Strategy for sensor based on fluorescence emission red shift of conjugated polymers: Applications in pH response and enzyme activity detection

Article abstract of DOI:10.1021/ac302840t

A new strategy was developed and applied in monitoring pH response and enzyme activity based on fluorescence emission red shift (FERS) of the conjugated polymer PPP-OR10 induced by the inner filter effect (IFE) of nitrobenzene derivatives. Neutral poly(p-

Full text of DOI:10.1021/ac302840t

Technical Note
pubs.acs.org/ac
Strategy for Sensor Based on Fluorescence Emission Red Shift of
Conjugated Polymers: Applications in pH Response and Enzyme
Activity Detection
Yanli Tang,* Yue Liu, and Ali Cao
Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life
Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, P. R. China
S
* Supporting Information
ABSTRACT: A new strategy was developed and applied in
monitoring pH response and enzyme activity based on
fluorescence emission red shift (FERS) of the conjugated
polymer PPP-OR10 induced by the inner filter effect (IFE) of
nitrobenzene derivatives. Neutral poly(p-phenylenes) func-
tionalized with oligo(oxyethylene) side chains (PPP-OR10)
was designed and synthesized by the Suzuki cross-coupling
reaction. Nitrobenzene derivatives display different light
absorption activities in the acidic or basic form due to
adopting different electron-transition types. When environ-
mental pH is higher than their pKa values, nitrobenzene derivatives exhibit strong absorbance around 400 nm, which is close to
the maximal emission of polymer PPP-OR10. As a result, the maximal emission wavelength of PPP-OR10/nitrobenzene
derivatives red shifts with the pH value increasing. Apparently, the IFE plays a very important role in this case. A new method has
been designed that takes advantage of this pH-sensitive platform to sensor α-chymotrypsin (ChT) based on the IFE of p-
nitroaniline, since the absorption spectrum of p-nitroaniline, the ChT-hydrolyzed product of N-benzoyl-L-tyrosine-p-nitroaniline
(BTNA), overlaps with the emission spectrum of PPP-OR10. In addition, the present approach can detect α-chymotrypsin with a
detection limit of 0.1 μM, which is lower than that of the corresponding absorption spectroscopy method. Furthermore, the pH
response and enzyme detections can be carried out in 10% serum, which makes this new FERS-based strategy promising in
applications in more complex conditions and a broader field.
n recent years, conjugated polymers (CPs) have attracted
shown that the nonspecific interactions especially electrostatic
Imuch attention owning to their unique photo- and interactions between conjugated polymers and targets are more
difficult to control than anticipated and are inevitably disturbed
by experimental conditions such as ionic strength.3,36
electrocharacteristics. CPs are comprised of a large number of
repeated conjugated units.1,2 The long conjugation within the
molecular backbone allows the excitation energy along the main
chain transfer to an energy or electron acceptor quickly after
excited by UV light, which accounts for the fluorescence signal
amplification.3 In the past few years, we and others have taken
advantage of its fluorescence signal amplification to design a
variety of biosensors and chemosensors.4−11 The detections of
DNA, RNA, ligand (sugar, lectin, biotin)-protein specific
interaction, enzyme (ALP, kinase, protease) activity, protein
fibrillation, as well as toxic metal ions, etc.12−26 have been
reported with good specificity and high sensitivity. Recently,
water-soluble conjugated polymers present unexpectedly
promising applications in antimicrobial susceptibility testing,
drug screening, cell imaging, and barcoding.27−30 It is
interesting to note that the sensing systems are designed and
realized mainly by means of fluorescence resonance energy
transfer (FRET),31 polymer aggregation or target-induced
conformational change,10,32,33 and photoinduced electron
transfer (PET) or charge transfer.34,35 The electrostatic
interactions and hydrophobic interactions play an important
part among these techniques. However, some reports have
In this study, we present an alternative approach to design
sensors based on the fluorescence inner filter effect (IFE),
resulting in fluorescence emission red shift of conjugated
polymers. The IFE is stemmed from absorption of the incident
light or absorption of the emitted light by absorbers in the
detection system.37 At present, some novel fluorescent assays
have been reported by researchers based on the IFE, although it
is usually thought of as an adverse factor in spectrofluorom-
etry.38−43 Notably, distance and interactions are dominant in
the techniques such as FRET, PET, etc.; however, this
approach does not require approximity and strong interactions
between the fluorescent probe and the target, which provides
considerable flexibility and broad applications in sensors.
