A New Covalent Organic Framework of Dicyandiamide-Benzaldehyde Nanocatalytic Amplification SERS/RRS Aptamer Assay for Ultratrace Oxytetracycline with the Nanogold Indicator Reaction of Polyethylene Glycol 600

In this paper, dicyandiamide (Dd) and p-benzaldehyde (Bd) were heated at 180 ∘C for 3 h to prepare a new type of stable covalent organic framework (COF) DdBd nanosol with high catalysis. It was characterized by molecular spectroscopy and electron microscopy. The study found that DdBd had a strong catalytic effect on the new indicator reaction of polyethylene glycol 600 (PEG600)-chloroauric acid to form gold nanoparticles (AuNPs). AuNPs have strong resonance Rayleigh scattering (RRS) activity, and in the presence of Victoria Blue B (VBB) molecular probes, they also have a strong surface-enhanced Raman scattering (SERS) effect. Combined with a highly selective oxytetracycline (OTC) aptamer (Apt) reaction, new dual-mode scattering SERS/RRS methods were developed to quantitatively analyze ultratrace OTC. The linear range of RRS is 3.00 × 10−3 –6.00 × 10−2 nmol/L, the detection limit is 1.1 × 10−3 nmol/L, the linear range of SERS is 3.00 × 10−3–7.00 × 10−2 nmol/L, and the detection limit is 9.0 × 10−4 nmol/L. Using the SERS method to analyze OTC in soil samples, the relative standard deviation is 1.35–4.78%, and the recovery rate is 94.3–104.9%.

Aptamer Turn-On SERS/RRS/Fluorescence Tri-mode Platform for Ultra-trace Urea Determination Using Fe/N-Doped Carbon Dots

Sensitive and selective methods for the determination of urea in samples such as dairy products are important for quality control and health applications. Using ammonium ferric citrate as a precursor, Fe/N-codoped carbon dots (CDFeN) were prepared by a hydrothermal procedure and characterized in detail. CDFeN strongly catalyzes the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 to turn on an indicator molecular reaction, forming an oxidized tetramethylbenzidine (TMBox) probe with surface-enhanced Raman scattering, resonance Rayleigh scattering, and fluorescence (SERS, RRS, and FL) signals at 1,598 cm−1, 370 nm, and 405 nm, respectively. The urea aptamer (Apt) can turn off the indicator reaction to reduce the tri-signals, and the addition of urea turns on the indicator reaction to linearly enhance the SERS/RRS/FL intensity. Thus, a novel Apt turn-on tri-mode method was developed for the assay determination of ultra-trace urea with high sensitivity, good selectivity, and accuracy. Trace adenosine triphosphate and estradiol can also be determined by the Apt-CDFeN catalytic analytical platform.

Visual binary testing of methanol contained in ethyl alcohol

A method for control of methanol traces in rectified ethyl alcohol and alcoholic drinks based on visual binary testing using one reference sample was proposed. An indicator reaction of formaldehyde interaction (product of methanol oxidation) with chromotropic acid disodium salt was chosen for methanol screening. The conditions of indicator reaction proceeding are analogous as for the spectrophotometric technique of methanol determination: methanol was oxidized to formaldehyde with potassium permanganate in an acidic medium; the formaldehyde then reacts with chromotropic acid in the presence of hot concentrated sulfuric acid and forms a violet product (color of this product is stable for 12 hours). It was established that the absorption spectrum of the reaction product does not change on going from 96% ethanol to aqueous-ethanol solutions with a volume fraction of 40% ethanol. The maximum light absorption of the reaction product corresponded to 570 nm. All further studies were carried out in water-ethanol solutions with a volume fraction of ethanol of 40%. According to regulatory documents the normalized limiting content of methanol (clim) in ethyl alcohol of the “Lux” grade (the most common in the alcoholic industry) and alcoholic beverages is 0.01% by by volume counted upon anhydrous alcohol. The comparison sample (the solution of colored reaction product of indicator reaction) had to be less than the normilized level on the value which providing the risk of false-negative test result not more than 5%. To determination the threshold concentration of methanol in the comparison sample was applied the statistics of observation. For the aim the solution of colored product corresponding to the normalized limiting methanol concentration clim = 0.01% by volume was prepared and comparison samples with lower methanol concentrations were also prepared. The interval of unreliability was discovered with the help of observers. The frequency of detecting of the difference in the color of comparison samples and normalized sample (P(c)) changed from 0 to 1 in this interval. The value of methanol concentration 0.0072% by volume counted upon anhydrous alcohol was taken for the lower boundary of the interval and the value of methanol concentration 0.01% by volume counted upon anhydrous alcohol was chosen the upper border of the interval. This interval was divided on eight concentrations with step Dс = 0.0004% by volume. Three parallel series of solutions were prepared and 48 observations for each concentration were received. The experimental efficiency curve obtained was checked for compliance with the mathematical functions of the known distributions: normal, logistic, lognormal, exponential and Weibull distribution function using the statistical criterions c2 and Kolmogorov-Smirnov λ. The efficiency curve was described by the theoretical functions of the lognormal and Weibull distributions. Calculated at a confidence level of 0.95 estimation of the threshold concentration for the comparison sample was 0.0073% by volume fraction corresponding to anhydrous alcohol. The visual binary testing of methanol trace in alcoholic drinks was carried out. The accuracy of visual binary testing of methanol was confirmed by gas chromatography.

