Fluorometric probing of the lipase level as acute pancreatitis biomarkers based on interfacially controlled aggregation-induced emission (AIE)

Article abstract of DOI:10.1039/c7sc02189e

As a sudden inflammation of the pancreas, acute pancreatitis presents severe complications and a high mortality rate, despite treatment. Lipase in serum serves as an essential biomarker of acute pancreatitis and even pancreatic cancer. Therefore, developing robust, convenient and sensitive probing of lipase levels is greatly needed. In this work, we present glutamate functionalized tetraphenylethylene (TPE) as a "turn-on" fluorescent probe (S1) based on the aggregation-induced emission (AIE) mechanism for lipase levels with new recognition units. In heterogeneous media, the hydrophilic amino and carboxyl groups in the probe were specifically introduced to facilitate its full access to lipase at the oil-water interface and achieve an interfacially controlled AIE process. The linear response of fluorescence ranging from 0 to 80 U L^-1, which included the concentration range of the lipase level in human serum, considering the dilution factor if necessary, the limit of detection as low as 0.13 U L^-1, and the fast response time (7 min) were determined. The value of the apparent Michaelis-Menten constant (K_m) was obtained as 4.23 μM, which indicated superior affinity between lipase and the probe molecule. The selectivity, photostability, dynamic monitoring of the enzymatic reaction, and preliminary commercial enzyme activity screening were summarized. As far as we know, this is the fastest, easiest and most sensitive method for lipase level probing in the reported literature. Finally, probing the lipase level for the first time in real human serum samples was also conducted successfully.

Full text of DOI:10.1039/c7sc02189e

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We demonstrate a sensitive, easy and fast method for fluorometric probing lipase levels as acute pancreatitis
biomarker based on a novel interfacial controlled aggregation-induced emission (AIE) mechanism.

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DOI: 10.1039/C7SC02189E
Journal Name
ARTICLE
Fluorometric probing lipase levels as acute pancreatitis biomarker
based on interfacial controlled aggregation-induced emission (AIE)
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
a
b
Jie Shi, Qianchun Deng, Chuyun Wan, Mingming Zheng, Fenghong Huang* and Bo Tang*
DOI: 10.1039/x0xx00000x
As a sudden inflammation of the pancreas, acute pancreatitis has severe complications and high mortality despite
treatment. Lipase in serum is served as an essential biomarker of acute pancreatitis even pancreatic cancer. Therefore,
developing robust, convenient and sensitive probing of lipase levels is greatly needed. In this work, we present glutamate
functionalized tetraphenylethylene (TPE) as “turn-on” fluorescent probe (S1) based on an aggregation-induced emission
www.rsc.org/
(AIE) mechanism for lipase levels with new recognition units. In heterogeneous media, hydrophilic amino and carboxyl in
probe were specifically introduced to facilitate its full access to the lipase at the oil-water interface to achieve an
interfacial controlled AIE process. A linear response of fluorescence ranging from 0 to 80 U/L, which is included in the
concentration range of lipase level in human serum considering the dilution factor if necessary, limit of detection as low as
0
m
.13 U/L and a fast response time (7 min) can be determined. The value of the apparent Michaelis-Menten constant (K )
was obtained as 4.23 μM, which indicated superior affinity between lipase and the probe molecule. On the other hand,
selectivity, photostability, dynamic monitoring of enzymatic reaction, preliminary commercially enzyme activity screening
were summarized. As far as we know, this is the fastest, easiest and most sensitive method for lipase levels probing in the
reported literature. Finally, the first application of probing lipase levels in real human serum samples was also conducted
successfully.
development of robust, convenient and sensitive detection of lipase
level is of great significance in terms of both clinical diagnoses and
Introduction
1
pathological analysis towards acute pancreatitis.
Acute pancreatitis is a sudden inflammation of the pancreas. In
severe cases, acute pancreatitis can result in bleeding into the gland,
So far, to our knowledge, very rare methods have been
established for lipase probing. Traditional methods such as
colorimetric or turbidimetric methods usually require sample
processing and complex instrumentation and indicate various
limitations. Therefore, some new methods emerged to overcome
these obstacles. For instance, Tian's group developed some gold
nanoparticles (GNPs) based biosensors for lipase assays,
colorimetric probes by using Tween 20 functionilized GNPs and one-
2
serious tissue damage, infection, and cyst formation. The mortality
for severe acute pancreatitis can be 10 to 30 %, sufferers often
endure pain and malnutrition, and are most likely left with higher
3
risk of pancreatic cancer (mortality rate > 99 %). Diagnosis of
pancreatic problems is often difficult and treatments are therefore
4
delayed because the organ is relatively inaccessible. However,
lipase in serum, which is an exocrine enzyme from pancreas, has
been employed as an essential biochemical indicator for early
1
8, 19
pot nanoparticle growth as reporters.
