A dual-mode fluorescence turn-on biosensor based on an aggregation-induced emission luminogen

Article abstract of DOI:10.1039/c3tb21576h

A novel dual-mode fluorescence turn-on probe is developed based on a phosphorylated tetraphenylethene (TPE) derivative bearing aggregation-induced emission (AIE) characteristics. The probe is weakly emissive in aqueous solution but its fluorescence is significantly enhanced in the presence of protamine or alkaline phosphatase (ALP). The cationic protamine interacted with the anionic phosphate group of the amphiphilic probe via electrostatic interaction and induced micelle formation. This micelle aggregates the hydrophobic TPE core and results in fluorescence enhancement. The detection limit for the protamine assay reached as low as 12 ng mL^-1. On the other hand, ALP hydrolysed the fluorescent probe and led to self-aggregation of insoluble fluorescent residues. The linear light-up response of the probe enables ALP quantification in the range of 10-200 mU mL^-1, which covers the physiological level of ALP activity in human serum. Moreover, the two activation modes could be differentiated by distinct responses to protamine and ALP. The Royal Society of Chemistry 2014.

Full text of DOI:10.1039/c3tb21576h

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Materials Chemistry B
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PAPER
A dual-mode fluorescence “turn-on” biosensor
based on an aggregation-induced emission
luminogen†
Cite this: J. Mater. Chem. B, 2014, 2,
1717
a
a
a
a
b
*
Zhegang Song, Yuning Hong, Ryan T. K. Kwok, Jacky W. Y. Lam, Bin Liu
acd
*
and Ben Zhong Tang
A
novel dual-mode fluorescence “turn-on” probe is developed based on a phosphorylated
tetraphenylethene (TPE) derivative bearing aggregation-induced emission (AIE) characteristics. The probe
is weakly emissive in aqueous solution but its fluorescence is significantly enhanced in the presence of
protamine or alkaline phosphatase (ALP). The cationic protamine interacted with the anionic phosphate
group of the amphiphilic probe via electrostatic interaction and induced micelle formation. This micelle
aggregates the hydrophobic TPE core and results in fluorescence enhancement. The detection limit for
the protamine assay reached as low as 12 ng mLꢀ1. On the other hand, ALP hydrolysed the fluorescent
probe and led to self-aggregation of insoluble fluorescent residues. The linear light-up response of the
probe enables ALP quantification in the range of 10–200 mU mLꢀ1, which covers the physiological level
of ALP activity in human serum. Moreover, the two activation modes could be differentiated by distinct
responses to protamine and ALP.
Received 8th November 2013
Accepted 13th January 2014
DOI: 10.1039/c3tb21576h
www.rsc.org/MaterialsB
assayed enzymes, which catalyses the hydrolysis of mono-
phosphate and has broad substrate adaptability.6 ALP activity in
Introduction
blood serum is oen regarded as a diagnostic indicator of
several diseases such as osteoporosis, achondroplasia,
cretinism, severe anemia, myelogenous leukemia and hepatic
diseases.7 So far, a few methods have been established for
protamine quantication and ALP activity assay. Umezawa and
coworkers developed voltammetric detection of protamine by
using self-assembled monolayers of thioctic acid.5 Kim's and
Yu's groups realized ALP activity assay based on iminocou-
marin–benzothiazole-based probes and dsDNA-templated
copper nanoparticles, respectively.8 However, these methods
are relatively insensitive or involve complicated synthesis
procedures of the probes. Thus, development of a simple and
sensitive uorescent biosensor for targeting protamine and ALP
is essential and signicant.
