Continuous fluorometric assays for acetylcholinesterase activity and inhibition with conjugated polyelectrolytes

Article abstract of DOI:10.1002/anie.200701724

(Figure Presented) Quick but sensitive: A super-quenched fluorogenic complex of a cationic acetylcholine (ACh) derivative with an energy-acceptor tag and an anionic water-soluble conjugated polymer forms the basis of a highly effective fluorescence turn-on assay for studying the enzyme kinetics and inhibition of acetylcholinesterase (AChE; see schematic representation of the assay). FRET = fluorescence resonant energy transfer.

Full text of DOI:10.1002/anie.200701724

Communications
DOI: 10.1002/anie.200701724
Biosensors
Continuous Fluorometric Assays for Acetylcholinesterase Activity and
Inhibition with Conjugated Polyelectrolytes**
Fude Feng, Yanli Tang, Shu Wang,* Yuliang Li, and Daoben Zhu
Alzheimerꢀs disease (AD) is a chronic, progressive neuro-
degenerative disorder that affects a significant proportion of
the population worldwide.[1] The hydrolytic breakdown of
acetylcholine (ACh) in the brain by acetylcholinesterase
(AChE)[2] can accelerate the assembly of amyloid b peptides
into amyloid fibrils, a process that results in AD.[3] Currently,
the inhibition of AChE with the aim of increasing the levels of
ACh is proving to be an effective strategy for AD therapies.[4]
AChE activity and inhibition are monitored traditionally by
UV/Vis spectroscopy.[5] However, a disadvantage of this
method is its low sensitivity. The most common assays for
AChE involve an enzyme cascade in which AChE converts
ACh into choline, which is then oxidized by choline oxidase
with the production of H2O2. In the presence of horseradish
peroxidase (HRP), the H2O2 produced is detected by
fluorescence, chemiluminescence, electroanalysis, or nano-
technology.[6–9] With these methods, two or more chemical
reactions and several additional enzymes are involved in the
detection process, which makes the assays complex, expen-
sive, and time-consuming. Furthermore, the assays are not
very suitable or precise enough for enzymological studies
because of the indirect and discontinuous methods used for
the measurement of reaction kinetics. Therefore, a new,
sensitive, simple, and continuous pathway for AChE assays is
urgently needed.
ing is mediated by the charge reversal of modified ACh upon
hydrolysis.
Our fluorescence assay for AChE is illustrated in
Scheme 1a. An ACh derivative labeled with the widely used
quencher dabcyl was synthesized as a substrate for AChE.
The resulting cationic compound ACh–dabcyl (see
Scheme 1b for its chemical structure and synthesis) can
form a complex with the anionic polymer poly{1,4-phenylene-
[9,9-bis(4-phenoxybutylsulfonate)]fluorene-2,7-diyl} (PFP-
ꢀ
SO3 )[22] through electrostatic interactions. The fluorescence
ꢀ
of PFP-SO3 is quenched efficiently by the dabcyl moiety
through the transfer of fluorescence resonance energy.[23]
Upon the addition of AChE, ACh–dabcyl undergoes catalytic
hydrolysis to produce choline and a negatively charged
residue that contains the dabcyl moiety. As a result of the
Conjugated polymers have attracted attention as optical
platforms in highly sensitive bioassays for proteins and
nucleic acids,[10–18] as well as nucleases, proteases, and
kinases.[19–21] In contrast to bioassays with small-molecule
counterparts, the transfer of the excitation energy along the
whole backbone of the conjugated polymer to the reporter
results in the amplified fluorescence signal.[10] Herein we
describe a new fluorescence turn-on assay for AChE activity
and inhibition based on the reversible fluorescence quenching
of an anionic conjugated polymer. The fluorescence quench-
[*] F. Feng, Y. Tang, Prof. S. Wang, Prof. Y. Li, Prof. D. Zhu
National Laboratory for Molecular Sciences
Key Laboratory of Organic Solids
Institute of Chemistry
Chinese Academy of Sciences
Beijing, 100080 (P.R. China)
Fax: (+86)10-6263-6680
E-mail: wangshu@iccas.ac.cn
[**] We are grateful for the financial support of the “100 Talents”
program of the Chinese Academy of Sciences, the National Natural
Science Foundation of China (20601027, 20574073, and 20421101),
the National Basic Research Program of China (No.2006CB806200),
and the Major Research Plan of China (No. 2006CB932100).