Moreover, some
5
-13
drawbacks still exist for the reported lipase assays, some specific
substrates and indicator have been used as the main screening tool,
however, all of which possessed relatively complicated production
process and suffer from lower sensitivity. For that, their application
to the detection of lipase level in real biological samples and early
warning of acute pancreatitis cannot be implemented. Up to now,
fluorometric assays are receiving remarkably attention owing to
their convenience, unparalleled sensitivity, simplicity, rapid
implementation, noninvasive monitoring capability and usability in
diagnosis and condition assessment of acute pancreatitis.
measuring serum lipase levels associate with pancreatic tumor
Also,
1
4,15
marker ca 19-9 can diagnose pancreatic cancer.
Under
conditions of pancreatic injury, pancreatic autolysis induces
significant elevation of the serum lipase level, high level of lipase
1
6, 17
strongly suggest acute pancreatitis.
Therefore, the
a.
Hubei Key Laboratory of Lipid Chemistry and Nutrition, Oil Crops and Lipids
Process Technology National & Local Joint Engineering Laboratory, Key
Laboratory of Oilseeds Processing, Ministry of Agriculture, Oil Crops Research
Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China. E-
mail: shij@whu.edu.cn
2
0-37
biological samples.
Nevertheless, fluorescent probe about lipase
38
level detection is a virgin land area until our work published. In
our previous work, an aggregation-induced emission (AIE)-based
fluorescence probe bearing characteristics to rapidly visualize lipase
activities has been constructed with compound P1, unfortunately,
however, the sensitivity and detection limit were significantly worse
b.
College of Chemistry, Chemical Engineering and Materials Science, Collaborative
Innovation Center of Functionalized Probes for Chemical Imaging in Universities of
Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education,
Shandong Normal University, Jinan 250014, China. E-mail: tangb@sdnu.edu.cn
Electronic Supplementary Information (ESI) available: [Synthesis, characterisation,
test conditions and other supplementary data.]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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Journal Name
detection limit and low sensitivity of lipase probeViaewndArtbiculeiOltnlainne
DOI: 10.1039/C7SC02189E
analytical method for early warning of acute pancreatitis. We
believe that the research of screening lipase level would not only
broaden the spectrum of AIE luminogens-based enzyme assays but
also provide a new approach on improving catalytic efficiency of the
interface.
Experimental
Materials
The All chemicals and reagents were commercially available
and used as received without further purification. Superdry
tetrahydrofuran (THF) were purchased from J&K Scientific.
Titanium tetrachloride (TiCl4), 4-dimethylaminopyridine
Scheme 1 Schematic illustration of sensing mechanisms and
structure of probe S1 in lipase detection.
(DMAP), 4-hydroxybenzophenone, n-butyllithium were
purchased from Aldrich. Dicyclohexylcarbodiimide (DCC), p-
toluene sulfonic acid (PTSA) monohydrate, Boc-L-glutamic acid
and the range of detection is outside the concentration range of
lipase level in human serum. Therefore, in order to obtain a probe
of excellent performance towards lipase, the affinity between
probe and enzyme must be greatly improved, by considering of the
interfacial reaction characteristics of lipase, the improvement of the
interfacial catalytic efficiency is the key factor to the limit of
detection and sensitivity of probe molecule.
1
-tert-butyl ester (Boc-Glu-OtBu) were purchased from Acros
Organics. Lipases from Pseudomonas cepacia (PCL), Candida
rugosa (CRL), Candida Antarctica B (CALB), Pseudomonas
fluorescens (PFL), Pocine pancreas (PPL), Aspergillus niger (ANL)
were all ordered from Amano Enzymes. Glucose oxidase,
alkaline phosphatase, acylase, laccase and α-amylase were all
purchased from Sigma-Aldrich. Lipase Assay Kit was purchased
from Nanjing Jiancheng Bioengineering Institute. Human
serum samples were supplied by Union Hospital, Tongji
Medical College, Huazhong University of Science and
Technology; and the serum samples were from both healthy
people and acute pancreatitis patients. The water used in the
texts was triple distilled, treated with an ion exchange column,
and then treated with Milli-Q water Purification system.
