Homogeneous probing of lipase and α-amylase simultaneously by AIEgens

Article abstract of DOI:10.1039/c9cc01959f

An AIE dual-reactive supramolecular probe has been devised for the first time to simultaneously measure endogenous lipase and α-amylase activity in a homogeneous system. Fluorescence quantitative analysis of lipase and α-amylase in real biological samples enables rapid and accurate diagnosis of diseases.

Full text of DOI:10.1039/c9cc01959f

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc
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DOI: 10.1039/C9CC01959F
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Homogeneous probing lipase and α-amylase simultaneously by
the AIEgens
Received 00th January 20xx,
Accepted 00th January 20xx
ab
a
a
c
a
b
a
Jie Shi, Qianchun Deng,* Ya Li, Zhe Zheng, Huijuan Shangguan, Lu Li, Fenghong Huang* and
b
Bo Tang*
DOI: 10.1039/x0xx00000x
An AIE dual-reactive supermolecular probe has been firstly
devised to simultaneously measure endogenous lipase and α-
amylase activity in homogeneous system. Fluorescent quantitative
analysis of lipase and α-amylase in real biological samples enables
rapid and accurate diagnosis of diseases.
Lipase (LPS) and α-amylase (AMY) have a wide range of
applications in the food and pharmaceutical industries, and
they are also the core enzymes in the human digestive
1
system. In our body, α-amylase and lipase are mainly secreted
by the pancreas and emerged in in digestive juices as well as
2
other body fluids. Under the conditions of pancreatic injury, Scheme 1 Schematic illustration of fluorescent response mechanisms of probe S4 to
pancreatic autolysis induces significant elevation of lipase and lipase, α-amylase, and lipase/α-amylase mixture.
α-amylase levels in blood, which is the symptom of acute
pancreatitis. The mortality for severe acute pancreatitis is up
3
emission (AIE) properties for detecting sole lipase or α-
amylase, which brings improved sensitivity and shortened
to 30%, sufferers often endure pain and malnutrition and are
most likely left with a higher risk of pancreatic cancer. Because
the pancreas is relatively inaccessible, problems are often
diagnosed by computed tomography (CT) and clinico-radiology
7
testing time. However, in clinical diagnosis of acute
pancreatitis, depending on the cause and onset time, case of
illness that solely elevated lipase or α-amylase levels could be
found, so that the misjudgments may occur if we only measure
4
and timely treatments are therefore delayed. Thence, as the
8
one of them, combining lipase and α-amylase determinations
most pivotal indicators in the clinical diagnosis of acute
pancreatitis, the rapid detection and sensitive monitoring the
simultaneously is highly demanded for improvement of
9
5
positive diagnosis rate of acute pancreatitis. Unfortunately,
activity of lipase and α-amylase will be greatly desirable.
the techniques for combining the two tests have been
generally limited, for example, two substrates of enzymes
should be selected for each test, lipase can only exert
Recently, some innovative strategies are being developed
for lipase or α-amylase detection to replace the conventional
6
turbidimetric and colorimetric methods, but some specific
1
0
maximum activity at the two-phase interface, making the
test is conduct in two-phase medium and so on. Up to date, no
breakthrough concept has emerged for design single small
molecule probe test system to overcome these obstacles.
Hence, to bypass these barriers, in this work, we engineered
a host-guest inclusion S4 (Scheme 1) for the simultaneously
detection of lipase and α-amylase, which comprises one
tetraphenylethene (TPE) conjugated with γ-cyclodextrin (CD)
by esterification (TPE-γ-CD) and one orange emissive AIEgen
substrates and tool enzymes have been used, possessing
relatively complicated processes and suffer from lower
sensitivity. Subsequently, our group synthesized some small-
molecule fluorescent probes with aggregation-induced
a. Hubei Key Laboratory of Lipid Chemistry and Nutrition, Key Laboratory of Oilseeds
Processing, Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy
of Agricultural Sciences, Wuhan 430062, China. E-mail: shijie@caas.cn
b.
College of Chemistry, Chemical Engineering and Materials Science, Collaborative
1
1
(TPE-H). In the light of the AIE principle, the TPE-γ-CD was
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
soluble and very weakly emissive in aqueous media, for TPE-H,
it’s not soluble in water, and as a strong emitter in aqueous
mixtures. When they are mixed with 1:1 molar ratio, TPE-H will
c.