Scheme 1. a) Assays of AChE activity. b) Chemical structure of the
anionic conjugated polymer PFP-SO3ꢀ, and the synthesis of cationic
ACh–dabcyl. CDI=1,1’-carbonyldiimidazole, DBU=1,8-diazabicyclo-
[5.4.0]undec-7-ene, DMSO=dimethyl sulfoxide, FRET=fluorescence
resonant energy transfer.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Chemie
ꢀ
charge reversal, the dabcyl moiety is repulsed by PFP-SO3
and moves away from the electrostatic complex. Therefore,
ꢀ
the fluorescence intensity of PFP-SO3 is recovered, and the
AChE-catalyzed hydrolysis can be monitored in a continuous
and real-time manner. Furthermore, the inhibition of AChE
can also be analyzed with the PFP-SO3ꢀ/ACh–dabcyl com-
plex.
To determine the sensitivity of the protocol, the fluores-
cence quenching of PFP-SO3ꢀ (2.0 mm in repeat units (RUs))
was examined with ACh–dabcyl in phosphate buffer solution
(25 mm, pH 8.0; see the Supporting Information). The
quenching efficiency is related to the Stern–Volmer constant
(Ksv) and is determined by monitoring measurable fluores-
cence changes by using the Stern–Volmer equation
[Eq. (1)][24] in which F0 is the fluorescence-emission intensity
in the absence of the quencher, F is the fluorescence-emission
intensity in the presence of the quencher, and [Q] is the
concentration of the quencher.
F0=F ¼ 1 þ Ksv½QŠ
ð1Þ
At low concentrations, a linear Stern–Volmer plot (F0/F
versus [ACh–dabcyl]) is obtained with a Ksv value of 1.84 ”
107 mꢀ1 (see the Supporting Information). Thus, the dabcyl
moiety shows superquenching behavior with respect to PFP-
SO3ꢀ. At a remarkably low concentration of 0.4 mm, ACh–
Figure 1. a) Emission spectra of PFP-SO3ꢀ/ACh–dabcyl as a function of
reaction time for AChE-catalyzed hydrolysis; [PFP-SO3ꢀ]=2 mm, [ACh-
dabcyl]=2 mm, [AChE]=2.0 unitsmLꢀ1. The experiment was carried
out ꢀwith a fixed concentration of AChE b) Emission intensity of PFP-
SO3 at 424 nm versus reaction time in the hydrolysis of ACh–dabcyl
with varying concentrations of AChE; [PFP-SO3ꢀ]=2 mm, [ACh–dab-
cyl]=2 mm, [AChE]=0.2–2.0 unitsmLꢀ1. Fluorescence measurements
were made in phosphate buffer solutions (25 mm, pH 8.0) with
excitation at 376 nm.
ꢀ
dabcyl quenches the fluorescence of PFP-SO3 (2.0 mm in
RUs) with an efficiency of about 92% upon excitation at
376 nm. One equivalent of ACh–dabcyl could quench the
ꢀ
fluorescence of PFP-SO3 with more than 99% efficiency.
The AChE assay shows very high sensitivity because of the
extremely low background noise.
The results of the fluorescence-monitored hydrolysis of
ACh–dabcyl by AChE are presented in Figure 1a. Upon the
addition of AChE to the PFP-SO3ꢀ/ACh–dabcyl complex
from the Stern–Volmer curve at pH 8.0 for each data point.
The initial reaction rate (V0) was calculated as the slope of the
plot of the concentration change of ACh–dabcyl versus
reaction time for the first five data points. The plot of 1/V0
versus 1/[S] obeys the Lineweaver–Burk equation and yields
the Michaelis constant[25] Km = 4.0 mm (Figure 2a), which is of
the same order of magnitude as those reported.[5,6] This result
shows that the modification of the substrate with the dabcyl
group does not affect AChE activity. It is possible to detect
AChE activity with a substrate concentration of 0.2–0.6 mm
with a common fluorometer within 2 min, which emphasizes
the high sensitivity and rapid response of the PFP-SO3ꢀ/ACh–
dabcyl complex.