Instrumentation
Herein, we report new fluorometric “turn-on” detection for lipase
level by manipulating the deaggregation and aggregation of probe
S1. The whole design rationale is illustrated in Scheme 1 and
explained as follows: (1) probe S1 is derived from
tetraphenylethylene (TPE) core with two glutamate units, TPE
derivatives were known as their fantastic AIE behavior; (2) as a
heterogeneous catalytic enzyme, it is well known that the best
catalytic performance of lipase can be achieved at the oil-water
interface, therefore, we could, by coupling the glutamate units onto
TPE core, on one hand, notably improved the solubility of the AIE-
1
13
active probe in hydrophobic medium as the existence of alkyl chains, H and C NMR spectroscopy study was conducted with a
on the other hand, significantly increased the hydrophilicity of S1 Bruker Avance 400 MHz NMR spectrometer using
with the absence of amino and carboxyl groups, facilitating its full tetramethylsilane (TMS; δ = 0 ppm) as internal standard.
access to the lipase in the aqueous phase at the oil-water interface, Fluorescence spectra were performed on a Hitachi F-7000
thus, an interfacial control effect can be conducted simply via the fluorescence spectrophotometer with a Xe lamp as the
hydrophilic group. (3) as shown in Scheme 1, lipase-catalyzed excitation source. ESI-MS spectra were recorded with a
cleavage of the ester group in probe S1 will induce the Thermo TSQ QUANTUM ULTRA EMR system. Dynamic laser
transformation of S1 into 4, 4’- dihydroxy tetraphenylethylene (see scattering (DLS) measurements were performed on
a
scheme S1, TPE-2OH), the glutamate units will be cut off, which commercial laser light scattering instrument (Malvern
may release an insoluble TPE residue (TPE-2OH) into the solvent. ZEN3690, Malvern Instruments) at 25 °C.
Accordingly, aggregation of 4, 4’- dihydroxy tetraphenylethylene in Synthesis
solvent will occur and resulting in strong blue emission, that is to
Synthesis of 4,4'-(1,2-diphenylethene-1,2-diyl)diphenol (TPE-
2
say, turn on the fluorescence; (4) since presence of interfacial
OH, E/Z isomers) Zinc dust (1.45 g, 22.0 mmol) and 4-
control effects, very lower lipase levels in some real biological
samples can be probed, and glutamate units are the specific
substrate for lipase hydrolysis reaction, S1 could serve as a
selectively probe for lipase levels in real biological samples like
serum. To this end, a simple fluorescence turn-on assay for lipase
probing can be established with S1, through this pathway, the
catalytic rate of reaction can be significantly improved by this AIE-
based interfacial control effect, therefore, the detection of lipase
with higher sensitivity and lower detection limit are achieved. As far
as we know, no reports to date have been published on the
application of a fluorescent probe in testing lipase level of real
human serum samples. This work solved the drawbacks of high
hydroxybenzophenone (1.0 g, 5.0 mmol) were placed in a two-
necked, round-bottomed flask fixed with a condenser. The
flask was evacuated under vacuum and purged with dry
nitrogen for three times. After addition of 60 mL superdry THF,
the mixture was cooled to -78 °C and TiCl (1.25 mL, 11.0 mmol)
4
was injected dropwise. After that, the mixture was slowly
warmed to room temperature, stirred for 0.5 h, and then
refluxed overnight. The reaction was quenched by 10%
aqueous K CO solution. The mixture was extracted with
2
3
diethyl ether three times and the combined organic layer was
washed with brine twice and dried over anhydrous sodium
sulfate. After solvent evaporation, the crude product was
2
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further purified by a silica gel column to obtain TPE-2OH (0.77 measurements which scanned at intervals of 1 mViienwfArroticmle O0nlitnoe
DOI: 10.1039/C7SC02189E
1
g, 84.9%, E/Z isomers) as a light yellow solid. H NMR (400 MHz, 10 min after incubation with 0, 5, 10, 30, 50, 70 and 80 U/L of
CDCl ): δ (TMS, ppm) 6.93-7.04 (m, 10H), 6.75-6.80 (m, 4H), lipase. To investigate the photobleaching of reaction product
3
6
.46-6.51 (m, 4H).
aggregates, the enzymatic product mixture solution was
Synthesis of compound TPE-BGO (E/Z isomers). To a 30 mL exposed to an ultraviolet lamp (254 and 365 nm
DCM solution, 4,4'-(1,2-diphenylethene-1,2-diyl)diphenol (2.0 simultaneously) for up to 150 min.
mmol, 0.728 g), DCC (3.0 mmol, 0.619 g), DMAP (0.4 mmol, Lipase levels detection in real samples
0
.048 g), PTSA monohydrate (0.4 mmol, 0.076 g) and Boc-L- For lipase detections in serum, human blood samples were
glutamic acid 1-tert-butyl ester (Boc-Glu-OtBu) (8.0 mmol, 2.43 obtained from Union Hospital, Tongji Medical College,
g) were added. The reaction mixture was stirred and refluxed Huazhong University of Science and Technology. The writing of
for 24 h. After solvent evaporation, the crude product was informed consent was obtained from human subjects in
further purified by a silica gel column to afford the compound compliance with relevant laws and institutional guidelines.
