A new label-free continuous fluorometric assay for trypsin and inhibitor screening with tetraphenylethene compounds

Article abstract of DOI:10.1021/ol100626x

A new label-free continuous assay with the ensemble of compound 1 and Arg₆ for trypsin and inhibitor screening was successfully developed by taking advantage of the aggregation-induced emission feature of tetraphenylethene compounds. Both CLSM and DLS studies confirm the formation of the aggregation complex of compound 1 and Arg₆ and disassembly after further addition of trypsin.

Full text of DOI:10.1021/ol100626x

ORGANIC
LETTERS
2010
Vol. 12, No. 10
2274-2277
A New Label-Free Continuous
Fluorometric Assay for Trypsin and
Inhibitor Screening with
Tetraphenylethene Compounds
Wangxin Xue, Guanxin Zhang, Deqing Zhang,* and Daoben Zhu
Beijing National Laboratory for Molecular Sciences, Organic Solids Laboratory,
Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
dqzhang@iccas.ac.cn
Received March 17, 2010
ABSTRACT
A new label-free continuous assay with the ensemble of compound 1 and Arg6 for trypsin and inhibitor screening was successfully developed
by taking advantage of the aggregation-induced emission feature of tetraphenylethene compounds. Both CLSM and DLS studies confirm the
formation of the aggregation complex of compound 1 and Arg6 and disassembly after further addition of trypsin.
As one class of protease, trypsin is the most important
digestive enzyme produced by the pancreas.1 It is involved
in the digestive enzyme activation cascade, which induces
the transformation of other pancreatic proenzymes into their
active forms within the intestine and then initiates autodi-
gestion. Therefore, trypsin plays a key role in controlling
pancreatic exocrine function. It is known that trypsin level
is increased with some types of pancreatic diseases.2 Obvi-
ously, convenient and continuous assays for trypsin and
inhibitor screening may lead to new diagnostic methods and
also have therapeutic implications for these pancreatic
diseases. Assays based on solid-phase cleavage,3 turbidity,4
and radiometry5 were described for trypsin, but these
methods are either time-consuming or require specific
instruments. Ionescu et al.6 reported a sensitive amperometric
biosensor for the detection of trypsin. However, most of these
biosensors with enzyme-modified electrodes respond slowly.
Fluorometric assay methods based on doubly labeled sub-
strate peptides were also reported for trypsin.7 A label-free
fluorescent assay for trypsin was established with a water-
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A.; Retzinger, R. Anal. Biochem. 2006, 351, 114–121.
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10.1021/ol100626x  2010 American Chemical Society
Published on Web 04/20/2010

soluble conjugated polymer.8 Nevertheless, convenient label-
free fluorometric assays for trypsin and inhibitor screening
still remain limited. In this paper, we report a new label-
free continuous assay for trypsin and inhibitor screening by
taking advantage of the abnormal fluorescent behavior of
tetraphenylethene (TPE) compounds which are nonfluores-
cent in solution but become strongly fluorescent after
aggregation.9
trypsin and as a result the aggregation complex of 1 and
Arg6 would be disassembled. Accordingly, the fluorescence
of 1 will be turned off. Therefore, it is possible to establish
a new label-free continuous fluorometric assay for trypsin
with the ensemble of compound 1 and Arg6. (4) The
hydrolysis of Arg6 catalyzed by trypsin will be retarded in
the presence of the respective inhibitors. As a result, a smaller
amount of the aggregation complex of 1 and Arg6 will be
disassociated; the fluorescence decrease due to the presence
of trypsin will become small. In this way, the ensemble of
1 and Arg6 can be employed for screening the inhibitors of
trypsin.
The design rationale for this label-free fluorometric assay
is schematically illustrated in Scheme 1 and is explained as
Compound 1 was synthesized similarly according to the
reported procedure,10 and the synthetic details including
characterization are provided in the Supporting Information.