Department of Modern Physics, University of Science and Technology of China
Hefei 230026, China.
Electronic Supplementary Information (ESI) available: [Synthesis, characterisation, drill into the hydrophobic cavity of the TPE-γ-CD, the
test conditions and other supplementary data.]. See DOI: 10.1039/x0xx00000x
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Journal Name
7
00
700
600
500
400
300
200
100
0
restriction of intramolecular rotation (RIR) effect is weakened,
A
B
I
View Article Online
DOI: 10.1039/C9CC01959F
[Lipase ] +7.3277
6
00
00
00
00
00
1
2
-1
=6.743
namely quenching the TPE-H fluorescence. When the lipase
is present, the ester bond linked insoluble TPE-COOH will be
released and aggregated to produce blue fluorescence, if α-
amylase was incubated, it will act on the α-1,4-glycosidic
bonds of the γ-CD, resulting in this host molecule disintegrate
and release the insoluble orange-emitting TPE-H again. And so
forth, if probe S4 encounters the lipase/α-amylase mixture,
mixed color of fluorescence will be captured. Thus, the double
emission turn-on results could be used for selective detection
of lipase and α-amylase simultaneously. More importantly,
first, the two emissive peaks were separated by roughly 150
nm upon the same excitation wavelength (390 nm) to avoid
spectral interference and caused sensitive visual changes;
Second, a micro hydrophilic-hydrophobic interface between
the hydrophobic cyclodextrin cavity and the aqueous solution
was constituted, which fascinates the interface activation
effect of lipase and abandons the tedious two-phase test
system. To our best knowledge, no reports to date have been
published on the application of one fluorescent probe in
testing lipase and α-amylase levels of real human serum
samples, this is the first report on “one pot” assay for lipase
and α-amylase simultaneously.
Lipase (U L
)
460
5
2
R
=0.996
72
50
4
4
8
3
36
3
1
26
9
0
2
100
0
4
00
460
520
580
640
700
0
20
40
Lipase
60
80
-
1)
Wavelenth (nm)
(
U L
500
400
C
D
I610=2.938
2
00 R =0.994
[-Amylase ] +1.5992
4
00 -Amylase
U L-1
( )
3
110
9
0
300
70
6
4
200
5
0
200
42
3
0
18
100
0
1
4
100
1
0
0
0
430
490
550
610
670
0
30
60
90
1)
120
-
Wavelenth (nm)
-Amylase
(
U L
800
00
00
00
00
00
200
00
35
30
25
20
15
10
5
E
F
7
Probe S4 Solution
Probe S4+AMY
Probe S4+LPS
Probe S4 Solution
-1
-1
6
47 U L LPS+ 54 U L AMY
-1
-1
6
2 U L LPS+ 65 U L AMY
-1 -1
2 U L LPS+ 96 U L AMY
5
3
4
3
1
The preparation of supramolecular S4 is quite simple, just
need to mix TPE-γ-CD and TPE-H with a 1:1 molar ratio in
DMSO. The supramolecular complex S4 were characterized
using NMR and mass spectrometry (Fig. S1, S2), satisfactory
0
400
0
480
560
640
720
1
10
100
1000
Wavelenth (nm)
Diameter (nm)
Fig. 2 Fluorescence spectra and of 10 μm S4 with the incubation of lipase (A) and α-
results were obtained corresponding to their molecular amylase ( C) in PBS buffer (pH 6.6, 0.1 M) for 30 min; B) Region of the calibration curve
structures. As illustrate in Fig. S3, compared to free TPE-H, of lipase (B) and α-amylase (D); E) Fluorescence spectra upon mixing 10 μm S4 and
lipase and α-amylase; F) Dynamic light scattering (DLS) results for the solution of probe
S4 (10 μM) in PBS buffer in the presence of lipase (100 U L ) or α-amylase (150 U L )
for 30 min. Excitation: 390 nm; the insets are the corresponding photographs under
firstly, two groups of new peaks (7.55-7.51 and 7.37 ppm) that
-1
-1
were ascribed to aromatic protons appeared, indicating that
most of the phenyl in TPE-H is encapsulated in TPE-γ-CD while
the former multiplets (7.15-7.11, 7.03-6.97 ppm) from
3
65 nm UV lamp.