Poly(p-phenylenes) substituted with oligo(oxyethylene) side
chains (PPP-OR10) (Scheme 1) are designed and synthesized
through a palladium-mediated Suzuki cross-coupling reaction
Received: June 12, 2012
Accepted: December 14, 2012
Published: December 14, 2012
© 2012 American Chemical Society
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Technical Note
nitrophenol, 2,4-dinitrophenol, 2,4,6-trinitrophenol, or p-nitro-
aniline) (2.0 × 10−4 M) was added in 5 mM PBS (pH = 3.5)
solution, and the absorption spectra were recorded at room
temperature. Then, the absorption spectra of nitrobenzene
derivatives in 5 mM PBS at pH 8.5 were obtained using a UV-
2450 spectrophotometer.
Scheme 1. The Chemical Structure of PPP-OR10
Fluorescence Spectra of PPP-OR10/Nitrobenzene
Derivatives at Various pH. A series of solutions of PPP-
OR10 ([PPP-OR10] = 8.0 × 10−6 M in RUs) and nitrobenzene
derivatives (2.0 × 10−4 M) were prepared with the total volume
of 3 mL in 5 mM PBS at different pH (from 2.35 to 12.11).
Then, the fluorescence spectra were measured at room
temperature using a Hitachi F-7000 spectrofluorometer.
Fluorescence Spectra of PPP-OR10/Nitrobenzene
Derivatives at Various pH in Serum. A series of solutions
of PPP-OR10 ([PPP-OR10] = 8.0 × 10−6 M in RUs) and
nitrobenzene derivatives (2.0 × 10−4 M) were prepared with
the total volume of 3 mL in 5 mM PBS with 10% serum
(volume %) at different pHs (from 2.35 to 12.11). Then, the
fluorescence spectra were measured at room temperature.
Reversibility of Maximal Emission Wavelength of
PPP-OR10/p-Nitrophenol. The solution of PPP-OR10/p-
nitrophenol ([PPP-OR10] = 8.0 × 10−6 M in RUs, [p-
nitrophenol ] = 2.0 × 10−4 M) was prepared in 5 mM PBS at
pH 3. All of the fluorescence spectra were recorded at room
temperature, unless otherwise specified. After adjusting the pH
value from 3 to 11 with 2 M NaOH, the fluorescence spectrum
was obtained. Then, the pH value was adjusted from 11 to 3
with 2 M HCl. The fluorescence spectrum was measured. The
measurements were cycled ten times according to the same
procedure.
Assay of α-Chymotrypsin by Fluorescence Spectros-
copy. A solution of PPP-OR10/BTNA ([PPP-OR10] = 8.0 ×
10−6 M in RUs, [p-nitrophenol] = 2.0 × 10−4 M) was prepared
in 5 mM PBS at pH 8.5. After addition of various amounts of α-
chymotrypsin ([ChT] = 0−62 μM), the fluorescence spectra
were measured at 30 °C every minute for 15 min. The assay of
α-chymotrypsin in the presence of PPP-OR10 in 5 mM PBS
with 10% serum (volume %) were carried out using the same
procedures described above.
Assay of α-Chymotrypsin by Absorption Spectrosco-
py. A solution of BTNA ([p-nitrophenol] = 2.0 × 10−4 M) was
prepared in 5 mM at pH 8.5 at room temperature. After
addition of different concentrations of α-chymotrypsin ([ChT]
= 0−62 μM), the absorption spectra were measured every
minute in the range from 0 to 15 min at 30 °C.
(Scheme S1 of the Supporting Information), and its monomer
can readily be synthesized according to previous literature.44
The alkoxy side chains significantly improve the water solubility
of PPP-OR10, which makes it feasible to develop assays in
aqueous media. Because conjugated polymers PPP-OR10 have
no charge, the electrostatic interactions will hardly affect the
sensor system. In the presence of targets, such as nitrophenol
and nitroaniline, even if they present in basic form, there are no
obvious electrostatic interactions between PPP-OR10 and the
targets. As we know, some compounds such as p-nitrophenol
and 2,4-dinitrophenol45 exhibit pH-dependent absorbance.