The aqueous solution of 1 (60.0 µM) in PBS buffer (2.0 mM,
pH ) 8.5) was prepared for the following fluorescent spectral
investigations. As anticipated, the aqueous solution of 1 was
almost nonfluorescent. As shown in Figure 1, the fluores-
Scheme 1. Illustration of the Formation of the Heteroaggregate
between Arg6 Peptide and Compound 1 (TPE Derivative) and
the Disassembly of the Aggregate in the Presence of Trypsin
follows: (1) Compound 1 is a tetraphenylethene derivative
with one sulfonate (-SO3 ) unit which will enable it to be
Figure 1. Fluorescence spectra of 1 (60.0 µM) in PBS buffer
-
solution (2.0 mM, pH ) 8.5) in the presence of different amounts
of Arg6 peptide (from 0.0 to 10.0 µM); the insets show (1) the
photos of the corresponding buffer solutions of 1 (60.0 µM) in the
absence (A) and presence(B) of Arg6 peptide (10.0 µM) under UV
light (365 nm) illumination and (2) variation of the fluorescence
intensity at 475 nm vs the concentration of Arg6.
dissolved in aqueous solution. It is expected that compound
1 shows an aggregation-induced emission (AIE) feature;
namely, it is weakly fluorescent in solution and its fluores-
cence is turned on after aggregation.9 (2) Arg6, a positively
charged peptide, is selected as the substrate for trypsin. We
assume that the presence of Arg6 in the solution would trigger
the aggregation of compound 1 because of the electrostatic
cence intensity of 1 increased gradually after addition of Arg6
to the solution. For instance, the fluorescence intensity at
475 nm of 1 was enhanced by 47 times when the concentra-
tion of Arg6 reached 10.0 µM in the solution. In fact, the
fluorescence quantum yield of the solution of 1 (60.0 µM)
increased from 0.005 to 0.156 (by reference to quinine
hemisulfate monohydrate) after Arg6 (10.0 µM) was intro-
duced to the solution. Such fluorescence enhancement
observed for 1 after addition of Arg6 can be distinguished
by the naked eye as depicted in the inset of Figure 1, where
photos (under UV light irradiation) of two PBS buffer
solutions of 1 in the absence and presence of Arg6 were
shown. Interestingly, the fluorescence intensity of the
ensemble solution of compounds 1 and Arg6 increased almost
linearly with the concentration of Arg6 in the range of 0-10
µM as displayed in the inset of Figure 1 (I475 nm ) 38.4[Arg6]
- 1.28, r ) 0.99).
-
interaction among the sulfonate (-SO3 ) unit in 1 and the
positive arginine residues. The corresponding hydrophobic
interaction among molecules of 1 and Arg6 may also facilitate
the aggregation. As anticipated, the fluorescence will increase
after the coaggregation of compound 1 and Arg6. (3) Arg6
will be hydrolyzed into small fragments in the presence of
(8) (a) An, L.; Tang, Y.; Feng, F.; He, F.; Wang, S. J. Mater. Chem.
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Org. Lett., Vol. 12, No. 10, 2010
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Both confocal laser scanning microscopic (CLSM) and
dynamic light scattering (DLS) studies confirm the formation
of the aggregation complex of 1 and Arg6, which induces
the fluorescence enhancement of 1. As displayed in Figure
S1 (Supporting Information), no detectable fluorescence
aggregates were observed for the solution of 1 (0.1 mM)
with CLSM. However, fluorescence aggregates with sizes
ca. 1.0 µm were observed for the buffer solution of 1 (0.1
mM) and Arg6 (20.0 µM) (see Figure S1, Supporting
Information). DLS data indicated that aggregates of around
1000 nm were formed in the solution of 1 (60.0 µM) and
Arg6 (10.0 µM) (see Figure S2, Supporting Information).11
Next, we want to demonstrate that the ensemble of 1 and
Arg6 can be employed to establish a label-free continuous
fluorometric assay for trypsin. As mentioned above, Arg6 is
hydrolyzed in the presence of trypsin. It is expected that
hydrolysis of Arg6 will destabilize the aggregation complex
of 1 and Arg6; as a result, the fluorescence of the ensemble
will decrease. As discussed above, the initial PBS buffer
solution of 1 (60.0 µM) and Arg6 (10.0 µM) showed strong
fluorescence. But, the fluorescence of the ensemble started
to decrease after introduction of trypsin into the solution,
and more fluorescence reduction was observed by prolonging
the incubation time as shown in Figure 2. The fluorescence
(0.2, 0.8, 1.6, 3.2, 8.0 µg/mL) were prepared to react
separately with Arg6 (10.0 µM). The hydrolysis reaction was
followed by measuring the fluorescence spectrum of 1 in
the reaction mixture. Figure 3 shows the variation of the
Figure 3. Variation of the fluorescence intensity at 475 nm vs the
reaction time for the ensemble of 1 (60.0 µM) in PBS buffer solution
(2.0 mM, pH ) 8.5, containing CaCl2 (10.0 µM)) and Arg6 peptide
(10.0 µM) in the presence of different concentrations of trypsin
(0.0, 0.2, 0.8, 1.6, 3.2, 8.0 µg/mL); the excitation wavelength was
340 nm.
