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## Studies on immobilization of acid phosphatase and its use for the flow injection analysis with potentiometric detection

### Anna Bronowska

#### Abstract

Flow injection analysis (FIA) is the widely used technique for quantitative chemical and biochemical analysis. The main advantages of the FIA analysis are: simple construction of the system, consumes minimal amounts of reagents, low cost, possibility to automate analysis and short analysis time. The enzymes are used in many routine analyses, for example in clinical, environmental or industrial analysis due to the following advantages: enzymes can increase the speed of only chosen reactions and these reactions usually proceed under mild conditions of temperature, pH and pressure. The other advantage is the possibility to automate the analysis with the use of enzymes, which facilitates the monitoring process. The enzymes can be used in two configurations in FIA systems. One solution is to construct a biosensor and use it as a detector for flow injection system. The second way is to use an enzymatic reactor in flow injection system with a detector capable of determination of analyte or product of enzymatic reaction in flow conditions. The aim of this thesis was to design the flow injection system with potentiometric detection, using an immobilized acid phosphatase. These studies are an introduction to further studies – the effect of inhibitors on the activity of acid phosphatase. There are many compounds, which have negative influence on biosystem, because they block the activity of enzymes. The use of flow injection analysis in measurements of activity of enzymes ensures constant and controlled conditions of temperature and pH, which are important in studies of this type. In the designed flow injection system the immobilized acid phosphatase was used, so it was necessary to choose a suitable method of immobilization of this enzyme. For the acid phosphatase immobilization three different methods were used: covalent binding to the polymeric F--selective membrane surface, covalent binding to Sepabeads carrier or cross-linking of enzyme aggregates (CLEAs). The immobilized enzymes were used as a receptor layer of detector (ion-selective electrodes) or inside of the reactor in flow injection system. The ion-selective electrode with polymeric membrane containing chloro-2,7,12,17-tetra-tert-butyl-5,10,15,20-tetraazaporphine aluminium (Al(TAP)) was used as a detector in flow injection analysis with an enzymatic reactor. In presented research the activity of immobilized enzyme in flow injection analysis was studied by determination of fluoride ions which are product of the enzymatic hydrolysis of sodium monofluorophosphate. The study revealed that the immobilized acid phosphatase was washed out from polymeric membrane. It is also shown, that the obtained immobilizates have too low activity to be used for flow injection analysis. This may be due to: application of contaminated enzyme, small amount of amino groups on the enzyme surface (covalent bonds are formed between the amino groups of enzymes and chemical groups on surface of carrier), low stability of the covalent bonds in the applied pH 4,5 or very low levels of native enzyme activity (4,33 U·mg-1 protein). It means that the goal to design the flow injection system for potentiometric determination of acid phosphatase inhibitors was not reached.
Record ID
WUT621d58f1a4224da493aabf7130ddd9aa
Diploma type
Master of Science
Author
Anna Bronowska Anna Bronowska,, Undefined Affiliation
Title in Polish
BADANIA METOD IMMOBILIZACJI FOSFATAZY KWAŚNEJ DO ZASTOSOWAŃ W UKŁADZIE PRZEPŁYWOWO – WSTRZYKOWYM Z DETEKCJĄ POTENCJOMETRYCZNĄ
Supervisor
Mariusz Pietrzak (FC/CMB) Mariusz Pietrzak,, Chair of Medical Biotechnology (FC/CMB)Faculty of Chemistry (FC)
Certifying unit
Faculty of Chemistry (FC)
Affiliation unit
Department Of Microbioanalytics (FC/CMB)
Study subject / specialization
, Mikrobioanalityka
Language
(pl) Polish
Status
Finished
Defense Date
10-07-2012
Issue date (year)
2012
Keywords in Polish
-
Keywords in English
-
Abstract in Polish
urn:pw-repo:WUT621d58f1a4224da493aabf7130ddd9aa