Research on biosensors has been booming in recent years
because of their wide applications in the area of healthcare and
diagnosis.1 Among them, uorescent biosensors have attracted
much attention as they offer many outstanding advantages such
as low background noise, high sensitivity and facile sample
preparation.2 Monitoring of physiological protein concentra-
tions or enzyme activities using uorescent bioprobes is of
clinical importance. For example, protamine, a highly positively
charged polypeptide with an isoelectric point of 12–13, is widely
used to reverse the anticoagulant activity of heparin aer
cardiovascular surgical procedures.3 Despite its universal use in
clinical practice, protamine can induce adverse effects such as
hypotension to idiosyncratic fatal cardiac arrest.4,5 On the other
hand, alkaline phosphatase (ALP) is one of the most commonly
Fluorophores with aggregation-induced emission (AIE)
characteristics have recently emerged as promising materials
for biosensing and imaging applications.9 Unlike conventional
uorophores suffering from uorescence quenching in high
concentration owing to strong p–p interaction, AIE luminogens
such as tetraphenylethene (TPE) and its derivatives enjoy high
emission efficiency when their molecules are aggregated or
bind to specic analytes.10 To date, a great number of AIE-based
“turn-on” biosensors have been reported to target proteins,
nucleic acids and glucose with superior sensitivity.11 To the best
of our knowledge, most of these AIE bioprobes are monofunc-
tional.8 It is more desirable to develop dual-mode or multi-
functional bioprobes with advantages of material saving, less
aDepartment of Chemistry, Institute for Advanced Study, Division of Biomedical
Engineering, Division of Life Science, State Key Laboratory of Molecular
Neuroscience and Institute of Molecular Functional Materials, The Hong Kong
University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
bDepartment of Chemical and Biomolecular Engineering, National University of
Singapore, 4 Engineering Drive 4, 117585, Singapore. E-mail: cheliub@nus.edu.sg
cGuangdong Innovative Research Team, SCUT-HKUST Joint Research Laboratory,
State Key Laboratory of Luminescent Materials and Devices, South China University
of Technology, Guangzhou 510640, China
dHKUST Shenzhen Research Institute, No. 9 Yuexing 1st RD, South Area, Hi-tech Park,
Nanshan, Shenzhen, China 518057. E-mail: tangbenz@ust.hk
† Electronic supplementary information (ESI) available: Deprotonation processes
and characterization data of TPE-TEG-PA. See DOI: 10.1039/c3tb21576h
This journal is © The Royal Society of Chemistry 2014
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synthetic work, cost-effectiveness and the potential for multi-
plex monitoring.12
Instrumentation
1H, 13C and 31P NMR spectra were recorded on a Bruker ARX 400
NMR spectrometer using CDCl3, methanol-d4, or acetone-d6 as a
solvent and tetramethylsilane (TMS; d ¼ 0 ppm) was chosen as
an internal reference. UV absorption spectra were recorded on a
Biochrom spectrophotometer. Photoluminescence (PL) spectra
were recorded on a Perkin–Elmer LS 55 spectrouorometer.
High-resolution mass spectra (HRMS) were obtained on a GCT
Premier CAB 048 mass spectrometer operated in MALDI-TOF
mode. Particle sizes and zeta potential of micelles were deter-
mined using a Zeta-Plus Potential Analyser. Morphologies of
micelles were examined using a JEOL 100CX transmission
electron microscope (TEM).
In this work, a dual-mode uorescence “turn-on” probe based
on a phosphorylated TPE derivative (TPE-TEG-PA) has been
developed for quantifying protamine concentration and ALP
activities by two different activation modes (Scheme 1). With the
aid of tetraethylene glycol (TEG) and phosphate (PA) groups, the
probeis water-solubleandemits weaklyin aqueous buffer. Owing
to their amphiphilic feature, the probe molecules form micelles
when interacting with protamine.13 The micelle aggregates the
hydrophobic TPE core and results in uorescence enhancement.
On the other hand, the ALPcanhydrolysethephosphate group on
the probe and release an insoluble TPE residue (TPE-TEG-OH)
into thebuffer solution resultingin strong emission. Through the
two different pathways, the detection of protamine and ALP
activity with high sensitivity and selectivity is achieved.
