Detection of lipase activity in human serum based on a ratiometric fluorescent probe

Article abstract of DOI:10.1039/d1nj01155c

Acute pancreatitis results in major mortality and morbidity. Lipase is a vital biomarker of acute pancreatitis in the early stage. Developing a simple and convenient methodology for monitoring the lipase activity is of significance to the early diagnosis of acute pancreatitis. In this work, we designed a novel ratiometric fluorescent probe (CARA) based on FRET for detecting lipase activity in human serum. The energy donor group coumarin derivative (CA) and the energy acceptor group rhodamine derivative (RA) were linked for the construction of a coumarin-rhodamine platform through an ester bond that is easily cleaved by lipase, breaking the FRET process between CA and RA. CARA shows a high selectivity to lipase over other biological substances and a good linearity between the fluorescence ratio and lipase activity in serum samples. CARA has the potential to be applied in the clinical diagnosis of acute pancreatitis.

Full text of DOI:10.1039/d1nj01155c

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Detection of lipase activity in human serum based
on a ratiometric fluorescent probe†
Cite this: DOI: 10.1039/d1nj01155c
Jiajie Luo,‡a Hongyi Zhang,‡a Jialiang Guan,b Baoshuai An,a Junli Peng,a Wei Zhu,a
a
Ningning Wei*a and Yanru Zhang
*
Acute pancreatitis results in major mortality and morbidity. Lipase is a vital biomarker of acute pancreatitis
in the early stage. Developing a simple and convenient methodology for monitoring the lipase activity is of
significance to the early diagnosis of acute pancreatitis. In this work, we designed a novel ratiometric
fluorescent probe (CARA) based on FRET for detecting lipase activity in human serum. The energy donor
group coumarin derivative (CA) and the energy acceptor group rhodamine derivative (RA) were linked for
the construction of a coumarin–rhodamine platform through an ester bond that is easily cleaved by lipase,
breaking the FRET process between CA and RA. CARA shows a high selectivity to lipase over other biological
substances and a good linearity between the fluorescence ratio and lipase activity in serum samples. CARA
has the potential to be applied in the clinical diagnosis of acute pancreatitis.
Received 11th March 2021,
Accepted 21st April 2021
DOI: 10.1039/d1nj01155c
rsc.li/njc
probes need to be used in a two-phase (lipid and water) solution
for meeting the interfacial catalysis requirement of lipase, which
1. Introduction
Acute pancreatitis is a common gastrointestinal disease with requires tedious operation. The lipid solution is also harmful to
high morbidity, and nearly 20% severe acute pancreatitis is the environment. Therefore, there is still demand for developing
associated with organ failure, which results in high mortality. an environment friendly fluorescent probe that has a built-in
The serum lipase level of an acute pancreatitis patient increases correction for the detection of lipase activity.28–31
dramatically within 24 h and is three times higher than the
In this paper, we designed and synthesized a novel ratio-
upper limit of normal.1,2 Therefore, lipase is a crucial biomarker metric fluorescent probe for the detection of lipase activity
in the early diagnosis of acute pancreatitis.3–5 Thus, the detection based on fluorescence resonance energy transfer (FRET). The
of serum lipase activity is necessary for the diagnosis and probe was named N-(6-(diethylamino)-9-(2-(((3-(6-(diethylamino)-
assessment of acute pancreatitis.6,7
2-oxo-2H-chromene-3-carboxamido)propanoyl)oxy)methyl)phenyl)-
Lipase is secreted from the pancreas for hydrolyzing triglycerides 3H-xanthen-3-ylidene)-N-ethylethanaminium (CARA).32,33 Energy
into glycerol and fatty acid. The conventional methodologies for the donor group 3-(6-(diethylamino)-2-oxo-2H-chromene-3-carboxamido)
detection of lipase activity are colorimetry and titrimetry. These propanoic acid (coumarin group) and energy acceptor group (E)-3-
methods undergo tedious sample processing and render damage (diethyliminio)-N-ethyl-N-ethylidene-9-(2-(hydroxymethyl)phenyl)-3H-
to lipase activity, affecting the measurement accuracy.8–11 xanthen-6-aminium (Rhodamine group) are linked by an ester bond
Compared with these methods, fluorescent probes have attracted (Scheme 1). The CARA probe can monitor lipase activity in aqueous
widespread attention due to their convenience, rapid response, solution through hydrolyzing the ester bond to interrupt the FRET
high sensitivity and noninvasiveness.12–22 However, only several process. CARA possesses two fluorescence emission peaks at 405 nm
single emission fluorescent probes were developed for the and 585 nm, and exhibits a high selectivity to lipase and a
measurement of lipase activity.17,23,24 These single emission good linearity between the fluorescence intensity ratio and the
probes are vulnerable to the variations in the environment, concentration of lipase. CARA also can detect the lipase activity in
concentration, and excitation intensity.25–27 In addition, some serum, providing a potential tool for diagnosis in pancreatic
diseases.