2
1
(0.82 g, 43.7%, E/Z isomers) as a light yellow solid. H NMR Human intravenous blood samples were obtained through
(
(
(
400 MHz, CDCl ): δ (TMS, ppm) 7.21-7.25 (m, 12H), 7.01-6.98 venipuncture and allowed to clot for 1 h at room temperature.
3
m, 6H), 4.49 (d, 1H), 4.47 (d, 1H), 2.34-2.31 (m, 4H), 2.18-2.15 After removing the clot part by centrifuging at 3000 rpm for 5
1
3
m, 2H), 1.95-1.91 (m, 2H), 1.51 (s, 18H), 1.49 (s, 18H).
C
min, the obtained serum samples were ten-fold diluted with
NMR(100 MHz, CDCl ): δ (TMS, ppm) 172.25, 171.78, 156.17, 0.1 M potassium phosphate buffer (pH 7.4). Standard recovery
3
1
1
53.36, 140.89, 139.91, 137.44, 128.92, 128.75, 128.40, 128.11, experiments were performed by adding a known quantity of
23.46, 81.92, 79.74, 53.17, 28.71, 28.36, 27.68, 26.12. MS lipase to diluted human serum samples of healthy people. The
+
(ESI): m/z 935.82 [M+H] .
percent recovery referred to the difference in lipase levels
Synthesis of compound S1 (E/Z isomers). Compound TPE-BGO determined before and after the addition of lipase in
0.47 g, 0.5 mmol) was dissolved under stirring in 10 mL DCM, comparison with the added amount. The fluorescence assay
(
then hydrogen chloride generated from the reaction of NaCl was performed as above to determine the amount of lipase.
and H SO was passed into the solution for 1 h. The organic For comparison, lipase levels were determined by using a
layer was washed with water. Afterwards the solvent was commercially lipase assay kit.
2
4
evaporated under vacuum to get the product (0.29 g, 92.1%,
1
E/Z isomers). H NMR (400 MHz, CDCl ): δ (TMS, ppm) 7.21-
3
Results and discussion
7
.26 (m, 12H), 7.00-6.96 (m, 6H), 4.49-4.46 (m, 2H), 2.38-2.33
1
m, 4H), 2.18-2.16 (m, 2H), 1.94-1.92 (m, 2H). C NMR(100 Design, synthesis and sensing mechanism of probe S1
3
(
MHz, CDCl ): δ (TMS, ppm) 173.91, 172.18, 153.41, 141.54, The sensing mechanism is illustrated in Scheme 1. As is known
3
1
2
39.97, 136.51, 128.74, 128.26, 127.91, 127.52, 126.63, 52.98, to all, lipases can catalyze the hydrolysis of ester bond, hence
+
9.07, 27.74. MS (ESI): m/z 623.71 [M+H] .
this mode of stimuli can activate the AIE characteristic, giving
fluorescent response through a simple reaction. Soluble
×10 M) was prepared by dissolving glutamate groups were attached onto TPE core, so as to make
Preparation of the solution of probe S1 and lipase
-3
A stock solution of S1 (1
the S1 solid (6.22 mg) in hexane (10 mL). The solution of S1 (10 the probe molecules are well dissolved in some media. The
μM) in hexane were prepared by placing 0.1 mL of S1 stock hydrolytic cleavage of the ester bond in S1 catalyzed by lipase
solution, then diluting the solution to 10 mL with hexane. For a and compound TPE-2OH was produced, then, the rapid
series of commercial purchased lipase samples solutions with aggregation of TPE-2OH will be triggered due to its poor
concentration gradients (5 to 90 U/L) and other commercial solubility which caused by the loose of glutamate units, thus
lipases for preliminary screening (80 U/L) were dissolved in 0.1 strong blue fluorescence can be “turn on” because of AIE
M potassium phosphate buffer (pH 7.4) which were prepared effect. The probe S1 was synthesized using the synthetic
by K HPO and KH PO .
protocol described in Scheme S1 and starting from 4-
Lipase detection and dynamic monitoring of enzymatic hydroxybenzophenone via a Mcmurry coupling reaction
hydrolysis leading to compound TPE-2OH, which was esterificated with
2
4
2
4
Different concentrations of lipase stock solutions (5~90 U/L) 3 two equivalents Boc-Glu-OtBu to yield intermediates, after
mL were mixed with 10 μM S1 solution 3 mL in a 10 mL flask hydrogen chloride was bubbled into the DCM solution of
and stirred at room temperature for 20 min. Then the organic compound TPE-BGO for 1 h, the desired product S1 was
1
layer was taken to proceeded fluorescence measurements obtained. The structure and purity of S1 were confirmed by H
1
3
with an excitation wavelength of 360 nm. To examine the NMR, C NMR and ESI-MS.