A kinetic analysis of coupled (or auxiliary) enzyme reactions

<div>As a case study, we consider a coupled (or auxiliary) enzyme assay of two reactions obeying the Michaelis-Menten mechanism. The coupled reaction consists of a single-substrate, single-enzyme non-observable reaction followed by another single-substrate, single-enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction is the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the coupled reaction is described by a pair of interacting Michaelis-Menten equations. Moreover, we show that when the indicator reaction is slow, the quasi-steady-state dynamics are governed by two fast variables and two slow variables, and when the indicator reaction is fast, the dynamics are governed by three fast variables and one slow variable. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis-Menten equations are derived. The theory can be extended to deal with more complex sequences of enzyme catalyzed reactions.</div>

A kinetic analysis of coupled (or auxiliary) enzyme reactions

<div>As a case study, we consider a coupled (or auxiliary) enzyme assay of two reactions obeying the Michaelis-Menten mechanism. The coupled reaction consists of a single-substrate, single-enzyme non-observable reaction followed by another single-substrate, single-enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction is the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the coupled reaction is described by a pair of interacting Michaelis-Menten equations. Moreover, we show that when the indicator reaction is slow, the quasi-steady-state dynamics are governed by two fast variables and two slow variables, and when the indicator reaction is fast, the dynamics are governed by three fast variables and one slow variable. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis-Menten equations are derived. The theory can be extended to deal with more complex sequences of enzyme catalyzed reactions.</div>

A kinetic analysis of coupled sequential enzyme reactions

<div>As a case study, we consider a coupled enzyme assay of sequential enzyme reactions obeying the Michaelis--Menten reaction mechanism. The sequential reaction consists of a single-substrate, single-enzyme non-observable reaction followed by another single-substrate, single-enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction becomes the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the sequential reaction is described by a pair of interacting Michaelis--Menten equations. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis--Menten equations are derived. The theory can be extended to deal with more complex sequences of enzyme catalyzed reactions.</div>

A kinetic analysis of coupled sequential enzyme reactions

<div>As a case study, we consider a coupled enzyme assay of sequential enzyme reactions obeying the Michaelis-Menten reaction mechanism. The sequential reaction consists of a single-substrate, single enzyme non-observable reaction followed by another single-substrate, single enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction becomes the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the sequential reaction is described by a pair of interacting Michaelis-Menten equations. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis-Menten equations are derived. The theory can be extended to deal with more complex sequences of enzyme catalyzed reactions.</div>

A Kinetic Photometric Method For Benzalkonium Chloride Determination In Eye Drops

A novel sensitive kinetic photometric method for the Benzalkonium Chloride (BAC) determination has been developed. The method is based on the ability to inhibit the reaction of Acetylcholine hydrolysis by cholinesterase. The reaction rate is evaluated by the non-hydrolysed Acetylcholine residue, which is determined by the amount of Peracetic acid, produced during the interaction with the excess of H 2 O 2 . Indicator reaction is an interaction of p-phenetidine with Peracetic acid that leads to the formation of 4,4'-azoxyphenetole with λ max = 358 nm (lg ε = 4.2). The conditions affecting the reaction (reagents concentration, pH, order of addition of reagents, stability in time) have been optimized. The linear dependence has been obeyed in the range of (1.4-8.4)·10 -6 mol L -1 of BAC with correlation coefficient of 0.999. The assay LOQ (20 % of the inhibition degree) has been 1.9·10 -6 mol L -1 . The proposed method has been successfully applied to the analysis of the eye drops and has shown an accuracy and reliability of the results obtained.

Phase-Plane Geometries in Coupled Enzyme Assays

The determination of a substrate or enzyme activity by coupling of one enzymatic reaction with another easily detectable (indicator) reaction is a common practice in the biochemical sciences. The dynamical behavior of couple enzyme catalyzed assays is studied by analysis in the phase plane. Usually, the kinetics of enzyme reactions is simplified with singular perturbation analysis to derive rate or time course expressions valid under the quasi-steady-state and reactant stationary state assumptions. In this paper, we analyze two types of time-dependent slow manifolds that occur in asymptotically autonomous vector fields that arise from enzyme coupled assays. We show that the motion of the slow manifolds relative to the motion of the solution must be taken into account in order to formulate accurate leading order asymptotic solutions. We also develop a rigorous mathematical framework from which to analyze enzyme catalyzed indicator reaction from couple enzyme assays.