Synthesis
4-(1,2,2-Triphenylethenyl)benzoic acid (TPE-COOH). 1-(4-
Bromophenyl)-1,2,2-triphenylethene (TPE-Br) was synthesized
according to a previous report.14 To a solution of TPE-Br
(6 mmol, 2.466 g) in distilled THF (80 mL), 4.1 mL of n-butyl-
Experimental section
Materials
All chemicals and reagents were commercially available and lithium solution (1.6 M in cyclohexane) was added dropwise
used as received without further purication. Tetrahydrofuran under N2 at ꢀ78 ꢁC. The reaction mixture was stirred and kept at
ꢁ
(THF) and dichloromethane (DCM) were distilled from sodium ꢀ78 C for two hours. Excess dry ice was added in one portion
benzophenone ketyl and calcium hydride respectively under followed by stirring over six hours at room temperature. The
nitrogen immediately prior to use. 4-Dimethylaminopyridine resulting solution was acidied with an excess amount of 1 M
(DMAP), 4-bromobenzophenone, n-butyllithium, protamine hydrochloric acid and extracted with DCM several times. The
sulphate and trypsin were purchased from Aldrich. p-Toluene organic layer was combined and dried using MgSO4. Aer
sulfonic acid (PTSA) monohydrate was obtained from Meryer. simple ltration, the organic solvent was removed under
Dicyclohexylcarbodiimide (DCC), tetraethylene glycol and tri- reduced pressure. The crude product was puried by silica gel
ethylamine were purchased from Acros Organics. Phosphorus chromatography with DCM–ethyl acetate (10 : 1) as eluent to
oxychloride was purchased from International Laboratory. afford the desired product (2.0 g, 5.3 mmol) as a white solid.
1
Alkaline phosphatase (ALP) from calf intestine was purchased Yield ¼ 88%. H NMR (400 MHz, CDCl3): d (TMS, ppm) 7.85–
from Invitrogen. Buffer solutions (pH ¼ 1–13) were purchased 7.83 (d, 2H), 7.15–7.10 (m, 11H), 7.05–7.00 (m, 6H). 13C NMR
from Aldrich. All the other reagents used in selectivity tests were (100 MHz, CDCl3): d (TMS, ppm) 171.39, 149.15, 142.53, 142.41,
purchased from Aldrich.
142.37, 142.08, 139.22, 130.80, 130.65, 130.63, 129.00, 127.26,
Scheme 1 Schematic illustration of sensing mechanisms of TPE-TEG-PA in protamine detection and ALP activity assay.
1718 | J. Mater. Chem. B, 2014, 2, 1717–1723
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127.09, 126.39, 126.34, 126.15. HRMS (MALDI-TOF), m/z calcd immediately with an excitation wavelength of 330 nm. In the
for C27H20O2: 376.1463. Found 376.1466 (M+).
selectivity study, dextran, calf DNA, trypsin, cytochrome C,
bovine serum albumin (BSA) and human serum albumin (HSA)
2-(2-(2-(2-Hydroxyethoxy)ethoxy)ethoxy)ethyl-4-(1,2,2-triphe-
nylvinyl)benzoate (TPE-TEG-OH). To a DCM solution, TPE- with a concentration of 0.002 mg mLꢀ1 in distilled water were
COOH (1.5 mmol, 565 mg), DCC (2.25 mmol, 464 mg), DMAP examined under the same conditions.