kinetics of the enzyme reaction, the enzymatic hydrolysis Fluorescent response toward lipase
process was monitored by the fluorescence spectral
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seen upon excitation even in a dark room. Next,ViweweAraticdledOendlinae
DOI: 10.1039/C7SC02189E
9
00
A
series of fixed concentration lipase to solution of probe S1,
Lipase (U/L)
8
00
00
00
00
00
00
00
00
after the incubation, excitingly, the blue fluorescence emission
displayed apparent enhancement. As shown in Fig. 1A, when
the concentration of lipase was increased in the test system,
the intensity centered at about 453 nm increased
correspondingly. In fact, even at the concentration of lipase as
low as 20 U/L, the emission intensity at about 453 nm
increased to about 15 times the original one. When the
concentration of lipase reaches 80 U/L, the fluorescence
intensity has increased to the maximum by 42-fold compared
to the blank. At last, lipase concentration reaches to 90 U/L,
the fluorescence depicts no significant enhancement and the
intensity reaches a plateau. This phenomenon verified our
design idea mentioned above: by accompanying with the
leakage of the alkoxy chain and hydrophilic groups of the
glutamate group linked to TPE core after incubated by some
lipase, its aggregation behavior changed to show detectable
photophysical signal in response to the analytes. On the other
hand, MS analysis (Fig. S4) also suggests that the product of
the enzymatic reaction has the same molecular structure as
TPE-2OH. Ideally, the change of a fluorometric probe should
be from one extreme case to another, just like in our case,
from the “off” state to “on”. According to the calibration curve
9
85
0
7
8
0
7
0
6
6
0
5
0
5
4
0
3
0
4
20
1
5
1
0
3
5
0
2
1
0
3
80
420
460
500
540
580
620
Wavelenth (nm)
1
000
B
9
00
00
00
00
00
00
00
00
00
0
I4 =10.6496 [Lipase]+18.7494
53
2
R =0.9985
8
7
6
5
4
3
2
1
(Fig. 1B), it was also remarkable that the relative fluorescence
intensity at about 453 nm gives a linear increase with the
2
concentration of lipase in the range from 0 to 80 U/L (R =
0
.9985). The photograph was taken from the solution of probe
S1 itself and the probe after incubation with lipase in hexane
solution under UV illumination at 365 nm. It is obvious that
blue fluorescence appeared after treatment with lipase, so
distinct fluorescence “turn on” was observed. More
importantly, the limit of detection (LOD) is as low as 0.13 U/L,
which is much better compared with previous reports and
sensitive enough for determining lipase levels in biological
assays considering the dilution factor, given that for normal
adults age 60 and younger, the normal of lipase level in blood
is 10-140 U/L, and that for adult older than age 60 is 18-180
0
10 20 30 40 50 60 70 80 90
Lipase (U/L)
Fig. 1 (A) The fluorescence emission spectra of probe S1 (10 μM)
after incubation with different concentrations of lipase-buffer
(
0.1 M PBS, pH 7.4) solution at 37 °C; (B) calibration curve versus
the different concentration (0-80 U/L) of lipase; inset shows the
photo of the corresponding solutions of probe S1 (10 μM)
without (left) and with (right) lipase (90 U/L) after incubation at
3
9
U/L.
To further verify the formation of aggregates of probe S1
was caused by lipase, particle sizes were performed by the DLS
3
7 °C for 15 min under UV light (365 nm) illumination. Excitation
wavelength: 360 nm.
(
Dynamic Laser Scattering) analysis, and the results were
To check the feasibility of our design, firstly, we studied the
photophysical properties of TPE-2OH when it aggregates. The
results in Fig. S1 suggest that TPE-2OH is an AIE-active
substance. Secondly, we investigated the fluorescence change
when the probe S1 was treated with lipase-buffer solution in
hexane. First, the effect of pH values and temperatures on the
reaction was investigated, with the results summarized in Fig.
S2 and Fig. S3, respectively. The pH value of 7.4 (PBS buffer)
and 37 °C were chosen in order to ensure sensitive and rapid
detection. A hexane solution of probe S1 (10 μM) was
prepared for the fluorescent spectral studies and Pocine
pancreas lipase (PPL) was selected to evaluate the application
of probe S1. In Fig. 1, as we expected, the fluorescent intensity
of probe S1 (10 μM) displays almost no emission under the
testing conditions, and nearly no photoluminescence could be
displayed in Fig. S5. In the stock solution of probe S1 (10 μM)
with no lipase added, the nanoparticles could be observed in
the range of 3-20 nm, while the mean diameter of S1 is
approximately
210
nm
after
incubated
with
determine concentration of lipase 80 U/L, which uncovered
that aggregates are indeed occurred in the solution.
Transmission Electron Microscopy (TEM) also confirms
compound TPE-2OH in the reaction mixture were produced
(
Fig. S6), and the length of formed aggregates appeared to be
00-400 nm, which was well consistent with the DLS analysis.
