A Universal Luminescent Probe for Enzymatic Reactions
FULL PAPER
concentration of NADH would result in an increase of the
fluorescence intensity because the complex remains in its
oxidised state.
Increasing amounts of inhibitor will result in an increase in
the fluorescence intensity because the enzyme will produce
smaller amounts of thiocholine.
To demonstrate that this universal method is not only lim-
ited to assays of redox enzymes, we applied our method to
monitor the hydrolase activity of AChE. The main substrate
of AChE is the neurotransmitter acetylcholine, which is hy-
drolysed at neuromuscular synapses and causes the termina-
Conclusion
We have developed an innovative biosensor system by using
[25]
III/II
tion of the synaptic transmission. AChE can also hydro-
lyse ATCh to yield the thiol-containing compound thiocho-
line and acetic acid. We have used the alternative substrate
ATCh to demonstrate that the SH group of any compound
can also act as a reducing agent for the osmium complex.
By using this method we were also able to follow the inhibi-
tion of AChE by 1,5-bis(4-allyldimethylammoniumphenyl)-
pentan-3-one dibromide (BW284c51), a potent inhibitor of
the electron-transfer mediator [Os
ACHTUNGTRNENUN(G bpy) Cl ACHTUNGTRENUNN(G PyCOOH)]
2
as a novel sensitive fluorescent probe. We have shown that
this complex simultaneously demonstrates a fully reversible
change in its redox state with an alteration of its emission
spectrum. By simply monitoring the changes in fluorescence
intensity, we have been able to detect micromolar concen-
trations of glucose and a-ketoglutarate. We have also ap-
plied this method to study the inhibition of AChE by ana-
logues of nerve gases, and have detected concentrations of
[26]
AChE which mimics the action of nerve gases.
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À11
Figure 5A illustrates the mechanism of AChE in our
assay. When AChE hydrolyses ATCh it yields acetic acid
and thiocholine, which in turn is able to reduce the osmium
complex. When we analysed the effect of thiocholine in our
system we observed that its SH group can act as a potent re-
inhibitor between 10 and 10 m. The examples described
herein demonstrate the versatility of this assay, and show
that other biocatalytic processes such as those including oxi-
doreductases or cofactor-dependent proteins may also be
followed by fluorescence spectroscopy.
III
ductant by donating one electron to the oxidised form [Os -
2
+
ACHTUNGTRENNUNG( bpy) Cl ACHTUNGTRENNUNG( PyCOOH)] . Figure 5B shows the emission spec-
2
tra of the osmium complex in the presence of AChE, ATCh
and variable concentrations of the inhibitor BW284c51. As
the concentration of BW284c51 increases, the emission peak
intensity at l=707 nm also increases. The spectra clearly in-
dicate that in the absence of an inhibitor large amounts of
thiocholine are produced, and consequently, the osmium
complex is reduced and the fluorescence intensity decreases.
Experimental Section
Materials: Potassium hexachloroosmiate(IV) was purchased from Alfa
Aesar, anhydrous d-(+)-glucose and hydrogen peroxide (30% v/v) from
Panreac, and enzymes and other chemicals were obtained from Sigma–
Aldrich and were used as supplied. Absorbance and fluorescence spectra
of the osmium complex were recorded on a ND-1000 spectrophotometer
(
Nanodrop Technologies) and a FluoroLog (Horiba Jobin Yvon) fluor-
ometer, respectively.
IIA
Synthesis of [Os (bpy)
cording to the literature procedure. ACHUTNGRNENUG( ES ) calcd for [Os -
C
T
N
T
E
N
N
2
Cl ACHTUNGERTNNUNG( PyCOOH)]Cl: This complex was prepared ac-
[17,27,28] + II
MS
2
ACHUTNGRENNU(G bpy) Cl ACHTUGNERNTNU(G PyCOOH)] : 662.0999; found: 662.0751.
+
Glucose oxidase assay: Variable concentrations of glucose were incubat-
À1
ed with glucose oxidase type VII from Aspergillus niger (1.72 UmL ),
À1
horseradish peroxidase type VI (0.52 UmL ), 3-mercaptopropionic acid
(
0.15 mm) and the Os complex (0.30 mm) in an acetate buffer (50 mm,
pH 5.0) at room temperature. The emission spectra of the resulting solu-
tions were recorded after 1 h.
Peroxidase assay: Variable concentrations of hydrogen peroxide were in-
À1
cubated with horseradish peroxidase (0.52 Uml ), 3-mercaptopropionic
acid (0.15 mm) and the Os complex (0.30 mm) in an acetate buffer
(
50 mm, pH 5.0) at room temperature. The emission spectra of the result-
ing solutions were recorded after 10 min.
Glucose dehydrogenase assay: Variable concentrations of glucose were
+
incubated with NAD (0.33 mm) and glucose dehydrogenase from Pseu-
À1
domonas sp. (4.01 Uml ) in a Tris-HCl buffer (50 mm, pH 7.6) to give a
final volume of 950 mL at room temperature for 1 h. A solution (50 mL)
containing the Os complex (5.98 mm) and H O (8.82 mm) in an acetate
2 2
buffer (50 mm, pH 5.0) was added to each and the emission spectra of the
resulting solutions were recorded.
Glutamic dehydrogenase assay: Variable amounts of a-ketoglutarate
were incubated with NADH (0.37 mm) and l-glutamic dehydrogenase
À1
Figure 5. A) AChE assay in the presence of the Os complex. B) Emission
spectra of the Os complex in the presence of AChE (1.75 mUmL ), ace-
from bovine liver (0.45 Uml ) in a Tris-HCl buffer (50 mm, pH 7.6) and
À1
ammonium acetate (53 mm) to give a final volume of 950 mL at room
temperature for 1 h. A solution (50 mL) containing the Os complex
(5.98 mm) and H O (8.82 mm) in an acetate buffer (50 mm, pH 5.0) was
2 2
added to each and the emission spectra of the resulting solutions were re-
corded.
tylthiocholine chloride (0.40 mm) and different concentrations of
À11
À10
À10
BW284c51: a) 0, b) 8.82ꢅ10
,
c) 1.76ꢅ10
,
d) 3.53ꢅ10
,
e) 7.06ꢅ
À10
À9
À9
À9
1
0
, f) 1.41ꢅ10 , g) 2.82ꢅ10 and h) 5.64ꢅ10 m. Inset: calibration
curve of BW284c51 at l=707 nm (BW=BW284c51).
Chem. Eur. J. 2009, 15, 6194 – 6198
ꢄ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
6197