530
Chemiluminescence and Bioluminescence, Analysis by
in
reaction
kinetics,
signal
intensity,
and signal-to-background ratio can be
achieved both in solution and on mem-
branes using certain water-soluble macro-
molecules
[e.g.
polyvinylbenzyl
(ben-
zyldimethylammonium) chloride]. Several
highly sensitive sustained signal CL assays
for horseradish peroxidase are available.
These are based on (a) an acridan sub-
strate that is converted by peroxidase into
an unstable acridinium ester intermediate
or (b) an enhanced chemiluminescent as-
say that uses luminol in conjunction with
an enhancer molecule (e.g. 4-iodophenol,
4-hydroxycinnamic acid) (Fig. 5). In addi-
tion, sensitive CL or BL detection reac-
tions of varying complexity are available
for glucose oxidase, glucose 6-phosphate
dehydrogenase, xanthine oxidase, and
β
-
galactosidase labels (Table 4).
CL is also useful in DNA sequencing. For
example, it is used to detect biotinylated
DNA fragments transferred onto a mem-
brane from a polyacrylamide sequencing
gel. The biotin-labeled DNA is reacted
with a streptavidin–alkaline phosphatase
conjugate, and then the bound enzyme is
detected using a 1,2-dioxetane substrate
(e.g. CSPD
). Light emission from the
membrane surface is imaged onto ±lm or
using a CCD camera as shown in Fig. 6.
4.3
Enzymes, Substrates, and Cofactors
The luminol, ±refly luciferase, and marine
bacterial luciferase reactions are sensi-
tive indicator reactions for the production
or consumption of peroxide, ATP, and
NAD(P)H respectively. They can be cou-
pled to other reactions involving oxidases,
kinases, and dehydrogenases, and can be
used to measure any component of the
coupled reaction (enzyme, substrate, co-
factor). Examples include the assays for
alcohol and glucose, shown in Eqs. (1)
and (2). Coimmobilization of a luciferase
with other enzyme reagents improves
the ef±ciency of the reaction because in-
termediates are effectively channeled to
successive enzymes in the coupled en-
zyme system.
Tab. 4
Chemiluminescent and bioluminescent detection reactions for enzyme labels.
Glucose
+
O
2
glucose oxidase label
−−−−−−−−−−−−−−−→
gluconolactone
+
H
2
O
2
H
2
O
2
+
isoluminol
microperoxidase
−−−−−−−−−−−→
light
Xanthine
+
O
2
xanthine oxidase label
−−−−−−−−−−−−−−−−→
hypoxanthine
+
H
2
O
2
H
2
O
2
+
luminol
+
Fe-EDTA
−−−−→
light
o
-nitrophenylgalactoside
β
-galactosidase label
−−−−−−−−−−−−−−−→
galactose
galactose
+
NAD
+
+
galactose dehydrogenase
−−−−→
galactonate
+
NADH
NADH
NADH : FMN oxidoreductase
+
bacterial luciferase
+
FMN
+
decanal
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
light
glucose 6-phosphate
+
NADP
+
glucose 6-phosphate dehydrogenase label
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
glucose
+
NADPH
NADPH
NADPH : FMN oxidoreductase
+
bacterial luciferase
+
FMN
+
decanal
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
light
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