, fully automated, and remotely controllable.     

Applicability of the quasi-adiabatic enthalpimetric method for the study of chemical kinetics

The thermal quasi-adiabatic method and the non-adiabatic one are compared for the study of chemical kinetics. With thermistors as temperature-sensitive elements, and a very simple apparatus, moderately fast reactions (with t(1 2 ) down to 2 sec) and consecutive reactions can be followed, and mixtures analysed with good accuracy. The reproducibility is satisfactory over a wide range of experimental conditions. The calculation of the kinetic parameters, for both first-and second-order kinetics is very simple and requires no calibration of the system. Application of the technique to the study of the alkaline hydrolysis of some alkyl acetates is described.     

A method for studying swelling kinetics based on measurement of electrical conductivity

A novel method was applied to the study of swelling kinetics of pH-responsive hydrogels. This technique is based on the pH-dependent electrical conductivity of these materials, which is measured by coating planar interdigitated electrode arrays with thin hydrogel membranes. To demonstrate the utility of the method, the swelling kinetics of a well-characterized pH-responsive hydrogel were studied. Cross–linked copolymers of 2-hydroxyethyl methacrylate (HEMA) with up to 20 mol% dimethylaminoethyl methacrylate (DMA) were studied as a function of copolymer composition in phosphate or triethanolamine buffer at buffer concentrations from 1 to 100 mM. The experiments consisted of measuring the change in electrical resistance of a hydrogel-coated electrode array following a small pH change in the external buffer medium. The characteristic response time to reach a new equilibrium following a pH change was proportional to the concentration of DMA within the polymer and was inversely proportional to the buffer concentration. The characteristic response times for devices tested in phosphate buffer were a function of the magnitude of the pH step, increasing from 2.6 to 5.6 min as the step size increased from 0.2 to 0.57 pH units. However, the response times for devices tested in triethanolamine were independent of step size. The observed dependences upon the values of the dissociation constant (pK_a) of the buffering ion, the apparent pK_a of DMA, and the pH of the external bath agreed with buffer-mediated diffusion–reaction theory, and as such this conductimetric method represents a powerful tool for the study of swelling kinetics of responsive hydrogels.     

Isofagomine lactams,synthesis and enzyme inhibition

The synthesis of isofagomine lactams (2-oxoisofagomines) corresponding to the biologically important hexoses is presented. The D-glucose/D-mannose analogue (3S,4R,5R)-3,4-dihydroxy-5-hydroxymethylpiperidin-2-one (9) was synthesised in 9 steps from D-arabinose, the D-galactose analogue (3S,4S,5R)-3,4-dihydroxy-5-hydroxymethylpiperidin-2-one (10) was synthesised in 11 steps from D-arabinose and the L-fucose analogue (3R,4R,5R)-3,4-dihydroxy-5-methylpiperidin-2-one (11) was synthesised in 12 steps from L-arabinose. The three lactams 9-11 were found to be glycosidase inhibitors with micro- to nanomolar inhibition constants. The lactam 10 showed slow onset inhibition of beta-galactosidase from A. Oryzae. The rate constants for this process were determined to be k(on) = 2.55 x 10(4) M-1 s-1 and k(off) = 1.7 x 10(-3) s-1. The activation energies and standard thermodynamic functions were also determined.     

Methyltransferase activity assay based on the use of exonuclease III,the hemin/G-quadruplex system and reduced graphene oxide on a gold electrode,and a study on enzyme inhibition

We describe an electrochemical bioassay for the detection of the activity of methyltransferase (MTase), and for screening this enzyme’s inhibitors. The assay is based on the conjugation of a hemin to a G-quadruplex that enables enzymatic signal amplification with the aid of exonuclease III (ExoIII). In the first step, double-stranded DNA containing the quadruplex-forming oligomer is assembled on the surface of a gold electrode and then methylated by DNA adenine methyltransferase (DAM). After cleaved by endonuclease DpnI, the methylated DNA is digested by ExoIII and the quadruplex-forming oligomers are liberated. This leads to the formation of a hemin/G-quadruplex (in presence of hemin and of potassium ions). The hemin/G-quadruplex catalyzes the oxidization of hydroquinone by H₂O₂ and the benzoquinone was formed to generate electrochemical signal. Finally, the gold electrode modified with reduced graphene oxide was used as working electrode for performing differential pulse voltammetry. The method has a detection limit of 0.31 unit · mL⁻¹. A study on the inhibition of MTase showed it was inhibited by epicatechin with an IC50 value of 157 μM.

We describe an electrochemical bioassay for the detection of the activity of methyltransferase and for screening for its inhibitors. Due to the conjugation of a hemin to a G-quadruplex, strong enzymatic signal amplification is enabled with the aid of exonuclease III.