1
It suggested that the aggregation size significantly influenced
the fluorescence of the solution, because the AIE effect could
be triggered by the larger aggregates.
4
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View Article Online
DOI: 10.1039/C7SC02189E
A
To investigate the possibility of quantitative analysis of
lipase levels, we carried out the kinetic studies of enzymatic
catalytic reaction. Fig. 2A displays the variation of the emission
intensity at 453 nm of probe S1 (10 μM) during incubation
with different concentrations of lipase (0-80 U/L) for different
times at room temperature. Evidently, the fluorescence
intensity increased gradually by prolonging the incubation time,
it was also found that the rate of fluorescence enhancement
was directly related to the concentrations of lipase, the
intensity of the probe increases gradually with different
magnitudes, more rapidly fluorescence enhancement were
observed when more lipase was used. This is understandable
by concerning the fact that a high concentration of lipase
would facilitate the hydrolysis of probe S1. The fluorescence
changes more clearly in the initial stage for all lipase
concentrations but becomes slower in the later period. The
fluorescence emission reaches the plateau after about 7 min,
illustrating that the test could be finished within 7 min, which
was more convenient and faster than conventional
turbidimetric methods. As displayed in Fig. 2A, such emission
enhancement was still detectable even when the
concentration of lipase was as low as 30 U/L within 1 min. It
reveals that the lipase turn-on mode could be applied
potentially for the real-time assay of lipase levels.
9
8
7
6
5
4
3
2
1
00 Lipase(U/L)
8
7
5
3
1
5
0
0
0
0
0
0
00
00
00
00
00
00
00
00
0
0
2
4
6
8
10
Time(min)
35
B
30
25
20
15
10
5
0
As mentioned above, in view of the fact that lipase catalytic
reactions only occurred at the oil-water interface, compared to
the reported probe P1, herein, carboxyl and amino groups
a b c d e f g h i j k l m n o p q r s t
Fig. 2 (A) Time-dependent fluorescence intensity of probe S1 which will improve the affinity of the probe towards the lipase
10 μM) in hexane solution at 453 nm versus the hydrolysis in the aqueous phase were specifically introduced into probe
(
reaction time in the presence of different concentrations of S1. Firstly, substrates dependence of the initial degradation
lipase in PBS (pH 7.4, 0.1 M) at 37 °C. Excitation wavelength: velocities for lipase were measured. (Fig. S7) The data of P1
3
60 nm. (B) Fluorescence response of 10 μM probe in PBS- and S1 for enzyme kinetics follow the Michaelis-Menten
buffered water (pH 7.4, 0.1 M) at 37 °C for 15 min in the equation, and the kinetic parameters have been determined as
presence of different biologically-relevant substances (red Vmax = 1.35 μM/min and Km = 28.66 μM for P1, Vmax = 1.42
bars); fluorescence response of 10 μM probe in PBS-buffered μM/min and K = 4.23 μM for S1. The K value of S1 is much
m
m
water (pH 7.4, 0.1 M) at 37 °C for 15 min in the presence of 80 smaller, indicating the high affinity between lipase and the
U/L lipase and with the addition of different biological probe S1 molecule. On the other hand, as depicted in Fig. S8,
substances respectively (green bars). (a) NaCl; (b) KCl; (c) the fluorescence response of P1 began to appear when lipase
MgCl ; (d) CaCl ; (e) AlCl ; (f) NaNO ; (g) Na CO ; (h) Na SO ; (i) level rose to 120 U/L, and the limit of detection is up to 44.23
2
2
3
3
2
3
2
4
arginine; (j) leucine; (k) glutamic acid; (l) phenylalanine; (m) U/L, which is 338 times higher than probe S1( LOD=0.13 U/L).
cysteine; (n) glucose oxidase; (o) alkaline phosphatase; (p) Also, the test could be finished within 7 min, which was faster
acylase; (q) laccase; (r) α-amylase; (s) lipase; (t) blank; The than that of probe P1 (13 min). Therefore, this hydrophilic
concentration of the ions and amino acids are 100 μM, while group modified interfacial control can successfully bring high
the concentration of protein enzymes are 80 U/L. Excitation sensitivity, low detection limit and short response time test
wavelength: 360 nm.
process to us.
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ARTICLE
Journal Name
denaturated. According to our hypothetiVciaewl ArtiscelenOsnilninge
DOI: 10.1039/C7SC02189E
mechanism, inactivated lipase could not hydrolyze the probe
40
35
30
25
20
15
10
5
0
S1 and no fluorescence “turn on” is expected. The result in Fig.
S9 indicates that inactivated lipase cannot bring the
enhancement of fluorescence, which reveals that it is the
catalysis effect of lipase that acting on this process, excluding
other interference effects such as electrostatic interaction and
hydrophobic interaction between probes and proteins.