a Departments of Pharmacology and Medicinal Chemistry, Qingdao University
School of Pharmacy, Qingdao 266071, China. E-mail: weiningning@qdu.edu.cn,
yanru.zhang@qdu.edu.cn
2. Experimental section
2.1. Materials and instrument
b Department of Emergency Internal Medicine, The Affiliated Hospital of Qingdao
University, Qingdao, 266000, Shandong, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
Unless stated otherwise, all reagents were purchased from
Macklin Company and used without further purification.
d1nj01155c
‡ These authors contributed equally to this work.
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Scheme 1 Synthesis of CARA.
Lipase was purchased from Macklin Company and the lipase washed with water (50 mL Â 3). The organic phase was dried
activity was determined by pNPB according to the reported with anhydrous MgSO4, filtered and concentrated in vacuo. The
method.34 Twice-distilled water was used throughout all residue was purified by column chromatography to give a light
1
experiments. The pH values were measured with a pH meter pink solid (RA) (38 mg, 45%). H NMR (500 MHz, CDCl3): d =
(PHS-3C). 1H NMR and 13C NMR spectra were acquired on a 7.39, (m, 1H), 7.25 (m, 3H), 6.67 (d, J = 8.6, 2H), 6.38 (d, J = 2.3,
Bruker Avance-500M and JNM ECZ 600R spectrometer, with 2H), 6.26 (dd, J = 8.5, 2.5, 2H), 5.34 (s, 1H), 4.55 (s, 2H), 3.31 (q,
used CDCl3 an d6-DMSO as solvents and tetramethylsilane J = 7.1, 8H), 1.15 (t, J = 7.2, 12H) ppm. 13C NMR (125 MHz,
(TMS) as an internal standard. Ultraviolet (UV) and fluorescence CDCl3): d = 151.70, 147.82, 144.83, 138.66, 131.48, 130.01, 129.69,
spectra were performed on an UV-2600 Spectrophotometer and 128.07, 127.00, 111.46, 107.47, 98.83, 63.12, 44.48, 40.12,
F-4600 Spectrophotometer, respectively. A HPLC system (Agilent 12.74 ppm. ESI+-HRMS (m/z): 429.2534 ([M]+ calculated: 429.2537).
1260 Infinity II, Agilent technologies, Germany) equipped with a
2.4. Synthesis of CA
Kromasil 100-5-C18 column (4.6 Â 250 mm).
b-Alanine ethyl ester hydrochloride (154 mg, 1.49 mmol) was
dissolved in dry CH2Cl2 (5 mL) and triethylamine (0.5 mL), and
2.2. Synthesis of compound 1
Compound 1 was synthesized according to the literature a solution of 7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl
methods.35 Briefly, rhodamine B (5 g, 10.44 mmol) was dissolved chloride (334 mg, 1.19 mmol) in CH2Cl2 (2 mL) was added
in THF (50 mL), and then LiAlH4 (2.5 M, 10 mL) was added slowly at room temperature. The mixture was stirred for 2 h and
slowly under the protection of nitrogen. After stirring overnight the organic solvents were removed under reduced pressure to
at room temperature, the reaction was quenched with H2O get the crude product. The crude product (1.0 g) was dissolved
(20 mL) and the mixture was extracted with CH2Cl2 (50 mL Â 3). in dry 1,4-dioxane (25 mL), and then HCl (25 mL, 3M) was
The organic phase was dried with anhydrous MgSO4, and added into the solution, and stirred overnight. The reaction
concentrated in vacuo to get the crude product. Then, it was solution was extracted with CH2Cl2 (50 mL Â 3) and the organic
purified by column chromatography to get a white solid phase was concentrated under reduced pressure. The residue
(compound 1, 1.32 g, 26%). 1H NMR (600 MHz, CDCl3): d = was purified by column chromatography to yield a yellow solid
7.42–7.38 (m, 1H), 7.25 (dd, J = 8.6, 3.6, 3H), 6.67 (d, J = 8.6, 2H), CA (350 mg, 73%).1H NMR (500 MHz, d6-DMSO): d = 12.30
6.38 (d, J = 2.3, 2H), 6.26 (dd, J = 8.5, 2.5, 2H), 5.34 (s, 1H), 4.55 (s, 1H), 8.80 (t, J = 5.6, 1H), 8.64 (s, 1H), 7.66 (d, J = 9.0, 1H), 6.78
(s, 2H), 3.31 (q, J = 7.1, 8H), 1.15 (t, J = 7.2, 12H) ppm. 13C NMR (d, J = 9.0, 1H), 6.59 (s, 1H), 3.47 (m, 6H), 2.48 (s, 5H), 1.12 (t, J =
(151 MHz, CDCl3) d = 151.70, 147.82, 144.83, 138.66, 131.48, 7.0, 6H) ppm. 13C NMR (125 MHz, d6-DMSO): d = 173.59,
130.01, 129.69, 128.07, 127.00, 111.46, 107.47, 98.83, 77.35, 162.58, 162.10, 157.68, 152.89, 148.23, 132.04, 110.56, 109.62,
77.14, 76.92, 63.12, 44.48, 40.12, 12.74 ppm.
