scholarly journals Real-Time Interferometric Refractive Index Change Measurement for the Direct Detection of Enzymatic Reactions and the Determination of Enzyme Kinetics

Sensors ◽  
2019 ◽  
Vol 19 (3) ◽  
pp. 539
Author(s):  
Søren Jepsen ◽  
Thomas Jørgensen ◽  
Henrik Sørensen ◽  
Søren Kristensen
Keyword(s):  
Refractive Index ◽  
Enzyme Kinetics ◽  
Adenosine Triphosphate ◽  
Direct Detection ◽  
Enzymatic Reaction ◽  
Refractive Index Change ◽  
Direct Determination ◽  
Initial Reaction ◽  
Enzymatic Reactions

Back scatter interferometry (BSI) is a sensitive method for detecting changes in the bulk refractive index of a solution in a microfluidic system. Here we demonstrate that BSI can be used to directly detect enzymatic reactions and, for the first time, derive kinetic parameters. While many methods in biomedical assays rely on detectable biproducts to produce a signal, direct detection is possible if the substrate or the product exert distinct differences in their specific refractive index so that the total refractive index changes during the enzymatic reaction. In this study, both the conversion of glucose to glucose-6-phosphate, catalyzed by hexokinase, and the conversion of adenosine-triphosphate to adenosine di-phosphate and mono-phosphate, catalyzed by apyrase, were monitored by BSI. When adding hexokinase to glucose solutions containing adenosine-triphosphate, the conversion can be directly followed by BSI, which shows the increasing refractive index and a final plateau corresponding to the particular concentration. From the initial reaction velocities, KM was found to be 0.33 mM using Michaelis–Menten kinetics. The experiments with apyrase indicate that the refractive index also depends on the presence of various ions that must be taken into account when using this technique. This study clearly demonstrates that measuring changes in the refractive index can be used for the direct determination of substrate concentrations and enzyme kinetics.

The direct determination of the temperature dependence of the refractive index of liquids and solids

1976 ◽  
Vol 9 (14) ◽  
pp. 1945-1951 ◽  
Author(s):  
G Abbate ◽  
A Attanasio ◽  
U Bernini ◽  
E Ragozzino ◽  
F Somma
Keyword(s):  
Refractive Index ◽  
Temperature Dependence ◽  
Direct Determination

Direct Fluorometric Determination of Total Cholesterol in Serum Using Derivatized Cholesterol Oxidase

2000 ◽  
Vol 54 (8) ◽  
pp. 1157-1162 ◽  
Author(s):  
Javier Galbán ◽  
José F. Sierra ◽  
José M. López Sebastian ◽  
Susana de Marcos ◽  
Juan R. Castillo
Keyword(s):  
Blood Serum ◽  
Total Cholesterol ◽  
Cholesterol Oxidase ◽  
Enzymatic Reaction ◽  
Direct Determination ◽  
Analytical Signal ◽  
Cholesterol Concentration ◽  
Serum Samples ◽  
Response Range

In this paper the use of cholesterol oxidase derivatized with a fluorescein derivative is proposed for the direct determination of total cholesterol in blood serum. The method is based on the changes in the fluorescence of the solution during the enzymatic reaction (λexe = 498 nm and λem 519 nm). A mathematical model which relates the analytical signal to the total cholesterol concentration was developed, and the model can also be used to obtain some of the thermodynamic constants of the process. The method has a linear response range up to 70 mg/L of cholesterol, a detection limit of 2.5 mg/L, and the precision was 1.0% (40 mg/L cholesterol, n = 10). The method was applied to total cholesterol determination in blood serum samples. The results were compared to those obtained by a commercial analyzer, and statistically similar results were obtained. The use of derivatized cholesterol oxidase makes it possible to simplify the methodology normally used in this type of determination (the indicator reaction is avoided and the number of reagents reduced), with the added advantage that the analytical signal is independent of the concentrations of O2 and cholesterol oxidase.