Generally, the maximal absorption of nitrobenzene derivatives
is shifted toward longer wavelength with the pH value
increasing. Thus, the variable fluorescence emission of PPP-
OR10/nitrobenzene derivatives is red-shifted with the pH value
increasing, which results from the IFE because of spectral
overlap of PPP-OR10 emission with the absorption of
nitrobenzene derivatives. Furthermore, a new pH sensor is
designed based on the fluorescence emission red shift (FERS)
of conjugated polymers, which is independent of electrostatic
interactions, and a sensitive method for enzyme detection is
developed by taking advantage of the sensor system as
described above.
EXPERIMENTAL PROCEDURES
■
Materials and Measurements. All chemicals were
purchased from Sigma-Aldrich or Alfa Aesar. All reagents
were of analytical grade and used without further purification.
N-Benzoyl-L-tyrosine-p-nitroanilide (BTNA) and α-chymotryp-
sin (from bovine pancreas) (ChT) were obtained from Sigma-
Aldrich. Fetal bovine serum was purchased from Sangon
Biotech Company, Ltd. (Shanghai, China). UV-vis absorption
spectra were taken on a UV-2450 (Shimadzu Company)
spectrophotometer. Fluorescence measurements were recorded
in 3 mL quartz cuvettes at room temperature using a Hitachi F-
7000 spectrofluorometer equipped with a xenon lamp
excitation source. All fluorescence spectra were measured at
an excitation wavelength of 340 nm. Water was purified by
using a Millipore filtration system.
RESULTS AND DISCUSSION
■
First, the basic photophysical properties of PPP-OR10 were
investigated in different solvents. The UV−vis absorption and
photoluminescence emission spectra of PPP-OR10 are
presented in Figure 1. With the increasing solvent polarity,
the maximal UV−vis absorption and photoluminescence
emission migrate toward longer wavelengths, which results
from the reduced gap between the π−π* energy level that is
induced by the solvent effect. Also the photoluminescence of
polymer is quenched significantly in water compared to the
other four organic solvents, which stems from the fact that the
fluorescence of PPP-OR10 is self-quenching because of the
π−π stacking effect. The alkoxy side chains were designed for
improving the water solubility of PPP-OR10 and making PPP-
OR10 neutral, which has some advantages, such as feasibility in
aqueous media and immunity to electrostatic interactions.
Absorption and Fluorescence Spectra of PPP-OR10. A
solution with a total volume of 3.0 mL H2O containing PPP-
OR10 ([PPP-OR10] = 1.0 × 10−6 M in RUs (repeated units)
for fluorescence and 5.0 × 10−5 M for absorption, respectively)
was prepared at room temperature. Then the absorption and
fluorescence emission spectra were measured respectively. The
excitation wavelength for the fluorescence emission spectra was
340 nm. The absorption and fluorescence emission spectra of
PPP-OR10 in other organic solvents (CH3OH, CHCl3, THF,
and DMSO) were measured according to the same procedure.
Absorbance of Nitrobenzene Derivatives under
Different pH. Nitrobenzene derivatives (o-nitrophenol, p-
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Figure 2a shows the emission spectra of PPP-OR10/p-
nitrophenol in a 5 mM PBS solution, from which we can see
Figure 1. (a) Normalized UV−vis spectra and (b) fluorescence
emission spectra of PPP-OR10 in various solvents. The excitation
wavelength is 340 nm.
Figure 2. (a) The fluorescence spectra of PPP-OR10/p-nitrophenol in
5 mM PBS buffer solution with various pH values. (b) The maximal
emission wavelength of PPP-OR10 in the presence of nitrobenzene
derivatives as a function of pH value. [PPP-OR10] = 8.0 × 10−6 M,
[nitrophenol] = [nitroaniline] = 2.0 × 10−4 M. The error bars
represent the standard deviations of three parallel measurements. The
excitation wavelength is 340 nm.
Figure S1 of the Supporting Information showed the
absorption spectra of nitrobenzene derivatives under different
pH values, which present that the absorptions of p-nitrophenol,
o-nitrophenol, and 2,4-dinitrophenol are pH-dependent while
those of 2, 4, 6-trinitrophenol and p-nitroaniline are pH-
independent in the experimental condition. The pKa values of
the five compounds used in the paper were shown in Table S1
of the Supporting Information. Under the detection conditions
(pH 2.35−12.11), the former three compounds (2.35 < pKa <
12.11) will change their forms from acidic to basic, along with
increasing pH, meanwhile, the absorptions red shift, due to
adopting different electron-transition types (π−π* → n−π*).