aromatic protons of free TPE-H disappeared, the broader Next, we try to quantify the α-amylase activity with a pure S4
peaks told that their chemical environment has changed. test system. As seen in Fig. 1C, the intensity centred at about
Secondly, compared to merely TPE-γ-CD, the peaks in the 610 nm increased correspondingly, and the fluorescence
-
range of 3.70-3.30 showed distinct changes, other new peaks depicted no enhancement when the enzyme reached 110 U L
were found in the range of 7.21-6.42 ppm, implying that the
1
. From the calibration curve (Fig. 1B, D), the relative
esterification linked TPE molecules on the γ-CD have been fluouorescence intensity at about 460 nm and 610 nm gave a
1
2
extruded to the outer cavity by TPE-H, which is free from the linear increase with the concentration of lipase as well as α-
-
1
2
-1
constraint of γ-CD. Furthermore, the peaks in the range of amylase in the range of 0-72 U L (R =0.996) and 0-110 U L
2
8
.42-7.77 ppm were almost showed no changes, implying that (R =0.994), respectively. The limit of detection (LOD) is
the double bond and benzothiazole units were out of the calculated to be as low as 0.11 U L for lipase, 0.17 U L for α-
cavity of γ-CD. The HRMS spectrum of S4 can be seen in Fig. amylase, which are the best ones compared with previous
-
1
-1
S2, the obvious molecular ion peak at m/z 2174.7642 was reports. On the other hand, we conducted HRMS to analyze
+
attributed to the [M-PF
6
] ion of S4 (calculated, 2174.7677).
the product of each enzymatic reaction. The results in Fig. S4
Afterwards, the supramolecular-based probe S4 buffer and S5 suggest that compound TPE-COOH and TPE-H appeared
solution (10 µM) was first incubated with different activity of after hydrolysis. The inserted photograph in Fig. 1A and Fig. 1C
lipase in a homogeneous buffer solution. Before the addition reflect the significant changes in fluorescence under various
of lipase, the solution of S4 was nearly no emission in the enzyme activities, which can be easily distinguished by the
aqueous solution. However, as depicted in Fig. 1A, when naked eye under UV light. To verify the possibility of
incubated with a growing activity of lipase, the homogeneous determination of lipase and α- amylase activity in chorus by
test system does not affect the arousal of fluorescence, an the S4 probe, we incubated it by the mixed lipase and α-
apparent emission band at 460 nm appeared and increased, amylase solution with a defined activity. The results in Fig. 1E
concurrently, the emission at 610 nm that ascribe to TPE-H is revealed that the emission at 460 nm and 610 nm is relatively
-1
quiet. After 72 U L lipase incubated, the intensity has reached independent and not affected by each other, the activity of the
to the maximum by about 24-fold compared to the initial one.
two enzymes can be simultaneously quantitatively analysed
2
| J. Name., 2012, 00, 1-3
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00
350
A
B
Lipase
0
(
U L-1
)
-Amylase
0
15
30
70
100
(
U L-1
)
300
5
00
00
00
00
00
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DOI: 10.1039/C9CC01959F
1
0
250
4
25
3
5
200
150
3
55
65
2
100
1
50
0
0
0
1
2
3
4
5
6
7
8
0
5
10
15 20 25 30
Time (min)
Time (min)
3
5
35
30
25
20
15
10
5
C
D
30
@460 nm, Lipase
610 nm, -Amylase
@
460 nm, Lipase
@
Fig. 2 Compound TPE-γ-CD was docked to the binding hydrophobic pocket of the lipase:
A) Detailed view; B) Total view.
25
@ 610 nm, -Amylase
20
15
through the fluorescence emission intensity and the curves.
