Choline is indispensable for a number of fundamental processes in the body.
Besides being the precursor of the acetylcholine, an important neurotransmitter,
choline is found in the cell membrane structure combining with fatty acids,
phosphate and glycerol. Its deficiency may result in nervous system disorders, fatty
acid build up in the liver, along with increased cholesterol levels, high blood pressure
and memory loss. Thus, rapid detection methods are required for the determination
of choline in biological fluids. In this study a choline oxidase biosensor was constructed for the determination
of choline. During construction of the biosensor, glucose oxidase was used as a
model enzyme, before choline oxidase used. The Teflon (PTFE) membrane of the
oxygen electrode was grafted with 2-hydroxyethyl methacrylate (HEMA, 15%, v/v)
in the presence of ferrous ammonium sulphate (FAS, 0.1%, w/v) by gamma
irradiation and ethyleneglycol dimethacrylate (EGDMA, 0.15 %, v/v) was used as a
crosslinker in a series of membranes. HEMA-grafted membranes were activated with
epichlorohydrin or glutaraldehyde to maintain covalent immobilization of enzyme.
The enzyme activity was measured with an oxygen electrode unit based on oxygen
consumption upon substrate addition.
Membranes were characterized in terms of grafting conditions and mechanical
properties. Membranes, gamma irradiated in a solution of HEMA (15%) and FAS
(0.1%) for 24 h, were found to be suitable for use in the further studies. Mechanical
test results revealed that HEMA grafting made Teflon membrane more flexible and
the presence of EGDMA made the grafted membrane stiffer. During optimization
stage, it was found that the immobilized enzyme amount was not sufficient to obtain
enzyme activity. Thus, the membrane preparation stage was modified to obtain
thinner membranes. The immobilized glucose oxidase and choline oxidase contents
on thin HEMA grafted membranes were determined by Bradford and Lowry
methods. The influence of EGDMA presence and the epichlorohydrin activation
duration on enzyme activity studies revealed that the membrane should be prepared
in the absence of EGDMA and 30 min activation duration is appropriate for
epichlorohydrin coupling. The study on the influence of membrane activation
procedures revealed that the membranes activated with glutaraldehyde had a higher specific activity than the membranes activated with epichlorohydrin. Upon stretching
membrane on the electrode directly rather than placing in the sample unit, the
response of the enzyme immobilized sensor improved with high specific activity.
The optimum choline oxidase concentration was found to be 2 mg/mL considering
the effect of immobilization concentration on enzyme activity. With the choline
oxidase biosensor, the linear working range was determined as 0.052-0.348 mM,
with a 40 ± / 5 µ / M minimum detection limit. The response of the sensor decreased
linearly upon successive measurements.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/4/1083021/index.pdf |
| Date | 01 January 2003 |
| Creators | Yucel, Deniz |
| Contributors | Hasirci, Vasif |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | M.S. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for public access |
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