Detection limit enzymatic reaction

Enzymatic determinations of the detection limit where the chromatograms are first sprayed with an enzyme solution Then after appropriate incubation the enzymatically altered components are detected by reaction with a suitable reagent... 

Lequea et al. used the activity of tyrosine apodecarboxylase to determine the concentration of the enzyme cofactor pyridoxal 5 -phosphate (vitamin B6). The inactive apoenzyme is converted to the active enzyme by pyridoxal 5 -phosphate. By keeping the cofactor the limiting reagent in the reaction by adding excess apoenzyme and substrate, the enzyme activity is a direct measure of cofactor concentration. The enzymatic reaction was followed by detecting tyramine formation by LCEC. The authors used this method to determine vitamin B6 concentrations in plasma samples. 

XOD is one of the most complex flavoproteins and is composed of two identical and catalytically independent subunits each subunit contains one molybdenium center, two iron sulfur centers, and flavine adenine dinucleotide. The enzyme activity is due to a complicated interaction of FAD, molybdenium, iron, and labile sulfur moieties at or near the active site [260], It can be used to detect xanthine and hypoxanthine by immobilizing xanthine oxidase on a glassy carbon paste electrode [261], The elements are based on the chronoamperometric monitoring of the current that occurs due to the oxidation of the hydrogen peroxide which liberates during the enzymatic reaction. The biosensor showed linear dependence in the concentration range between 5.0 X 10 7 and 4.0 X 10-5M for xanthine and 2.0 X 10 5 and 8.0 X 10 5M for hypoxanthine, respectively. The detection limit values were estimated as 1.0 X 10 7 M for xanthine and 5.3 X 10-6M for hypoxanthine, respectively. Li used DNA to embed xanthine oxidase and obtained the electrochemical response of FAD and molybdenum center of xanthine oxidase [262], Moreover, the enzyme keeps its native catalytic activity to hypoxanthine in the DNA film. So the biosensor for hypoxanthine can be based on... 

In the field of responsive agents, enzyme targeting has specific advantages. A small concentration of the enzyme can convert a relatively high amount of the probe in multiple catalytic cycles which considerably decreases the detection limit for the enzyme as compared to other biomolecules. Moreover, enzymatic reactions are usually highly specific therefore, the observed change... 

Willeman et al. [26] modeled the enzyme-catalyzed cyanohydrin synthesis in a stirred batch tank reactor. Assumption of a mass transfer limitation (Figure 9.3b) is made, which results in a low concentration of substrate in the aqueous phase, thus suppressing the non-enzymatic reaction. In a well-stirred biphasic system the enzyme concentration was varied, keeping the phase ratio constant A maximum rate of conversion is reached at the concentration where mass transfer of the substrate becomes limiting. Further increase of enzyme concentration does not enhance the reaction rate [27]. The different results achieved by the two groups are explained by the different process strategies. No mass transfer limitation could be detected by Hickel et al. because the stirring rate in the aqueous phase was not varied [26]. 

Another important set of observations is related to the detection limit dynamic range and sensitivity. For the expected values of the diffusion coefficient (in the gel) of approximately 10-6cm2 s and substrate molecular weights about 300, the detection limit is approximately 10 4M. This is due to the fact that the product of the enzymatic reaction is being removed from the membrane by diffusion at approximately the same rate as it is being supplied. The dynamic range of the sensor... 

The second limitation comes from the fact that, despite the exquisite selectivity of enzymatic reactions, the selectivity of the potentiometric enzyme sensors is very poor. The reason for this lies in the detection mechanism itself, when factors such as the buffer capacity of the sample seriously interfere. 

Enzymatic reactions coupled to optical detection of the product of the enzymatic reaction have been developed and successfully used as reversible optical biosensors. By definition, these are again steady-state sensors in which the information about the concentration of the analyte is derived from the measurement of the steady-state value of a product or a substrate involved in highly selective enzymatic reaction. Unlike the amperometric counterpart, the sensor itself does not consume or produce any of the species involved in the enzymatic reaction it is a zero-flux boundary sensor. In other words, it operates as, and suffers from, the same problems as the potentiometric enzyme sensor (Section 6.2.1) or the enzyme thermistor (Section 3.1). It is governed by the same diffusion-reaction mechanism (Chapter 2) and suffers from similar limitations. 

The objective is to describe a new non-enzymatic urea sensor based on catalytic chemical reaction. The sensor consists of screen-printed transducer (IVA, Ekaterinburg, Russia) and catalytic system which is immobilized on the transducer surface as a mixture with carbon ink. The sensor is used for measuring concentration of urea in blood serum, dialysis liquid. Detection limit is 0.007 mM, while the correlation coefficient is 0.99. Some analysis data of serum samples using the proposed sensor and urease-containing sensor (Vitros BUN/UREA Slide, Johnson Johnson Clinical Diagnostics, Inc.) are presented. 

NCE is a relatively new development in separation science, especially in proteomics and genomics. In the last two decades NCE has gained increasing importance, as can be seen from a good number of publications [17-20]. In addition to the above advantages, NCE is a suitable technique for samples that may be difficult to separate by NLC as the principles of separation are entirely different. Lower detection limits of NCE lead to the possibility of separating and characterizing small quantities of materials. Moreover, the enzymatic reactions for analytical purposes can be conducted within the capillary. 

Two disparate translation methods are investigated for the measurement of sulfur dioxide. Both involve interaction with an aqueous solution. In the first, collected S(IV) is translated by the enzyme sulfite oxidase to which is then measured by an enzymatic fluorometric method. The method is susceptible to interference from i CWg) efforts to minimize this interference is discussed. The second method involves the translation of SO2 into elemental Hg by reaction with aqueous mercurous nitrate at an air/liquid interface held in the pores of hydrophobic membrane tubes. The liberated mercury is measured by a conductometric gold film sensor. Both methods exhibit detection limits of 100 pptv with response times under two minutes. Ambient air measurements for air parcels containing sub-ppbv levels of SO2 show good correlation between the two methods. 

Brennan et al. used a method to detect the reaction of acetylcholineesterase with acetylcholine [46]. The method was based on the use of a monolayer, consisting of fatty acids having Ci6 chain lengths, which were covalently attached to quartz wafers and which contained a small amount of nitrobenzoxadiazole dipalmitoyl phosphatidylethanolamine (NBD-PE) (partitioned from water into the membrane). The enzyme substrate reaction produced a decrease in fluorescence intensity from the monolayer, and the detection system was sensitive to the changes in bulk concentration of as small as 0.1 pM, with a limit of detection of 2 pM of acetylcholine. The mechanism of transduction of the enzymatic reaction was investigated using spectrofluorimetric methods and fluorescence microscopy. 

A low detection limit directly influences the sensitivity of the enzyme-based assay. The final enzyme-substrate interaction must yield an ample amount of some end product which can be accurately monitored and, hopefully, quantitated. The authors experiences have been chiefly with enzymatic detection systems which culminate in a visible chromogenic reaction (e.g. alkaline phosphatase, nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate). 

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