ꢀ
([PFP-SO3 ] = 2.0 mm, [ACh–dabcyl] = 2.0 mm) in a phosphate
buffer solution (25 mm, pH 8.0) and equilibration at 378C, the
ꢀ
fluorescence of PFP-SO3 was observed to rise gradually
during the first 10 min until a plateau was reached after
12 min (final [AChE]: 2.0 unitsmLꢀ1). This result indicates
that AChE catalyzes the hydrolysis of ACh–dabcyl, which
leads to a “turn-on” response of PFP-SO3ꢀ fluorescence. The
hydrolysis of ACh–dabcyl by AChE enhanced the fluores-
cence of PFP-SO3ꢀ 130-fold: Thus, the PFP-SO3ꢀ/ACh–dabcyl
complex has an extremely high fluorescence turn-on response
for AChE. The fluorescence recovery of PFP-SO3ꢀ depends
on the concentration of AChE (Figure 1b). A decrease in the
concentration of AChE leads to a slow initial cleavage
reaction rate and a low level of fluorescence recovery. The
limit of detection (LOD) of this assay is 0.05 unitsmLꢀ1,
which is comparable to that of most sensitive chemilumines-
cence techniques.[6]
As the exciton can diffuse during its lifetime along the
backbone of the conjugated polymer, one would not expect to
ꢀ
see fluorescence recovery of PFP-SO3 until after the
hydrolysis of all of the ACh–dabcyl if an excessive amount
of ACh–dabcyl is bound to the polymer. Thus, the use of
heavily quenched polymers to determine enzyme kinetics
may underestimate initial reaction rates and affect the
To measure the enzyme reaction kinetics, the cleavage enzyme activity. We therefore performed measurements of
reaction was monitored as a function of substrate concen-
tration. A plot of the change in intensity of PFP-SO3
enzymatic activity with the polymer PFP-COOH, which is
only quenched slightly by ACh–dabcyl. A linear relationship
between the fluorescence response of PFP-COOH and the
concentration of ACh–dabcyl was found, with a Ksv value of
8.21 ” 105 mꢀ1 (see the Supporting Information). On the basis
of these results we can be confident that there is no overlap of
ꢀ
emission at 424 nm versus the AChE incubation time was
used to calculate the initial velocity (in m sꢀ1; see the
Supporting Information). The concentration of ACh–dabcyl
ꢀ
was calculated from the fluorescence intensity of PFP-SO3
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for 10 min to allow hydrolysis to occur. The inhibition ability
of an inhibitor is described by an IC50 value, which refers to
the inhibitor concentration required for 50% inhibition of
enzyme activity. The fluorescence intensity of PFP-SO3ꢀ in
the presence and absence of the inhibitor was converted into
the concentration of ACh–dabcyl for all data points by using
the Stern–Volmer curve at pH 7.4. The IC50 value was
obtained from the plot of inhibition efficiency versus inhibitor
concentration; the inhibition efficiency (IE) was determined
by monitoring concentration changes of ACh–dabcyl by using
Equation (2) in which C0 is the initial concentration of ACh–
dabcyl, and Ct(inhibitor) and Ct(no inhibitor) are the concentrations of
ACh–dabcyl during the hydrolysis reaction with AChE in the
presence and absence of the inhibitor, respectively.
C
tðinhibitorÞꢀC
IE ¼
tðno inhibitorÞ ꢁ 100 %
ð2Þ
C0ꢀCtðno inhibitorÞ
Galanthamine and donepezil were found to inhibit AChE
with IC50 values of 12 and 23 nm, respectively (Figure 3a,b).
The PFP-SO3ꢀ/ACh–dabcyl complex is more favorable for
screening inhibitors with IC50 values at or below the nm scale,
and such inhibitors are urgently required for AD treatment.
Thus, the methodology described herein provides a sensitive,
rapid, and convenient protocol for screening AD drugs.
In summary, we have taken advantage of the charge-
reversal mechanism of a novel quencher-modified substrate
combined with the light-harvesting properties of conjugated
polymers to develop an assay for AChE activity and inhibitor
screening. The new method has several significant features.