However, the absorption maximal wavelength of 2,4,6-
trinitrophenol and p-aniline do not shift because they stay the
same basic form at the same condition (pH > pKa). The
difference of absorption spectra will bring the different overlap
effect with absorption and fluorescence emission spectra of
PPP-OR10. As shown in Figure S2 of the Supporting
Information, when pH is far below pKa (pH 2.35, acidic
form), the absorbance of the p-nitrophenol compound is
relatively low, while no obvious absorbance appears in the
polymer emission region (350−500 nm), which overlaps
absorption partly but barely overlaps fluorescence spectra of
PPP-OR10. When environmental pH value is higher than the
pKa (pH 12.11, basic form), p-nitrophenol exhibits a broader
overlap band with both absorption and emission of PPP-OR10.
The 2,4-dinitrophenol contains a similar overlap effect with
PPP-OR10 (Figure S2b of the Supporting Information).
Undoubtedly, 2,4,6-trinitrophenol and p-nitroaniline possess
the unchanged spectral overlap range with PPP-OR10, during
the tested pH range. Notably, the absorption spectra of the
basic form of o-nitrophenol almost overlap with the emission
spectra of PPP-OR10 completely. As a result, the emission
spectrum of PPP-OR10 is modulated by nitrobenzene
derivatives due to IFE, which depends on the spectral overlap
effect. Thus, the change in the absorbance of nitrobenzene
derivatives can be translated into changes in the maximal
fluorescence emission wavelength.
the maximal fluorescence emission wavelength around 418 nm
in a relatively acidic environment (pH < 6) because the spectral
overlap mainly comes from the absorption of p-nitrophenol and
that of PPP-OR10. The IFE causes the fluorescence intensity
attenuation; however, the maximal emission wavelength is
affected a little. As the pH value continues to increase, the
maximal emission peaks of PPP-OR10/p-nitrophenol exhibit
gradual red shifts until the final emission wavelengths appear at
460 nm when the pH value increased to 12,46 which is mainly
attributed to the p-nitrophenol absorbing the partial emission
light of PPP-OR10 from the spectral-overlap band. With the
increasing of pH value from 2 to 12, the absorbance of p-
nitrophenol at 317 nm decreases while the peak at 400 nm
increases gradually (Figure S1 of the Supporting Information).
Thus, the spectral overlap of the absorption band of p-
nitrophenol with the emission spectra of PPP-OR10 changes to
the longer wavelength range. Therefore, the emission peak
wavelength of PPP-OR10 red-shifted due to the IFE. The
extreme change of the pH value indeed has a certain impact on
the fluorescence intensity of PPP-OR10, which leads to the
fluctuation in irradiation (Figure 2a) but will not affect the
maximal emission wavelength of PPP-OR10. From Figure 2b,
we can see that 2,4-dinitrophenol had a similar maximal
emission wavelength changing curve as p-nitrophenol, while
2,4,6-trinitrophenol and p-nitroaniline just kept nearly the same
maximal emission wavelength due to their constant basic forms
in our experimental conditions. If the assay was carried out in a
more acidic buffer (pH value lower than pKa of 2,4,6-
trinitrophenol and p-nitroaniline), an upward plot can also be
found for 2,4,6-trinitrophenol and p-nitroaniline, just like the
other two nitrobenzene derivatives (data not showed here).
However, the maximal emission wavelength of PPP-OR10
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Analytical Chemistry
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keeps around 419 nm in the presence of o-nitrophenol, even if
the pH increases from 2 to 12 because of the almost complete
spectral overlap of absorption of the basic form of o-
nitrophenol with emission of PPP-OR10. The corresponding
results are that the fluorescence intensity decreases while the
maximal emission wavelength remains unchanged due to the
IFE. These results demonstrated that the red shift of the
maximal emission wavelength depends on the spectral overlap
effect. In addition, some research groups have reported that the
IFE can be corrected mathematically.37,47−50 The corrected
emission wavelengths and intensities through the IFE equation
were compared with those observed from experiments. (see the
Supporting Information). All results indicated that the IFE
caused the fluorescence quenching and wavelength red shift. To
investigate the effect of the solution ionic strength on the
emission red shift, we carried out the experiments in 50 mM
PBS, as the same procedure. The results we obtained present
the same fluorescence emission red-shift trend (data not
showed here), which show the strategy based on the IFE could
avoid the interference to some extent from the ionic strength
that generally affects FRET or PET techniques.