fluorescence intensity at 475 nm with hydrolysis time for
the five concentrations of trypsin. It is obvious that the
fluorescence intensity of the solution of 1 and Arg6 decreased
rapidly with high concentration of trypsin. This fluorescence
reduction for ensemble of 1 and Arg6 after incubation with
trypsin is ascribed to the disassembly of the aggregation
complex of 1 and Arg6, which is supported by both CLSM
and DLS studies. The initial fluorescence images detected
for the solution of 1 (0.1 mM) and Arg6 (20.0 µM)
disappeared after the solution was incubated with trypsin (8.0
µg/mL) for 30 min (see Figure S1, Supporting Information).
The number of large aggregates around 1000 nm was
significantly reduced for the solution of 1 (60.0 µM) and
Arg6 (10.0 µM) after addition of trypsin (8.0 µg/mL) as
shown in Figure S2 (Supporting Information).
The hydrolysis of Arg6 catalyzed by trypsin was also
studied with different concentrations of Arg6. Four buffer
solutions of Arg6 with concentrations of 3.4, 4.7, 6.8, and
10.8 µM were prepared separately, and each solution
containing compound 1 (60.0 µM) and trypsin (3.2 µg/mL)
was incubated for different times at room temperature. The
fluorescence spectrum of each solution after certain incuba-
tion time was measured. In this way, the hydrolysis reaction
was followed by measuring the fluorescence intensity of 1
at 475 nm, and the concentration of Arg6 remained at certain
hydrolysis reaction time was deduced accordingly. The
corresponding initial reaction rate (V0, in µM·min-1) was
calculated for the hydrolysis reaction at 4.0 min with different
concentrations of Arg6. Figure S3 (Supporting Information)
shows the plot of 1/V0 vs 1/[Arg6], the corresponding
Lineweaver-Burk plot. The kinetic parameters for trypsin,
Km and Vmax, were estimated to be 2.17 µM and 13.8
µM·min-1, respectively, by fitting the plot with Michalis-
Menten equation. The Km value is close to those reported
for trypsin with other assay method.7a The above results
Figure 2. Fluorescence spectra of the ensemble of 1 (60.0 µM) in
PBS buffer solution (2.0 mM, pH ) 8.5, containing CaCl2 (10.0
µM)) and Arg6 peptide (10.0 µM) in the presence of trypsin (8.0
µg/mL) incubated at room temprature for different times; the inset
shows the photos of the corresponding solutions of 1 and Arg6
peptide without (A) and with (B) trypsin (8.0 µg/mL) after
incubation at room temperature for 20.0 min under UV light (365
nm) illumination.
reduction observed for the ensemble of 1 and Arg6 upon
addition of trypsin could also be detected with the naked
eye as indicated in the inset of Figure 2, where the photos
of the solution of 1 and Arg6 in the absence and presence of
trypsin were displayed. It should be noted that the fluores-
cence variation of 1 upon addition of only trypsin can be
neglected under the same conditions.
The hydrolysis of Arg6 should be dependent on the
concentration of trypsin used. Five concentrations of trypsin
(11) Aggregates of ca. 100 nm were detected with DLS for the solution
of 1 before addition of Arg6. Compound 1 is an amphiphilic compound,
and thus, it is understandable that molecules of 1 tend to assemble in aqueous
solution.
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Org. Lett., Vol. 12, No. 10, 2010

clearly indicate that a label-free fluorometric assay for trypsin
can be established with the ensemble of compound 1 and
Arg6. With the ensemble of 1 and Arg6, trypsin with a
concentration as low as 0.2 µg/mL can be analyzed.
decreases by prolonging the hydrolysis time, but in the
presence of BBI, the degree of the fluorescence reduction
for the ensemble is reduced gradually. Based on the plot of
the inhibition efficiency13 vs the concentration of BBI (Figure
S4, Supporting Information), the corresponding IC50 value
of BBI from soybean toward trypsin was estimated to be
1.53 µg/mL. This IC50 value is different from those deter-
mined with other assay methods.14 But, it is understandable
because the reported IC50 values are affected by several
parameters including the concentrations of trypsin and the
substrate. All these results obviously indicate that the
ensemble of 1 and Arg6 can be utilized not only for trypsin
activity assay but also for the corresponding inhibitor
screening.