Figure 2. a) Lineweaver–Burk plot for AChE-catalyzed hydrolysis in the
presence of PFP-SO3ꢀ; [PFP-SO3ꢀ]=2 mm, [AChE]=1.0 unitmLꢀ1
,
[ACh–dabcyl]=0.2, 0.25, 0.3, 0.4, 0.6 mm. Fluorescence measurements
were made in phosphate buffer solutions (25 mm, pH 8.0) with
excitation at 376 nm. b) Lineweaver–Burk plot for AChE-catalyzed
hydrolysis in the presence of PFP-COOH; [PFP-COOH]=2 mm,
[AChE]=0.25 unitsmLꢀ1, [ACh–dabcyl]=1, 1.5, 2.0, 2.5, 4.5 mm. Fluo-
rescence measurements were made in phosphate buffer solutions
(5 mm, pH 8.0) with excitation at 376 nm.
the quenching radius of multiple quenchers. The Km value of
8.7 mm found (Figure 2b) is in good agreement with that
obtained with PFP-SO3ꢀ. The results of this control experi-
ment combined with the instant fluorescence response of the
ꢀ
PFP-SO3 /ACh–dabcyl complex to AChE show that the PFP-
ꢀ
SO3 /ACh–dabcyl complex can be used to measure the
enzymatic kinetics of AChE as long as ACh–dabcyl is not
used in excess (that is, the charge ratio of the quencher to the
polymer should not be greater than 1.0).
The PFP-SO3ꢀ/ACh–dabcyl complex described herein can
also be used to screen AChE inhibitors. We selected for our
study the two most effective inhibitors currently employed
clinically for AD, galanthamine and donepezil.[26] As these
inhibitors are amino salts, all experiments were performed in
phosphate buffer (25 mm) at pH 7.4 to avoid their deproto-
nation and precipitation from solution. In the inhibition
assays, solutions of AChE and the inhibitors of varying
concentration were incubated at 258C for 15 min, then ACh–
dabcyl and PFP-SO3ꢀ were added, and the mixtures were left
Figure 3. Plots of the inhibition efficiency of a) galanthamine and
b) donepezil for AChE versus the concentration of the inhibitor; [PFP-
SO3ꢀ]=2 mm, [AChE]=1.0 unitmLꢀ1, [ACh–dabcyl]=0.3 mm, [galanth-
amine]=0.002, 0.01, 0.02, 0.1, 0.3 mm, [donepezil]=0.005, 0.01, 0.05,
0.1 mm. Fluorescence measurements were made in phosphate buffer
solutions (25 mm, pH 7.4) with excitation at 376 nm.
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Equation (3)[28] in which S0 is the standard deviation of the back-
ground and S is the sensitivity.
First, it offers a convenient “mix-and-detect” approach for the
rapid and sensitive detection of AChE activity and inhibition.
Second, the fluorescence intensity of PFP-SO3 is enhanced
ꢀ
S0
LOD ¼ 3
S
ð3Þ
130-fold through the hydrolysis of ACh–dabcyl, and therefore
the assay offers the benefits of extremely low background
noise and high detection sensitivity. Third, the method is more
favorable than existing methods for screening inhibitors with
IC50 values at or below the nm scale. Finally, this continuous
method is well suited for studies of the kinetics of enzyme
reactions. In principle, this sensor mechanism may prove to be
highly generalizable and provide a means to monitor the
activity of other enzymes (such as butyrylcholinesterase and
phosphatase). Furthermore, such fluorescence-assay systems
could be expanded to fluorescence-based high-throughput
screening assays.
Kinetic assay: ACh–dabcyl (0.2, 0.25, 0.3, 0.4, and 0.6 mm) and
then AChE (1.0 unitmLꢀ1) were added to PFP-SO3 (2.0 mm) in
ꢀ
phosphate buffer (1 mL, 25 mm, pH 8.0) in five 3-mL polystyrene
cuvettes. The cuvettes were incubated at 378C, and fluorescence
spectra were recorded at 20-s intervals over 400 s with excitation at
376 nm. The fluorescence intensity at 424 nm was plotted against the
incubation time of AChE. The substrate concentration was calculated
from the fluorescence intensities by using the Stern–Volmer curve.
The initial reaction rate was calculated by using the first five
acquisition points. A plot of 1/V0 versus 1/[S] was used to calculate the
Michaelis constant (Km).