(0.3 mmol, 36 mg), PTSA monohydrate (0.3 mmol, 57 mg) and
tetraethylene glycol (6 mmol, 1.164 g) were added. The reaction
mixture was stirred at room temperature for 1 day. Aer solvent
ALP activity assay and dynamic monitoring of enzymatic
hydrolysis
evaporation, the crude product was puried by silica gel chro-
A 10 mM TPE-TEG-PA probe solution was prepared by mixing
matography with DCM–methanol (10 : 1) as eluent to afford the
20 mL of the stock solution (1 mM) with 1.98 mL of Tris–HCl
desired product (340 mg, 0.62 mmol) as a gel-like solid. Yield ¼
1
buffer (10 mM, pH 9.6). Different amounts of ALP stock solu-
41%. H NMR (400 MHz, acetone-d6): d (TMS, ppm) 7.79–7.77
tions (35 000 mU mLꢀ1) were added into the Tris–HCl buffer
(d, 2H), 7.16–7.11 (m, 11H), 7.05–7.02 (m, 6H), 4.39–4.37 (q, 2H),
solution to yield nal ALP from 0 to 200 mU mLꢀ1. PL
3.79–3.77 (q, 2H), 3.62–3.54 (m, 11H), 3.49–3.46 (t, 2H). 13C
measurements were carried out aer the prepared solutions
ꢁ
were incubated at 25 C for 30 min. The enzymatic hydrolysis
process was monitored by the uorescence spectral measure-
ments which scanned at intervals of 90 s.
NMR (100 MHz, acetone-d6): d (TMS, ppm) 164.98, 148.73,
142.62, 142.43, 139.48, 130.55, 130.38, 130.32, 128.19, 127.21,
126.20, 126.09, 72.01, 69.74, 69.69, 69.58, 68.14, 63.33, 60.46.
HRMS (MALDI-TOF), m/z calcd for C35H36O6: 552.2512. Found:
552.2524 (M+).
2-(2-(2-(2-Phosphonooxy)ethoxy)ethoxy)ethyl-4-(1,2,2-triphe-
nylvinyl)benzoate (TPE-TEG-PA). Under a nitrogen atmosphere,
a solution of TPE-TEG-OH (120 mg, 0.22 mmol) and triethyl-
Results and discussion
Design and synthesis of dual-mode biosensor
To develop a dual-mode uorescence “turn-on” biosensor, two
modes of stimuli can activate the uorescent response through
different mechanisms respectively: one mode was for prot-
amine detection and the other was for ALP activity assay. Prot-
amine detection can be realized via electrostatic interaction
between the anionic TPE derivative and cationic protamine. The
design rationale is illustrated in Scheme 1. TPE-TEG-PA
comprises a hydrophobic TPE core and hydrophilic TEG and PA
groups. The synthesis route to TPE-TEG-PA is shown in Fig. 1.
The amphiphilic feature makes the probe partially soluble in
pure water or buffer solution. The probe shows weak uores-
cence in the aqueous solution when the concentration is low.
While increasing the concentration of TPE-TEG-PA to above its
critical micelle concentration (CMC), the uorescence is
enhanced signicantly due to the hydrophobic TPE core
aggregated in the micelles. Similarly, when the concentration of
TPE-TEG-PA is xed below its CMC, a uorescence enhance-
ment could also be achieved through addition of protamine
which induces the micelle formation through electrostatic
interaction. Because of the high density of positive charges in
amine (0.1 mL, 0.66 mmol, 3.0 equiv.) in dry THF (9 mL) was
added dropwise to a solution of distilled phosphoryl chloride
(0.21 mL, 2.2 mmol, 10 equiv.) in freshly distilled THF (1 mL) on
an ice bath with stirring over 4 h. The precipitates were removed
and the ltrate was evaporated under reduced pressure. The
crude product was puried by silica gel chromatography with
DCM–methanol (5 : 1) as eluent to afford the desired product
(50 mg, 0.08 mmol) as a white gel-like solid.15 Yield ¼ 36%. 1H
NMR (400 MHz, methanol-d4): d (TMS, ppm) 7.77–7.74 (d, 2H),
7.12–7.09 (m, 11H), 7.01–6.98 (m, 6H), 4.43–4.41 (m, 2H), 3.99–
3.96 (m, 2H), 3.82–3.79 (m, 2H), 3.68–3.61 (m, 10H). 13C NMR
(100 MHz, methanol-d4): d (TMS, ppm) 167.29, 149.93, 144.00,
143.84, 143.51, 140.84, 131.93, 131.77, 131.73, 129.52, 128.45,
128.26, 127.50, 127.33, 71.42, 70.97, 70.85, 70.84, 70.82, 69.72,
64.79, 64.74, 64.67. 31P NMR (162 MHz, methanol-d4): d (TMS,
ppm) 1.12. HRMS (MALDI-TOF), m/z calcd for C35H37O9P:
632.2175. Found: 633.2260 (M + H)+.
Determination of critical micelle concentration
A stock solution of TPE-TEG-PA (1 mM) was prepared by dis-
solving the gel-like solid (12.6 mg) in distilled water (20 mL)
under oscillation. For a series of TPE-TEG-PA solutions with
concentration gradients (0.1 to 400 mM), varying amounts of
stock solution and distilled water were mixed with a nal
volume of 2 mL. PL spectra of all the samples were recorded at
room temperature with an excitation wavelength of 330 nm. All
samples were freshly prepared before each test.
Fluorescence detection of protamine
A 10 mM TPE-TEG-PA probe solution was prepared by mixing 20
mL of the stock solution (1 mM) with 1.98 mL of distilled water.
Protamine solution (0.1 mg mLꢀ1 in distilled water) was added
intermittently to 2 mL of the probing solution and mixed well.
PL spectra were recorded aer each addition of protamine Fig. 1 Synthesis route to TPE-TEG-PA.
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protamine, the formation of micelles between TPE-TEG-PA and
protamine is expected. Thus, protamine detection can be real-
ized by measuring the magnitude of uorescence enhance-
ment. Subsequently, the uorescence of the protamine/probe
micelles will be diminished if the micelles are degraded. On the
other hand, the end group PA on the probe could be hydrolysed
by ALP and converted to a hydroxyl group. Because TPE-TEG-OH
has limited water solubility, the molecules would aggregate in
aqueous solution to trigger the AIE response. Such uorescence
enhancement enables assay of the ALP activity with high
sensitivity and selectivity.
Fluorescence property and CMC determination
The PL property of TPE-TEG-PA was investigated using THF and
hexane solvent mixtures (Fig. 2). TPE-TEG-PA is almost non-
emissive in pure THF solution. With increasing the hexane
content ranging from 0 to 50%, the uorescence intensity
remains at a low level. Further addition of hexane into the
mixture results in a sharp and dramatic enhancement of the
uorescence intensity. At the 90 vol% hexane content, the PL
intensity is about 150-fold higher than that in the pure THF
solution, suggesting that TPE-TEG-PA is AIE-active.
Fig. 3 (A) PL spectra of TPE-TEG-PA in aqueous solution at different
concentrations. (B) Plot of peak intensity in different concentrations of
TPE-TEG-PA (with logarithmic axis). (C) Particle size distribution of
Since the CMC value is an essential parameter for the prot-
amine detection, the CMC value of TPE-TEG-PA was determined TPE-TEG-PA (100 mM) in pure water. (D) TEM image of TPE-TEG-PA
(100 mM) in pure water. lex ¼ 330 nm.
by a uorometric assay. The PL spectra of the dye in aqueous
solutions at different concentrations are shown in Fig. 3A.
When the concentration of the dye is lower than 10 mM, only a
very weak uorescent signal is detectable. At the concentration
of 20 mM, the PL intensity enhances abruptly, suggesting that
the dye starts to form micelles. By further increasing the dye
concentration, the PL intensity increases. The plot of peak
intensity against TPE-TEG-PA concentration is shown in Fig. 3B.
An inexion at around 20 mM could be derived through the
intersection point of the two tangent lines, indicating that the
CMC value of the uorescent probe is about 20 mM. To further
verify the micelle formation, particle sizes and TEM measure-
ments were carried out. When the dye concentration is below
the CMC value, no detectable signal is recorded in both
measurements. However, a diameter of a micelle entity around
200 nm is found above the CMC value of the dye (Fig. 3C and D).