The photograph clarified that, compared to another
photographs in Fig. 1A and Fig. 1C, the emission of two
10
5
0
0
1
2
3
4
5
6
7
8
9
10
1 2 3 4 5 6 7 8 9 10
enzymes system shows obvious distinction to the sole enzyme. Fig. 3 Time-dependent fluorescence intensity of probe S4 (10 μM) at 460 nm and 610
nm in the presence of different activities of lipase (A) and α-amylase (B) in PBS (pH 6.6,
Moreover, dynamic light scattering (DLS) measurements was
used to determine the change of particle size after incubation
0
.1 M). Excitation wavelength: 390 nm. C) Fluorescence response of 10 μM probe S4
for 30 minutes in the presence of different species (PBS buffer, pH 6.6, 0.1 M); (1)
(
Fig. 1F). The average particle size of the S4 aggregates was alkaline phosphatase; (2) α-chymotrypsin; (3) typsin; (4) pepsin; (5)
lactatedehydrogenase; (6) thrombin; (7) alcohol dehydrogenase; (8) lysozyme; (9) AChE;
10) lipase or α-amylase; D) fluorescence response of 10 μM probe S4 for 30 minutes in
the presence of lipase or α-amylase and with the addition of different species
distinctly larger after incubation with lipase or α-amylase than
that of S4 only.
(
To explain the high activity of TPE-γ-CD against the lipase, respectively (PBS buffer, pH 6.6, 0.1 M); No. 1-No. 9 are the same as those of Fig. 3A;
-
1
(
10) all species; The concentration of lipase and α-amylase are 75 U L , other protein
the theoretical binding mode between them was performed.
In Fig. 2A, the four phenyl groups of the TPE-γ-CD located at
the big hydrophobic pocket, surrounded by the residues Ala-
-1
enzymes are greater than or equal to 100 U L . Excitation wavelength: 390 nm.
involved hydrolysis of γ-CD is slower than ester bond.
Therefore, all the assay procedure could be finished in about
5 minutes, considering whether the aggregated product of
5
2
1, Ala-151, Pro-178, Ile-179, Val-183, Leu-184, Leu-205, Leu-
13 and Leu-241, forming a strong hydrophobic binding.
2
Detailed analysis showed that one of the phenyl group of TPE-
γ-CD formed a anion-π interaction with the residue Asp-180.
Importantly, two key hydrogen bonds were observed between
the residues Gly-177 (bond length: 2.5 Å) and Arg-240 (bond
length: 3.2 Å) and the TPE-γ-CD, which was the main
interaction between them. All these interactions helped TPE-γ-
CD to anchor in the binding site of the lipase. Meanwhile, to
reveal that S4 can analyze lipase activity in a homogeneous
lipase is stable after 25 minutes, the stability of enzymatic
aggregates was also investigated and the results in Fig. S7 tell
us they are very stable. Again, we confirmed the photostability
of S4 system under continuous irradiation with a UV light (Fig.
S8). The influences of common proteins in the blood or human
body were all studied. From Fig. 3C, only lipase and α-amylase
can arouse two fluorescence emissive responses while the
nearly no reply were appeared for other enzymes, which
exhibits about 24-fold and 12-fold fluorescence enhancement
compared to the assay system with nothing added. We tested
the interference of these proteins in the probe S4 assay
system later, as shown in Fig. 3D, in the presence of other
interfering proteins, no more changes in fluorescence intensity
can be seen, and demonstrating that probe S4 is highly
selective to lipase and α-amylase.
7
solution, molecule S1, a heterogeneously reactive lipase
probe we reported before, was then docked into the binding
pocket of the lipase (Fig. S6). We can infer that, from Fig. 2B
and Fig. S6B, for TPE-γ-CD, the hydrophobic cavity of γ-CD can
drive the “lid” structure above the lipase active cavity to
open,10 making two hydrophobic regions connected, so that
the ester bond enters, but S1 can’t do this and still needs a
hydrophobic medium to open the “lid”, which rational explains
why S4 can react with lipase only in homogeneous media. In
The kinetics of lipase and α-amylase on the probe S4 are
summarized in Fig. S9. For lipase, herein, hydrophobic cavity of
γ-CD were used to build micro interfaces. Firstly, the substrate
dependence of the initial degradation velocities for lipase were
measured (Fig. S9A, B). The data of S4 for the enzyme kinetics
follow the Michaelis-Menten equation, and the kinetic
−
1
addition, the estimated binding energies were -7.6 kcal mol
for S1 and -8.4 kcal mol− for TPE-γ-CD, respectively,
suggesting TPE-γ-CD was more active to bind the lipase than
that of S1, which explained that TPE-γ-CD shows slight higher
sensitivity than S1.
