3. Results
When samples of River Thames water were filtered and the
disc filter placed on chromogenic culture media, the presence
and content of E. coli and coliforms was confirmed.
GUD positive E. coli appeared as green colonies after 18 h
incubation and were counted on triplicate plates. The
biomarker GUD activity can also be detected and quantified
using fluorescent substrates. To rapidly quantify E. coli we
exploited the activity of GUD using the soluble fluorescent
substrate MUG and measured the resulting fluorescence
with the new, miniaturised, hand-held fluorescence
detector. In order to achieve quantification of GUD activity,
and by this means the level of E. coli present in a sample,
we established a reference curve with 4MU. Linearity
between the measured fluorescence and the concentration
of the fluorophore was achieved between 4MU concentrations
from 10 nM to 20 mM using the hand-held sensor
(Fig. 1A). This was comparable with the performance of
a typical bench-top fluorescence reader (Fig. 1A) where the
linear range extended to 50 mM. The hand-held sensor was
then assessed for the fluorogenic MUG assay using a b-Dglucuronidase
preparation. The resulting graph for the
enzyme using a substrate concentration of 50 mM is shown
in Fig. 1B. Linearity was obtained for an enzyme activity
range from 1 m/mL to 2.5 m/mL. The assay range and sensitivity
for the enzyme using the hand-held sensor was
identical to the results obtained when measuring the same
samples with the bench-top reader (Fig. 1B). The fluorescent
signal was proportional to the enzyme concentration in the
range from 1 m/mL to 1 m/mL. The substrate and enzyme
concentrations, reaction time and detector sensitivity were