Compared with traditional amphiphilic molecules, the AIE
molecules show a self-reported CMC value through uorescence
signal change. TPE-TEG-PA could report whether the concen-
tration reached its CMC value by the aggregation behaviour of
the TPE unit. This unique feature would obviously benet the
subsequent protamine detection, making the probe solution
simple and easy to prepare.
As the phosphate group of TPE-TEG-PA can be protonated
under acidic conditions, such protonation could change the
solubility and thus the aggregation behaviour of the dye. We
thus examined the pH-dependent uorescence of TPE-TEG-PA.
As shown in Fig. S6,† the probe is highly emissive under strong
acidic conditions (pH < 3). When the pH is increased to 3, the
uorescence of the probe decreased signicantly. Increasing
the pH from 3 to 7 further decreases the uorescent intensity.
Under basic conditions with pH > 7, the emission remains
weak. Under acidic conditions, the phosphate group is
protonated, which is less soluble in water and the AIE response
is activated, while under basic conditions, the deprotonated
ionic species is highly water-soluble and thus less emissive.
Change of uorescence intensity versus pH revealed that there
are two deprotonation processes of TPE-TEG-PA involved with
the increase of pH (Fig. S6B and Scheme S1†). Since the probe
emits faintly with the pH range from 3–13, the probe could be
used for turn-on sensing of biomolecules in this broad pH
range.
Protamine detection
Fig. 2 (A) PL spectra of TPE-TEG-PA (10 mM) in THF–hexane mixtures
with different fractions of hexane (fH). (B) Plot of I/I0 at 480 nm versus
fH, where I0 is the PL intensity in pure THF solution. lex ¼ 330 nm.
To construct a “turn-on” biosensor with excellent signal to
noise ratio, the concentration of the probe was chosen to be
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10 mM for the protamine detection. At this concentration, the (HSA) and bovine serum albumin (BSA) was performed under
background emission of the probe is low. Meanwhile, the pH the same experimental conditions. As shown in Fig. 5, the
of the probing solution will also affect the performance of addition of protamine into TPE-TEG-PA causes the highest
protamine quantication due to the protonation/deprotona- uorescence enhancement (ꢂ13-fold). HSA and BSA present
tion processes of TPE-TEG-PA at different pHs. Under acidic tiny interference, which may attribute to hydrophobic inter-
conditions, the PA group of TPE-TEG-PA is protonated, which actions between the large proteins and the probe.16 Other
decreases the solubility and increases the background signal. biomolecules such as dextran, DNA, trypsin and cytochrome
Under neutral or basic conditions, the PA group is deproto- C do not induce obvious increases in the uorescence
nated, which increases the solubility of the dye and thus intensity. Based on the results, TPE-TEG-PA can work as a
lowers the background noise for the assay (Fig. S9–S11†). The “turn-on” uorescent probe for protamine quantitation with
signal increment for TPE-TEG-PA to protamine is more high sensitivity and selectivity.
signicant in pure water than under basic conditions, prob-
Considering that protamine is rich in lysine and arginine
ably because there is no interference from other ionic species residues, which are the enzymatic substrates for trypsin, we
in pure water to effect the interaction between TPE-TEG-PA hypothesize that the decomposition of protamine by trypsin will
and protamine. Therefore, pure water is selected as a probing disassemble the micelles. We investigated the effect of trypsin
medium.
on the uorescence intensity of the probe in the presence of
As shown in Fig. 4A, the probe emits weakly with a maximum protamine. The result reveals that the addition of trypsin to the
emission wavelength of 450 nm in the absence of protamine. probe/protamine complex solution almost quenches the uo-
The uorescence intensity of the probe increases gradually rescence (Fig. 6). It further supports the protamine-induced
upon the addition of protamine solution. When the concen- micelle formation process.
tration of protamine in the probe/protamine complex reaches
2.9 mg mLꢀ1, the uorescence intensity has increased by 12-fold
compared to the blank. This uorescence enhancement could
be attributed to the protamine-induced micelle formation and
aggregation of the TPE core. To further verify the protamine-
induced micelle formation, we performed particle size analysis.