nitroanilide (BTNA) is used here as a substrate to assay the α-
chymotrypsin in our system. From Scheme 2, we can see after
Scheme 2. Schematic Demonstration of α-Chymotrypsin
Hydrolysis Process
BTNA being hydrolyzed by ChT, p-nitroaniline is released in
our testing environment (pH = 8.5). It is undisputed that the
absorption spectrum of p-nitroaniline overlaps with the
emission spectrum of PPP-OR10, which leads to the maximal
emission wavelength of the PPP-OR10 red shift, as a result of
IFE. Thus, different fluorescence emission signals can be
obtained to monitor the enzyme activity before and after the
hydrolysis of BTNA by ChT.
After the addition of ChT (31.0 μM) to a working solution
containing PPP-OR10/BTNA ([PPP-OR10] = 8.0 × 10−6 M in
RUs, [BTNA] = 2.0 × 10−4 M) in 5 mM PBS at pH 8.5, Figure
4a was obtained by monitoring the enzymatic reaction time at
To detect the reversibility of this system, the maximal
emission wavelength of PPP-OR10/p-nitrophenol can be
switched back and forth by adjusting the pH value between 3
and 11. As shown in Figure 3, after experiencing a shuttling
Figure 3. The maximal emission wavelength of PPP-OR10/p-
nitrophenol upon cycling the pH value between 3 and 11 in a 5
mM PBS buffer solution. [PPP-OR10] = 8.0 × 10−6 M and [p-
nitrophenol] = 2.0 × 10−4 M. The error bars represent the standard
deviations of three parallel measurements. The excitation wavelength
is 340 nm.
between 3 and 11 (pH value) ten times, the system still exhibits
stable results: the maximal emission wavelength is situated
around 418 nm at pH 3 and 458 nm at pH 11, respectively. It
can work very well as long as the emission of the two
compounds partly overlapped without additional requirements,
such as distance and interactions, so a better cycling result can
be obtained.
It was reported that p-nitrophenol and p-nitroaniline have
been utilized to covalently attach to substrates as tags to
monitor enzymatic activity through a signal change of the UV−
vis spectra.51,52 However, when large amounts of samples are
required, high-throughput analysis is necessary and convenient,
which can be achieved mainly by fluorescence spectroscopy. So,
it is interesting to design a new assay method involving the
utility of p-nitrophenol and p-nitroaniline in the fluorescence
field. Here, we also investigate the feasibility of applying our
system to detect an enzyme. α-Chymotrypsin (from bovine
pancreas), a kind of protease, selectively catalyzes the hydrolysis
of peptide bonds on the C-terminal side of tyrosine,
phenylalanine, trytophan, and leucine. N-Benzoyl-L-tyrosine-p-
Figure 4. (a) Fluorescence emission spectra of polymer/BTNA as a
function of α-chymotrypsin (ChT) incubation time ([α-chymotryp-
sin] = 31 μM). (b) The maximal emission wavelength as a function of
α-chymotrypsin incubation time ([α-chymotrypsin] = 0−62 μM). The
measurements were performed in a PBS buffer solution (5 mM, pH =
8.5) at 30 °C, [PPP-OR10] = 8.0 × 10−6 M, and [BTNA] = 2.0 × 10−4
M. The error bars represent the standard deviations of three parallel
measurements. The excitation wavelength is 340 nm.
30 °C for an interval of 1 min for 15 min. The maximal
emission red-shifted about 30 nm, while fluorescence was
quenched gradually with the incubating time from 0 to 15 min.