In summary, we successfully developed a new label-free
continuous assay with the ensemble of 1 and Arg6 for trypsin
and inhibitor screening by taking advantage of the aggrega-
tion-induced emission feature of tetraphenylethene com-
pounds. Both CLSM and DLS studies confirm the formation
of the aggregation complex of 1 and Arg6, which is driven
by the electrostatic and hydrophobic interactions, and disas-
sembly after further addition of trypsin. Compared to the
reported assay methods for trypsin, this new fluorometric
assay has the following advantages besides being label-free
and having continuous features: (1) the assay can be carried
out in pure aqueous solution, (2) the response time is
acceptable, and (3) compound 1 is easily prepared and Arg6
is commercially available; thus, this new assay should be
cost-effective. Therefore, this fluorometric assay is potentially
useful for high-throughput screening of trypsin inhibitors that
may have implications for diagnostic methods for pancreatic
diseases and anticarcinogenic drug discovery.15
The hydrolysis of Arg6 catalyzed by trypsin will be
retarded when the corresponding inhibitors of trypsin were
present in the solution. Accordingly, less fluorescence
reduction for the ensemble of 1 and Arg6 containing trypsin
is expected after further addition of the inhibitors. Therefore,
the ensemble of 1, Arg6 and trypsin can be used to screen
the trypsin inhibitors. A Bowman-Birk inhibitor (BBI) from
soybean12 was selected to demonstrate the usefulness of the
ensemble to screen trypsin inhibitors. Similarly, the fluo-
rescence spectra of the ensemble of 1 (60.0 µM), Arg6 (10.0
µM), and trypsin (3.2 µg/mL) in the presence of different
amounts of BBI (0.0, 1.0, 2.0, 5.0 µg/mL) were recorded
after incubation for different times at room temperature.
Figure 4 shows the variation of the fluorescence intensity of
Figure 4. Plot of the fluorescence intensity at 475 nm vs the reaction
time for the ensemble of 1 (60.0 µM in PBS buffer solution (2.0
mM, pH ) 8.5, containing CaCl2 (10.0 µM))), Arg6 peptide (10.0
µM), and trypsin (3.2 µg/mL) in the presence of different
concentrations of BBI (0, 1.0, 2.0, 5.0 µg/mL); the excitation
wavelenth was 340 nm.
Acknowledgment. The present research was financially
supported by the NSFC, Chinese Academy of Science, and
State Key Basic Research Program.
Supporting Information Available: Synthesis and char-
acterization of compound 1; CLSM and DLS data; the
Lineweaver-Burk plot of the hydrolysis of Arg6 peptide for
trypsin; plot of the inhibition efficiency of BBI toward
trypsin. This material is available free of charge via the
Internet at http://pubs.acs.org.
the ensemble at 475 nm with the hydrolysis time in the
presence of four different concentrations of BBI. As ex-
pected, in the absence of BBI, the fluorescence intensity
(12) BBI is a protein of a molecular weight of 8 KDa with a well-
characterized ability to inhibit trypsin and chymotrypsin and has been studied
extensively as an anticarcinogenic protease inhibitor: (a) Kennedy, A. R.
Protease Inhibitors As Cancer ChemopreVentiVe Agents; Plenum Press: New
York, 1993. (b) Birk, Y. Int. J. Peptide Protein Res. 1985, 25, 113–131.
(c) Rawlings, N. D.; Tolle, D. P.; Barrett, A. J. Biochem. J. 2004, 378,
705–716. (d) Ye, X. Y.; Ng, T. B.; Rao, P. F. Biochem. Biophys. Res.
Commun. 2001, 289, 91–96.
OL100626X
(13) Ho, V. M.; Ng, T. B. J. Pept. Sci. 2008, 141, 278–282.
(14) Wang, H. X.; Ng, T. B. Biochem. Biophys. Res. Commun. 2006,
342, 349–353.
(15) Kennedy, A. R. Am. J. Clin. Nutr. 1998, 68, 1406S–1412S.
Org. Lett., Vol. 12, No. 10, 2010
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