As shown in Fig. S12,† uniform micelles with a diameter of
about 100 nm are detected when 0.002 mg mLꢀ1 of protamine is
added to the probe. Meanwhile, it is noted that the emission
peak of the complex is red-shied to 480 nm aer protamine
addition, which is the typical emission wavelength for TPE
aggregates.
It was also remarkable that the plot of I/I0 versus the
protamine concentration gives a linear curve with R2 ¼ 0.9999
in the range of 0–1000 ng mLꢀ1 (Fig. 4B). More importantly,
the limit of detection (LOD) is as low as 12 ng mLꢀ1, which is
Fig. 5 Change of PL intensities of TPE-TEG-PA (10 mM) at 480 nm in
much better compared with previous reports.11d To address
the presence of different biomacromolecules (0.002 mg mLꢀ1). lex
¼
the selectivity of TPE-TEG-PA toward protamine, a control
experiment with other biomacromolecules including dextran,
calf DNA, trypsin, cytochrome C, human serum albumin
330 nm.
Fig. 6 PL spectra of the probe, probe/protamine and probe/prot-
Fig. 4 (A) PL spectra of TPE-TEG-PA (10 mM) in the presence of amine/trypsin. Concentration: probe (10 mM); protamine (2 mg mLꢀ1);
different concentrations of protamine. (B) Plot of I/I0 versus protamine trypsin (0.01 mg mLꢀ1); lex ¼ 330 nm. Inset: photographs of corre-
concentrations, where I and I0 are the PL intensities in the presence or sponding solutions (a) probe only, (b) probe/protamine and (c) probe/
absence of analytes. lex ¼ 330 nm.
protamine/trypsin under UV light illumination at 365 nm.
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ALP activity assay
The obvious difference in water-solubility and emission
behaviours of TPE-TEG-PA and its precursor TPE-TEG-OH
(Fig. S15†) inspired us to utilize the probe for ALP activity assay.
ALP could catalyse the hydrolysis reaction of monophosphate to
a hydroxyl group in high efficiency. As the enzymatic product
TPE-TEG-OH is insoluble in aqueous buffer, it would form
aggregates to turn on the uorescence. Thus, TPE-TEG-PA can
work as an ALP activity reporter. To check the feasibility of this
design, we investigated the uorescence change when the probe
TPE-TEG-PA was treated with ALP in Tris–HCl buffer solution
(pH 9.6).
In the absence of ALP, the probe emits weakly in the buffer
solution. Upon addition of 200 mU mLꢀ1 ALP, the uorescence
increases gradually and the intensity reaches a plateau aer 40
minutes (Fig. 7), with about 8-fold increase of the PL intensity.
The photograph was taken from the solution of TPE-TEG-PA
itself and the probe aer incubation with ALP in buffer solution
under UV illumination at 365 nm. It is obvious that green
uorescence is turned on aer being treated with ALP, which is
12 nm red-shied when compared to the emission colour
induced by protamine (inset of Fig. 6). Although the difference
in wavelength was not so signicant, it could be distinguished
by the naked eye, which was essential for a dual-mode probe.
To examine the possibility of quantitative analysis of ALP
activity, we conducted the experiment of dynamic monitoring of
enzymatic reaction. Fig. 8A shows the uorescence intensity
changes of the probe as a function of the ALP concentrations at
different times. With different concentrations of ALP added, the
PL intensity of the probe increases gradually with different
magnitudes. The uorescence changes more obviously at the
Fig. 8 (A) Time-dependent PL intensity of 10 mM TPE-TEG-PA in 10
mM Tris–HCl buffer solution (pH 9.6) at 492 nm versus the
hydrolysis reaction time in the presence of different concentrations of
ALP at 25 ꢁC. (B) Plot of relative fluorescence intensity (I/I0) at 492 nm
after incubation with different concentrations of ALP for 30 min.
lex ¼ 330 nm.
beginning for all ALP concentrations but becomes slower in the
later period. The uorescence intensity reaches the plateau aer
about 60 min, indicating that TPE-TEG-PA is nearly completely
hydrolysed by ALP. These results reveal that the ALP turn-on
mode could be applied potentially for the real-time assay of ALP.