AChE inhibition: Inhibitors were preincubated at varying con-
centrations (0–0.3 mm for galanthamine, 0–0.1 mm for donepezil) with
AChE (1 unitmLꢀ1) in phosphate buffer solution (1 mL, 25 mm,
pH 7.4) at 258C for 15 min, then PFP-SO3ꢀ (2.0 mm) and ACh–dabcyl
(0.3 mm) were added, and the resulting solutions were incubated at
378C for 10 min. Fluorescence spectra were then measured, and the
fluorescence intensities of PFP-SO3 in the presence and absence of
the inhibitors were converted into the concentration of ACh–dabcyl
by using the Stern–Volmer curve at pH 7.4.
Experimental Section
PFP-SO3ꢀ was synthesized according to a procedure described in the
literature.[22] 6-(p-Methyl red)aminohexanoic acid was prepared from
p-methyl red according to a procedure described in the literature.[27]
The synthesis and characterization of PFP-COOH are described in
the Supporting Information. Choline bromide was prepared from 2-
bromoethanol by treatment with trimethylamine at room temper-
ature. Acetylcholinesterase was purchased from Sigma, and the
solution of the enzyme was cooled in ice before use. Stock solutions of
the enzyme in Tris-HCl buffer (20 mm, pH 7.8; Tris= tris(hydroxy-
methyl)aminomethane) were prepared immediately before use and
maintained below 48C. Enzyme assays were performed in phosphate
buffer solution (25 mm, pH 8.0 for kinetics assays, pH 7.4 for
inhibition assays). Fluorescence spectra were obtained at 378C by
using a Hitachi F-4500 fluorometer equipped with a Xeron-lamp
ꢀ
Received: April 18, 2007
Published online: September 4, 2007
Keywords: biosensors · conjugated polymers · drug screening ·
.
enzyme catalysis · fluorescent probes
excitation source. The sample (1 mL) was placed in
a 3-mL
polystyrene cuvette. The excitation wavelength was 376 nm. The
water was purified by using a Millipore filtration system.
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silica gel (eluent: CH2Cl2/MeOH/H2O 65:25:4) to give ACh–dabcyl
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(15 mg, 34%) as a red solid. H NMR (400 MHz, D2O): d = 7.70 (d,
J = 7.6 Hz, 2H), 7.55–7.51 (m, 4H), 6.57 (d, J = 8.3 Hz, 2H), 4.48 (t,
J = 2.8, 2.2 Hz, 2H), 3.65 (t, J = 3.7, 1.8 Hz, 2H), 3.31 (t, J = 3.8,
2.3 Hz, 2H), 3.14 (s, 9H), 2.88 (s, 6H), 2.41 (t, J = 7.4, 7.0 Hz, 2H),
1.60–1.57 (m, 4H), 1.35–1.25 ppm (m, 2H). 13C NMR (150 MHz,
D2O): d = 174.5, 167.5, 154.2, 152.1, 142.3, 133.3, 127.9, 125.3, 121.8,
111.0, 67.8, 58.1, 53.8, 39.9, 39.3, 33.4, 28.7, 26.0, 24.9 ppm. MS (ESI):
m/z 468.4 ([MꢀBr]+).
Fluorescence quenching: ACh–dabcyl was added in a number of
ꢀ
batches to the solution of PFP-SO3 ([PFP-SO3ꢀ] = 2.0 mm) in
phosphate buffer (25 mm, pH 8.0 or pH 7.4) at room temperature,
and the fluorescence spectra of the resulting mixtures were measured.
The Ksv value was calculated from the first five acquisition points by
using the Stern–Volmer equation [Eq. (1)].
Fluorescence recovery: AChE (2.0 unitsmLꢀ1) was added to a
solution of ACh–dabcyl (2.0 mm) and PFP-SO3ꢀ (2.0 mm) in phosphate
buffer (25 mm, pH 8.0). The resulting solution was incubated at 378C
for a certain period of time (from 0 to 30 min), and its fluorescence
spectrum was then measured. The fluorescence intensity of PFP-SO3ꢀ
at 424 nm was then plotted as a function of the incubation time of
AChE. The limit of detection (LOD) of this method is obtained from
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