1
-1
parameters have been determined as Vmax = 2.23 μM min and
The time-course analysis of enzymatic catalytic reaction was
addressed. In Fig. 3A, upon incubation with various activities of
K
m
= 4.29 μM, which is similar to probe S1 reported previously
7
(K
m
= 4.23 μM), validating the good affinity between the
-1
lipase (0-65 U L ), the intensity of fluorescence gradually
increased by prolonging the incubation time, and the rate of
fluorescence enhancement was directly corresponded to the
levels of lipase. At last, it reaches the plateau after about 7
minutes. For α-amylase in Fig. 3B, the activity gradient was 0-
probe and lipase. Soon after, the kinetics of α-amylase on the
probe S4 were also evaluated (Fig. S9 C, D), the related kinetic
−1
parameters have been determined as Vmax = 4.14 μM min
7
and K
m
= 4.82 μM, compared to the α-amylase probe S2, the
K
m
value of S4 is slightly larger, which also stats that the
-1
1
00 U L , the fluorescence intensities were stop to increase
hydrolysis of γ-CD is a relatively slow process.
after 25 minutes, under each activity. Since the α-amylase
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J. Name., 2013, 00, 1-3 | 3
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300
Blue: 36.18 300
Blue:54.08
Red:32.67 200
Blue: 62.59
Red: 54.98
analysis of lipase and amylase in a homogeneous system
within 25 minutes, showing selectivity and not interfered by
other biological components, depending on the color of
3
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DOI: 10.1039/C9CC01959F
Red: 21.76
B
2
200
100
100
700
600
1
0
0
0
20 40 60 80 100 0
0
20 40 60 80 100
0
30 60 90 120 150
Intensity
“lighting up”, bedside testing can be realized. On the basis of
Intensity
Intensity
FL @ 460 nm
500
the advantages above, this system can be popularized and
applied in sensing of lipase and α-amylase during clinical
diagnosis.
A
400
300
200
4
00
00
00
00
400
200
150
100
50
Blue: 81.37
Red: 76.05
Blue: 82.98
Red: 103.47
Blue: 150.83
Red: 112.97
FL @ 610 nm
3
300
FL @ 460 nm
2
200
We are grateful to the National Science Foundation of China
100
1
100
FL @ 610 nm
(no. 31872896, 21535004, 91753111 and 21390411), the
0
0
0
0
20
40 60 80 100 120
60 80 100 120 140
Intensity
0
100 200 300
Intensity
Normal Pople
Patients
Young Elite Scientists Sponsorship Program by CAST
Intensity
Fig. 4 A) Picture of probe S4 after incubation with 10-fold diluted real human serum (2017QNRC001) for financial support.
samples under UV light illumination at 365 nm. Upper row: normal people; lower row:
patients. The histograms show the intensity distributions of pixels in the red and blue
channels. The numbers in the figures represent the average intensity per pixel in the
blue and red color channel. B) Box plot representation of fluorescence intensity at 460
nm and 610 nm of detection system in response to lipase and α-amylase from diluted
serum specimens of normal people and acute pancreatitis patients.
Notes and references
1
F. K. Winkler, A. Darcy and W. Hunziker, Nature, 1990, 343,
7
71-774; (A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M.
Wubbolts and B. Witholt, Nature, 2001, 409, 258-268.
Finally, the probe was applied to simultaneously sensing
lipase and α-amylase in human serum at the point of care. We
use 10-fold diluted serum specimens from 9 acute pancreatitis
patients and 9 normal people. After the incubation, we took
a picture of the mixtures in 96-well plate with a digital camera,
selected the designated area, and quantified the signal using a
software script, determines the average intensity per pixel
2
3
M. Roxas, Altern. Med. Rev., 2008, 13, 307-314; P. G.
Lankisch, M. Apte and P. A. Banks, Lancet, 2015, 386, 85-96.
M. D. Johnson, Gut, 2005, 54, 1-9; J. L. Frossard, M. L. Steer
and C. M. Pastor, Lancet, 2008, 371, 143-152; P. A. Banks and
M. L. Freeman, Am. J. of Gastroenterol., 2006, 101, 2379-
2
400.