Figure 4b illustrates the maximal emission wavelength of PPP-
OR10 in different concentrations of α-chymotrypsin as a
function of incubation time. In these experiments, the BTNA
concentration was fixed (2.0 × 10−4 M) and the ChT
concentration was varied over a range of 0−62.0 μM. Control
tests show that BTNA itself has little effect on our detection
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Analytical Chemistry
Technical Note
system in limited time. It is noted that the maximal emission
wavelength of PPP-OR10 is obviously concentration-depend-
ent. When the concentration of α-chymotrypsin is 3.1 μM, the
increased maximal emission wavelength is around 12 nm after
incubation for 15 min. Interestingly, when the concentration of
α-chymotrypsin is 62.0 μM, the maximal fluorescence emission
wavelength red shifts 30 nm and it reaches the platform after an
incubation time of only 6 min. The limit of detection (LOD) of
this system is obtained as 0.1 μM. In comparison with the LOD
(0.3 μM) obtained by measuring the absorbance of p-
nitroaniline (Figure S3 of the Supporting Information), this
method is more sensitive than UV−vis absorption spectrosco-
py. The results in Figure 4b show that the maximal emission
wavelength is time- and enzyme concentration-dependent,
which indicates that the sensor is available in real-time
detection and the enzyme reactions are the rate-limiting
process in the assay.
unknown compounds in serum on α-chymotrypsin. In
conclusion, the method is feasible in the more complex
detection environment that the other techniques are usually
limited to.
CONCLUSIONS
■
In summary, we have developed a new strategy for pH response
and enzyme activity detection based on the inner filter effect
(IFE). The detection was based on the fluorescence emission
red shift that results from spectral overlap of neutral conjugated
polymer and nitrobenzene derivatives. Our detection is not just
confined to the polymers or absorbers we employed in this
contribution but can be applied to any system that satisfies the
basic requirements: the absorption of the target must overlap
with the emission of probe to a degree, and the target displays a
different absorption when the external environment changes. In
comparison with previous techniques, the IFE-based approach
yields greater flexibility and a larger breadth of applications in
sensors because no interactions between conjugated polymers
and targets were needed, and it worked very well in a more
complex environment, such as 10% serum. In addition, the limit
of detection of α-chymotrypsin obtained via this method was
lower than that obtained by UV−vis absorption spectrometry.
The system brings enzymatic analysis based on the p-
nitrophenol- and p-nitroaniline-tagged substrates to a new
level of field-fluorescence detection, which makes high-
throughput assays possible.
To investigate the application of the new method in complex
condition, the measurements of pH response and α-
chymotrypsin analysis based on the IFE were carried out in
10% serum. Figure 5a showed results similar to those observed
ASSOCIATED CONTENT
* Supporting Information
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
* E-mail: yltang@snnu.edu.cn. Tel: (086) 029-81530726. Fax:
(086) 029-81530727.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are grateful for the financial support from the
National Natural Science Foundation of China (Grants
21222509 and 21105061), Natural Science Basic Research
Plan in Shaanxi Province of China (Grant 2011JQ2008),
Fundamental Research Funds for the Central Universities
(Grant GK201103002), Specialized Research Fund for the
Doctoral Program of Higher Education of China (Grant
20110202120006), and the Program for Changjiang Scholars
and Innovative Research Team in University (IRT 1070).
Figure 5. (a) The maximal emission wavelength of PPP-OR10 in the
presence of nitrobenzene derivatives as a function of pH value in 10%
serum. [PPP-OR10] = 8.0 × 10−6 M, [nitrophenol] = [nitroaniline] =
2.0 × 10−4 M. (b) The maximal emission wavelength as a function of
the α-chymotrypsin incubation time in 10% serum. [α-chymotrypsin]
= 0−62 μM, [PPP-OR10] = 8.0 x10−6 M, [BTNA] = 2.0 × 10−4 M.
The error bars represent the standard deviations of three parallel
measurements. The excitation wavelength is 340 nm.
in 5 mM PBS solution. For example, the maximal emission
wavelength of p-nitrophenol/PPP-OR10 in 10% serum red-
shifted from 419 to 466 nm, along with the pH increasing from
2 to 12. The maximal emission wavelength of 2,4,6-
trinitrophenol stayed around 457 nm and that of o-nitrophenol
stayed at 420 nm in the detection pH range. Furthermore, the
BTNA hydrolysis by α-chymotrypsin was successfully realized
in 10% serum. As shown in Figure 5b, the maximal emission
wavelength of PPP-OR10 red shifts with increasing the
incubation time and concentration of α-chymotrypsin. The
enzyme reaction rate is higher to some extent in 10% serum
than in 5 mM PBS alone, which may come from the effect of
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