Improvement of enzyme carbon paste-based biosensor using carbon nanotubes for determination of water-soluble analogue of vitamin E

2015 ◽  
Vol 69 (1) ◽  
Author(s):  
Milan Sýs ◽  
Radovan Metelka ◽  
Tomáš Mikysek ◽  
Karel Vytřas
Keyword(s):  
Carbon Nanotubes ◽  
Vitamin E ◽  
Electrochemical Techniques ◽  
Enzymatic Reaction ◽  
Direct Determination ◽  
Water Soluble ◽  
Trolox Equivalent Antioxidant Capacity ◽  
Total Phenolic ◽  
Antioxidant Power

AbstractThe catalytic oxidation of a synthetic water-soluble analogue of vitamin E (α-tocopherol, Trolox) by tyrosinase enzyme in the presence of molecular oxygen was studied using electrochemical techniques. This specific enzymatic reaction was exploited for the preparation of a biosensor based on the amperometric reduction of the electroactive product (α-tocoquinone) formed. An electroactive surface of the transducers used was covered with a thin conductive layer of Nafion containing tyrosinase. Significant progress in sensitivity towards polyphenolic compounds such as Trolox was achieved at CPE with carbon nanotubes immobilised on its surface (CPE/CNTs) as electric transducers. The biosensor so developed can be used for the direct determination of total phenolic content (TPC). This important nutrition value can be expressed as the mass equivalent of Trolox, i.e. Trolox equivalent antioxidant capacity (TEAC), which could be used as an alternative to the evaluations currently used based on spectrophotometric methods such as total radical-trapping antioxidant parameter (TRAP), ferric reducing-antioxidant power (FRAP) or 1,1-diphenyl-2-picrylhydrazyl spectrometric assay (DPPH). The effects of the enzyme amount in the Nafion layer (3.0 μg), the influence of the nanoparticles present, the optimal pH value suitable for enzymatic activity (7.0), and the kinetics of enzymatic and electrochemical reactions were studied using cyclic voltammetry (CV). The determination of optimal conditions for amperometry in batch configuration (working potential, speed of stirring, volume of sample, calibration curve, etc.) was not a target of this electrochemical study.

scholarly journals Direct Determination of Coagulation Factor IIa and Plasmin Activities for Monitoring of Thrombotic State

2020 ◽  
Vol 5 (6) ◽  
pp. 1265-1276
Author(s):  
Junhua Zhang ◽  
Lihui Zou ◽  
Chengyang Liu ◽  
Chuanbao Li ◽  
Meng Wang ◽  
...  
Keyword(s):  
Direct Detection ◽  
Oral Anticoagulants ◽  
Direct Determination ◽  
Coagulation Factor ◽  
High Specificity ◽  
Coagulation Factors ◽  
Coagulation Cascade ◽  
Proteolytic Activation ◽  
Activated State

Abstract Background Current laboratory examinations for hypercoagulable diseases focus on the biomarker content of the activated coagulation cascade and fibrinolytic system. Direct detection of physiologically important protease activities in blood remains a challenge. This study aims to develop a general approach that enables the determination of activities of crucial coagulation factors and plasmin in blood. Methods This assay is based on the proteolytic activation of an engineered zymogen of l-phenylalanine oxidase (proPAO), for which the specific blood protease cleavage sites were engineered between the inhibitory and activity domains of proPAO. Specific cleavage of the recombinant proenzyme leads to the activation of proPAO, followed by oxidation and oxygenation of l-phenylalanine, resulting in an increase of chromogenic production when coupled with the Trinder reaction. Results We applied this method to determine the activities of both coagulation factor IIa and plasmin in their physiologically relevant basal state and fully activated state in sodium citrate–anticoagulated plasma respectively. Factor IIa and plasmin activities could be dynamically monitored in patients with thrombotic disease who were taking oral anticoagulants and used for assessing the hypercoagulable state in pregnant women. Conclusions The high specificity, sensitivity, and stability of this novel assay not only makes it useful for determining clinically important protease activities in human blood and diagnosing thrombotic diseases but also provides a new way to monitor the effectiveness and safety of anticoagulant drugs.

Derivative matrix-isopotential synchronous spectrofluorimetry: a solution for the direct determination of urinary δ-aminolevulinic acid

2019 ◽  
Vol 43 (46) ◽  
pp. 18092-18097
Author(s):  
Muhammad Ajmal ◽  
Ali Abbas Falih Shindi ◽  
Yi-Hong Liu ◽  
Yan Zhao ◽  
Ping-Ping Wu ◽  
...  
Keyword(s):  
Aminolevulinic Acid ◽  
Direct Detection ◽  
Direct Determination ◽  
Emission Spectra ◽  
Synchronous Fluorescence ◽  
Fluorescence Spectrometry ◽  
Synchronous Spectrofluorimetry ◽  
Synchronous Fluorescence Spectrometry ◽  
Derivative Matrix

The excitation and emission spectra formulated 3D contours, from which isopotential trajectory was selected for the direct detection of urinary δ-aminolevulinic acid, using derivative matrix isopotential synchronous fluorescence spectrometry.

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