As shown in Fig. 8B, the relative uorescence intensity change
(I/I0) presents a linear increase with the activity of ALP in the
range from 0 to 100 mU mLꢀ1, which covers the normal range of
ALP in serum samples. To address the selectivity of TPE-TEG-PA
toward ALP, a control experiment with other nonspecic
enzymes including esterase, deoxyribonuclease (DNase),
acetylcholinesterase (AChE) and trypsin was performed under
the same conditions. As shown in Fig. 9, about 8-fold PL
enhancement of the probe was recorded with the effect of ALP,
while negligible changes in emission intensity were observed
for other enzymes, showing that the probe is highly specic to
ALP. To further demonstrate that uorescence enhancement
was due to ALP-catalyzed hydrolysis rather than other effects, a
control experiment between ALP and denatured ALP was con-
ducted. It is well known that guanidine hydrochloride is one of
the traditional protein denaturants. According to our proposed
mechanism, denatured ALP could not hydrolyse the probe and
Fig. 7 Time-dependent emission spectra of 10 mM TPE-TEG-PA in 10
mM Tris–HCl buffer solution (pH 9.6) upon addition of 200 mU mLꢀ1 Fig. 9 Variation of the PL intensity of TPE-TEG-PA (10 mM) in Tris–HCl
ALP at room temperature; inset: photographs taken under UV light (10 mM, pH ¼ 7.4) buffer solution at 492 nm after incubation with
(365 nm) illumination of the corresponding solutions in the (a) absence esterase (200 mU mLꢀ1), DNase (200 mU mLꢀ1), AChE (200 mU mLꢀ1),
and (b) presence of ALP incubated for 30 min. lex ¼ 330 nm.
trypsin (0.002 mg mLꢀ1) and ALP (200 mU mLꢀ1). lex ¼ 330 nm.
1722 | J. Mater. Chem. B, 2014, 2, 1717–1723
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no uorescence enhancement is expected. The PL result shows
that denatured ALP is not able to turn on the uorescence
(Fig. S14†), which reveals that it is the catalysis effect of ALP that
functions on this process, excluding other interference effects
such as hydrophobic interaction and electrostatic interaction
between proteins and probes.
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Conclusions
A dual-mode uorescence “turn-on” probe TPE-TEG-PA for
protamine detection and ALP activity assay was established by
making use of the AIE technology. Both TEM and particle size
measurements conrmed the formation of micelles in high dye
concentration as well as in the presence of protamine. The
detection limit for the protamine assay was as low as 12 ng
mLꢀ1. On the other hand, ALP-catalysed hydrolysis of the
uorescent probe led to self-aggregation of insoluble products,
resulting in strong uorescence. The assay range of ALP was
located in 10–200 mU mLꢀ1. The uorescence “turn-on”
method provided a platform for the research on dual-mode
biosensors and it would potentially benet the development of
multiplex monitoring using a single probe.
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Acknowledgements
This work was partially supported by the National Basic
Research Program of China (973 Program; 2013CB834701), the
Research Grants Council of Hong Kong (604711, 604913,
HKUST2/CRF/10 and N_HKUST620/11), Innovation and Tech-
nology Commission (ITCPD/17-9), the University Grants
Committee of Hong Kong (AoE/P-03/08) and the Singapore
Ministry of Defence (R279-000-340-232). B. Z. T. is grateful for 11 (a) R. Hu, J. W. Y. Lam and B. Z. Tang, Macromol. Chem.
the support from the Guangdong Innovative Research Team
Program of China (201101C0105067115).
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