Additionally, the resistance to photobleaching for the
aggregates of the enzymatic products was also discussed after
a direct exposure to ultraviolet lamps (Fig. S10). The relative
fluorescence intensity change ratio (I /I ) remained almost
t
0
unchanged upon
a
continuous illumination, at last
fluorescence intensity of the reaction product aggregates only
reduce by 2.9 %, it was indicated that the reaction product
aggregates have good photostability.
PCL CALB RFL ANL CRL PPL Blank
We carried out a preliminary screening using the sensing
mechanism to evaluate the activity of some commercial lipase
samples (ANL, CALB, RFL, PPL, RNL and PCL). The fluorescence
intensity in the presence of a fixed concentration (80 U/L) of
lipase samples was monitored after certain time incubation
Lipase
Fig. 3 Plot of I/I intensity of probe S1 upon the addition of a fixed
0
concentration (80 U/L) of commercial lipase samples, respectively,
and within a interval of 20 min. (Inset) Corresponding visual
fluorescence under UV light illumination at 365 nm. Excitation
wavelength: 360 nm.
(20 min). As shown in Fig. 4, the variety of fluorescence
intensity was depending on the different lipase activities. We
concluded that the lipase activity increases in the following
Furthermore, to evaluate the selectivity of probe S1, the order: PCL < RFL < CRL < CALB < ANL < PPL. This sequence is
specific nature of this fluorometric assay for lipase was also consistent with the results detected with a pH-stat method
tested, a control experiment with other nonspecific biological (Table S1), demonstrating that this fluorometric assay protocol
+
+
2+
2+
3+
substances including ions (such as Na , K , Mg , Ca , Al , can be used as an alternative to conventional high throughput
2-
-
NO , CO and SO ), amino acids (such as leucine, glutamic screening technologies for lipase activity. The inserted
2-
3
3
4
acid, cysteine, arginine and phenylalanine) and other enzymes photograph in Fig. 4 was taken from the probe S1 after
such as alkaline phosphatase, acylase, glucose oxidase, α- incubation with different commercial lipase under UV
amylase and laccase) were performed with
fixed illumination at 365 nm. It is visible that different activity of
(
a
concentration. From the red bars in Fig. 2B, it can be seen that, lipases lead to the strong or weak blue fluorescence, namely, it
only lipase induces a prominent intensity enhancement, which could be easily distinguished by the naked eye under UV light.
brings about 32-fold fluorescence enhancement of the probe Lipase detection in real human serum samples
was recorded with the effect of lipase, while nearly no rise in
Afterwards, in order to explore the operability and efficacy of
emission intensity were observed for other biological
the probe system in complex biological fluids, the probe were
substances. Moreover, we also tested the interference of
applied to detecting the lipase levels in real samples, such as
these biologically-relevant substances towards probe. The red
human serum. Herein, we tried to detect the endogenous
bars tell us the presence of other background substances did
not show any obvious disturbance, indicating that S1 had
superior selective response toward lipase and these potentially
lipase levels in human serum samples by using the calibration
curve (Fig. 1B) as standard. Firstly, a standard recovery
experiment was performed by adding fixed amounts of lipase
interfering substances show negligible interference on the
in human serum, as depicted in Table S2, the recovery value
enzymatic reaction between lipase and probe. To further
was between 95.97% and 102.44%, SD values from these two
reveal that fluorescence enhancement was caused by lipase-
methods are also similar, suggesting the reliability and
catalyzed hydrolysis rather than other factors, a control
accuracy of our sensing system for lipase detection. For real
experiment between lipase and inactivated lipase was
serum samples, the procedures of detection were illustrate in
performed. (Fig. S9) Herein, deactivated lipase which treated
by high temperature (100 °C) was selected and it is well known
that high temperature environments can make the protein
6
| J. Name., 2012, 00, 1-3
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Page 8 of 9
Journal Name
ARTICLE
View Article Online
DOI: 10.1039/C7SC02189E
Table 1 Endogenous lipase levels detection in real human serum
samples by using lipase assay kit and probe S1.
People
Serum
no.
Detected by assay
kit (U/L)
Detected by
probe S1 (U/L)
134.06±1.38
1
129.79±2.09
2
3
119.36±3.17
152.18±1.63
123.64±2.67
157.97±1.85
Healthy
People
4
5
6
7
8
87.81±2.44
176.87±2.59
106.01±3.81
422.69±1.68
471.14±5.06
96.51±2.41
171.35±3.62
104.83±2.74
425.59±3.26
479.87±3.82
AP
Patients
9
517.07±3.47
441.89±2.84
584.41±3.31
549.62±4.01
510.20±2.11
443.43±4.97
581.05±2.06
557.03±3.45
1
1
1
0
1
2
B
Note: The serum samples were 10-fold diluted for
measurement; and the values (mean ± SD) herein represent
lipase levels in the undiluted serum samples, which are
calculated based on the measurements for the diluted
samples. Samples no.1 to no.6 were obtained from healthy
people; samples no.7 to no.12 were obtained from patients
with acute pancreatitis.
