Abstract:
Much research has been done on aflatoxins since their discovery in the 1960’s
where it was concluded that aflatoxins have carcinogenic, mutagenic, teratogenic
and immunosuppressive properties. Aflatoxin M1 exists in milk and since milk is a
major component of the diet of infants, the maximum permissible limit set by the
EU is 50 parts per trillion (ng L
-1
).
Current methods of analysis for aflatoxin M1 is primarily based around techniques
such as HPLC and TLC which require extensively trained operators and equipped
laboratories. Using antibodies as receptors in an enzyme linked immunosorbent
assay (ELISA), the analysis costs can be reduced and simplified, however, an
equipped laboratory is still required. Hence there is a need for a low cost, rapid,
portable instrument which is easy to use at the point of source for the detection of
aflatoxin M1.
This thesis describes the development of affinity sensors to meet these
requirements. Firstly the design and optimisation of an ELISA method was carried
out, utilising a commercially sourced monoclonal antibody.
Once the antibodies suitability for sensing aflatoxin M1 was determined the
antibody was successfully employed as the receptor for a screen printed HRP/TMB
based immunosensor. Upon the analysis of milk it was observed that milk caused
extensive interference and through a series of chemical extractions the
interference was attributed to whey proteins in the milk with suspicion towards a-
lactalbumin. A simple pre-treatment technique of adding calcium chloride was
performed and the interference from the whey proteins was removed. The resulting
immunosensor achieved a sensitivity of 39 ng L
-1
(Figure 3.26), however, poor
reproducibility was observed due to the screen printed electrode production (%CV
= 21% variance for screen printed electrode production). Gold cell on a chip microelectrode arrays were used to replace the screen printed
electrodes and the successful covalent attachment of the antibody to the
microelectrode array through PDITC cross linking compound was monitored using
atomic force microscopy and scanning electron microscopy. It was shown that the
majority of the antibodies during immobilisation orientate in a ‘side on’ orientation
and therefore a cheap capture polyclonal antibody was first immobilised before the
addition of the sensing anti-aflatoxin M1 monoclonal antibody. Using the
microelectrode array an improvement of the sensitivity as well as a reduction of the
milk interference was shown. Sensitivity was improved to 8 ng L
-1
in milk (Figure
4.23).
Further work was performed to substitute the fragile antibody used in the sensing
layer for a robust synthetic peptide receptor. Initially a virtual library of synthetic
peptides was created using de novo design techniques in silico. Further
computational techniques were performed to determine the best peptide from the
library. This peptide had a sequence of PVGPRP. From literature a peptide (LLAR)
was reported with affinity for aflatoxin B1. This sequence along with the de novo
design peptide was synthesised and tested using a host of techniques and
immobilisation chemistries such as optical waveguide lightmode spectroscopy
(OWLS), BIAcore and enzymatic techniques using EDC/NHS, glutaraldehyde and
BS
3
cross linking methods. The affinity of both peptides to aflatoxin M1 was
demonstrated however further work is required to quantify the affinity and to
incorporate the peptides into the microelectrode array.