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E. J. Balthazar, Radiology, 2002, 223, 603-613; M. Avanesov,
J. M. Weinrich, T. Kraus, T. Derlin, G. Adam, J. Yamamura and
M. Karul, Eur. J. Radiol., 2016, 85, 2014-2022; M. Avanesov,
A. Loeser, S. Keller, J. M. Weinrich, A. Laqmani, G. Adam, M.
Karul and J. Yamamura, Eur. J. Radiol., 2017, 95, 278-285.
D. Yadav, B. Ng, M. Saul and E. D. Kennard, Pancreas, 2011,
1
3
(
AIP) in the blue and red color channels. Fig. 4A and inset
photograph indicted that the patients’ AIP values of blue and
red color channels were significant higher than the normal
ones. Therefore, the AIP of normal people can be used to build
the threshold values for the point of care testing and diagnosis
of acute pancreatitis. Subsequently, the using of calibration
curve (Fig. 1B, D), as a standard, to the simultaneous
quantitative analysis of lipase and α-amylase levels in human
serum, the results in Fig. 4B and Table S1 indicate that the
lipase and α-amylase levels of normal people was in the range
5
6
4
0, 383-389; N. W. Tietz, Clin. Chim. Acta, 1997, 257, 85-98.
W. Zhang, Y. Tang, J. Liu, Y. Ma, L. Jiang, W. Huang, F. Huo
and D. Tian, J. Mater. Chem. B, 2014, 2, 8490-8495; J. Zhang,
J. Cui, Y. Liu, Y. Chen and G. Li, Analyst, 2014, 139, 3429-3433;
J. Shi, Q. Deng, C. Wan, M. Zheng, F. Huang and B. Tang,
Chem. Sci., 2017, 8, 6188-6195; J. Shi, Q. Deng, Y. Li, M.
Zheng, Z. Chai, C. Wan, Z. Zheng, L. Li, F. Huang and B. Tang,
Anal. Chem., 2018, 90, 13775-13782.
7
8
−1
of 88.49-168.57 and 37.23-141.25 U L , which were within the
normal range of enzymes. For patients, their lipase and α-
amylase levels were significantly higher, being in the range of
K. Satoh, T. Shimosegawa, A. Masamune, M. Hirota, K.
Kikuta, Y. Kihara, S. Kuriyama, I. Tsuji, A. Satoh, S. Hamada
and P. Res Comm Intractable Dis, Pancreas, 2011, 40, 503-
5
4
07; B. Frank, K. Gottlieb, Am. J. of Gastroenterol. 1999, 94,
63-469.
−1
4
4
60.13-702.16 and 420.01-729.75 U L . The photograph in Fig.
A, on one hand, it is clear that very weak fluorescence is
9
1
1
J. P. Corsetti, C. Cox, T. J. Schulz and D. A. Arvan, Clin. Chem.,
1993, 39, 2495-2499; T. Cartier, P. Sogni, F. Perruche, O.
Meyniard, Y. E. Claessens, J. F. Dhainaut and G. Der Sahakian,
Emerg. Med. J., 2006, 23, 701-702.
observed from normal ones’ samples and that for patients’
samples, significant double fluorescence emission “light-up”
can be noticed, demonstrating the possibility of rapid eyeball
diagnosis. On the other hand, if there are very few cases in
which lipase or α-amylase is elevated independently, our
probe system will exhibit monochromatic emission, affording
the early warning of pancreatic injury either. In addition, the
determined results were confirmed with those obtained by the
complex commercial lipase assay kit and α-amylase assay kit
methods (Table S2).
In summary, we have successfully designed and fabricated a
dual-reactive AIE probe that can respond to both lipase and α-
amylase with two differentiable fluorescent signals under the
same excitation wavelength. The combination with the
enzymatic hydrolysis function and decomposition of host-
guest inclusion triggered the AIE affect, and there is a 150 nm
way between two emission peaks, making the signal
distinguished quite easily by the naked eye. Importantly, this
simple test system can simultaneously perform quantitative
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DOI: 10.1039/C9CC01959F
Entry for the table of contents:
An AIE dual-reactive supermolecular probe has been devised to simultaneously measure endogenous lipase and
α-amylase activity in homogeneous system. Fluorescent quantitative analysis of lipase and α-amylase in real
biological samples enables rapid and accurate diagnosis of diseases.