Fig. 4 (A) Procedure schematic illustration of this simple
technique for lipase levels detection in real human serum
samples. (B) Corresponding visual fluorescence of probe S1
after incubation with 10-fold diluted real human serum
samples under UV light illumination at 365 nm.
relatively simple test process of samples except the pre-
treatment, and shorter test time (7 min) than assay kit (25
Fig. 4A, since the linear response of probe is ranging from 0 to min). It’s indicated that our fluorescence probe could act as a
0 U/L, the dilution factor of the serum sample must be taken quite convenient one-step assay for an accurate detection of
8
into account. Serum samples were obtained from human lipase levels in human serum and had great potential as an
venous blood via centrifugation, and then the diluted serum effective detection method for lipase levels in diagnostic
was incubated with the probe solution for a while, the related applications.
fluorescence emission of S1 probe will experience off-to-on
change, which can be monitored by FL spectra, as well as
naked eye under UV light illumination at 365 nm. The serum
the endogenous lipase levels of both healthy people (sample
Conclusions
In summary, for lipase fluorescent probes, the interfacial
no.1~no.6) and acute pancreatitis (AP) patients (sample
catalytic efficiency determines its sensitivity, we designed a
no.7~no.12) measured by this probe are close to those
measured by lipase assay kit. The results from probe S1 in
Table 1 indicate that, the lipase level in the serum samples of
novel fluorescence turn-on probe for lipase detection, which
functions through lipase-induced transformation of soluble to
insoluble features and the corresponding AIE effect. We adjust
no.1 to no.6 was in the range of 96.51-171.35 U/L, which were
the reaction behavior of AIE probe in the interface by the
within the normal level of serum lipase, however, for AP
introduction of hydrophilic groups, the highest sensitivity,
patients, their serum lipase level were significantly higher in
lowest detection limit and shortest response time in reported
the range of 425.59-581.05 U/L. The inserted photograph (Fig.
5
literature were obtained. The probe is more suitable for a
B) was taken from the probe S1 after incubation with diluted
linear detection of lipase levels ranging from 0 to 80 U/L,
which fits into the concentration range of lipase in human
serum considering the dilution factor if necessary, the
aggregates product also shows a good photostability against a
direct exposure to ultraviolet, few interference by other
biological components and good agreement with assay kits
results. To the best of our knowledge, this is the first report on
the detection of lipase levels in real human serum samples
using a fluorescent probe. Therefore, the probe would provide
a helpful approach for further elucidating the physiological
real human serum samples under UV illumination at 365 nm to
conduct eyeball detection. It is clear that, for serum samples
from healthy people (no.1~no.6), very weak fluorescence is
observed, and that for AP patients serum samples
(no.7~no.12), significant fluorescence emission enhancement
can be noticed. In addition, the determination results were
well consistent with those obtained by the lipase assay kit,
moreover, compared with commercial lipase assay kit, our
probe has the advantages of much lower limit of detection
(0.13 U/L), which is 38-fold lower than assay kit (5.0 U/L),
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Journal Name
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2
2
2
6 Y. Yuan, C. Zhang, R. T. K. Kwok, D. Mao, B. Z. TaVniegwaAnrtdicleBO. nLliiune,
Chem. Sci., 2017, , 2723-2728.
7 W. Zhang, Y. Chen, T. Shao and L. Fan, J. Mater. Chem. B,
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8 X. Zhang, X. Zhang, L. Tao, Z. Chi, J. Xu and Y. Wei, J. Mater.
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9 Y. Jia, X. Zuo, X. Lou, M. Miao, Y. Cheng, X. Min, X. Li and F.
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0 Y. Ding, L. Shi and H. Wei, Chem. Sci., 2015, 6, 6361-6366.
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3 L. Yan, H. Shi, X. He, K. Wang, J. Tang, M. Chen, X. Ye, F. Xu,
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4 W. Zhang, W. Liu, P. Li, H. Xiao, H. Wang and B. Tang, Angew.
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roles of lipase as well as for conducting pathological analysis
for diseases involving lipase.
8
DOI: 10.1039/C7SC02189E
2
2
Acknowledgements
2
We are grateful to the National Science Foundation of China
(no. 31501423 and no.31671820), the Director Fund of Oil
3
3
Crops Research Institute of CAAS (no. 1610172015006) and
Agricultural Science and Technology Innovation Project of
Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2013-
OCRI) for financial support.
3
3
3
3
3
3
3
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