     

Optimization of a direct spectrophotometric method to investigate the kinetics and inhibition of sialidases

Backgrounds

Streptococcus pneumoniae expresses three distinct sialidases, NanA, NanB, and NanC, that are believed to be key virulence factors and thus, potential important drug targets. We previously reported that the three enzymes release different products from sialosides, but could share a common catalytic mechanism before the final step of product formation. However, the kinetic investigations of the three sialidases have not been systematically done thus far, due to the lack of an easy and steady measurement of sialidase reaction rate.

Results

In this work, we present further kinetic characterization of pneumococcal sialidases by using a direct spectrophotometric method with the chromogenic substrate p-nitrophenyl-N-acetylneuraminic acid (p-NP-Neu5Ac). Using our assay, the measured kinetic parameters of the three purified pneumococcal sialidase, NanA, NanB and NanC, were obtained and were in perfect agreement with the previously published data. The major advantage of this alternative method resides in the direct measurement of the released product, allowing to readily determine of initial reaction rates and record complete hydrolysis time courses.

Conclusion

We developed an accurate, fast and sensitive spectrophotometric method to investigate the kinetics of sialidase-catalyzed reactions. This fast, sensitive, inexpensive and accurate method could benefit the study of the kinetics and inhibition of sialidases in general.     

A novel enzyme electrode method for the determination of nitrite based on nitrite reductase

The enzyme, nitrite reductase, can be extracted and purified from spinach leaves; the freeze-dried preparation is completely stable for at least 4 months if kept in a freezer. The enzyme catalyzes the reduction of nitrite to ammonia in the presence of reduced methyl viologen as electron donor. An assay of nitrite can be based on the measurement of the ammonia formation, with an air-gap electrode as sensor. Nitrite in the 10⁻⁴ M—5 · lO⁻² M range can be accurately determined with either soluble or immobilized enzyme, but the latter is stable for at least 3 weeks, is less susceptible to interferences during assay, and can be used repeatedly for about a hundred runs. These advantages make the method very simple, valuable and economical for the routine analysis of nitrite ion.     

Analysis method of the angiotensin-I converting enzyme inhibitory activity based on micellar electrokinetic chromatography.

A micellar electrokinetic chromatography (MEKC) method was developed for estimating the angiotensin-I converting enzyme (ACE) inhibitory activity by separating the hippuric acid liberated in the ACE reaction mixture in the presence of an inhibitor, captopril. The hippuric acid was successfully separated and detected by MEKC with a 25 mM sodium dodecyl sulfate solution in a 25 mM phosphate-50 mM borate buffer at pH 7.0; the total analysis took about 5 min. A good linear relationship was observed between the inhibitor and the peak area of hippuric acid release. No significant difference in the ACE inhibitory activity (IC50) of captopril (an antihypertensive medicine) or autolyzed-mushrooms (functional foods) was observed between the conventional method and the MEKC method. The MEKC method was found to be a useful technique for a rapid assay of the ACE inhibitory activity.     

NBS-荧光素-盐酸头孢吡肟化学发光体系的研究

盐酸头孢吡肟(Cefepime Hydrochloride)作为第四代头孢菌素类抗菌新药,具有高效、广谱、低毒、耐细菌β-内酰胺酶等特点。目前测定盐酸头孢吡肟的方法有液相色谱[1]、高效液相色谱法[2-7]、红外与X-射线[8]等。本文研究发现,在氢氧化钠介质中,盐酸头孢吡肟可被N-溴代琥珀酰亚胺(NBS)氧化产生微弱的化学发光,在荧光素共存时产生较强的化学发光,其发光强度与盐酸头孢吡肟的质量浓度在0·05mg·L-1~10mg·L-1范围内呈良好的线性关系。基于此,结合流动注射技术,建立了测定盐酸头孢吡肟的流动注射化学发光方法。该法的检出限(3σ)为29μg·L-…     

荷移光度法测定注射用头孢唑啉钠含量

以注射用头孢唑啉钠作为电荷给体,百里酚蓝作为电荷受体,用分光光度法研究了它们之间形成电荷转移配合物的条件。结果表明:在乙醇介质中,二者于室温即可形成1∶1的配合物,其最大吸收波长为450nm,4.67~93.32mg·L-1范围内呈线性关系,相关系数是0.9986,稳定常数是106.34,表观摩尔吸光系数ε=3.45×104L·mol-1·cm-1。对形成配合物的机理进行了探讨。应用拟订的方法对注射用头孢唑啉钠进行了含量测定,结果与文献方法一致,平均回收率为99.9%。     

A differential method for the analysis of chemical kinetics results based on reversed-flow gas chromatography

Summary The reversed-flow gas chromatographic technique has been applied to the determination of the apparent rate constant and the reaction order of a reaction between two gases or vapors. For seven hydrocarbons (ethane, ethene, ethyne, propene, butene, benzene and toluene) reacting with nitrogen dioxide, the above mentioned kinetic parameters have been determined. For these determinations, the necessary mathematical formulation of the problem has been written and solved, leading to simple expressions which describe the height of the chromatographic sample peaks as a function of time.