108.08, 96.29, 44.77, 35.30, 34.36, 12.76 ppm, ESI+-HRMS (m/z):
333.1403 ([M + H]+ calculated: 333.1406).
2.3. Synthesis of RA
2.5. Synthesis of CARA
Compound 1 (86 mg, 0.2mmol) and DDQ (67 mg, 0.3 mmol)
were dispersed in CH2Cl2 (10 mL), and then the mixture was Compound 1 (150 mg, 0.45 mmol) and CA (232 mg, 0.54 mmol)
stirred at room temperature for 4 h. The reaction solution was were dissolved in THF (5 mL). Then, to this above solution,
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2,4,6-trichlorobenzoyl chloride (343 mg, 1.4 mmol) and triethyl- all mixed solutions were recorded using a fluorescence spectro-
amine (101 mg) were added sequentially. After stirring for photometer after incubation for an hour. The concentration
5 min, 4-dimethylaminopyridine (DMAP, 122 mg, 1 mmol) dependence of lipase was measured by adding a certain
was added and the reaction solution was further stirred for amount of lipase to the CARA in PBS (pH 7.2, 2 mL). Then,
30 min. The organic solvents were removed under reduced the fluorescence spectra of CARA were recorded at various
pressure, and the crude product was purified by column concentrations of lipase. The fluorescence emission spectra
chromatography to yield a dark purple solid. The dark purple were recorded from 425 nm to 700 nm with excitation at a
solid (143 mg, 0.2 mmol) and DDQ (67 mg, 0.3 mmol) were wavelength of 405 nm. The UV absorption spectroscopy
dissolved in CH2Cl2 (10 mL) and stirred at room temperature spectrum was recorded from 300 nm to 800 nm.
for 4 h. The reaction solution was washed with water (50 mL Â 3).
2.8. Lipase activity assay in human serum (HS)
The organic phase was dried with anhydrous MgSO4, filtered
and concentrated in vacuo. The crude compound was purified
by column chromatography to give a dark purple solid (CARA,
67 mg, 46.8%). 1H NMR (500 MHz, CDCl3): d = 8.90 (s, 1H), 8.60
(s, 1H), 7.59 (d, J = 21.9, 3H), 7.41 (s, 1H), 7.31 (s, 1H), 7.09
(s, 2H), 6.85 (s, 3H), 6.65 (s, 1H), 6.46 (s, 1H), 4.84 (s, 2H), 3.62
(s, 8H), 3.45 (s, 6H), 2.37 (s, 2H), 1.31 (s, 12H), 1.23 (s, 6H) ppm.
13C NMR (125 MHz, CDCl3): d = 171.12, 163.29, 162.55, 157.70,
155.68, 152.75, 148.07, 133.89, 131.64, 131.21, 130.39, 129.67,
128.92, 114.26, 113.67, 110.15, 109.50, 108.19, 96.61, 96.42,
63.81, 46.24, 45.12, 34.97, 33.99, 12.68, 12.41 ppm, ESI+-
HRMS (m/z): 743.3802 ([M]+ Calculated: 743.3803).
The serum from a volunteer was collected by the Affiliated
Hospital of Qingdao University and informed consent was
obtained for the use of human serum. All experiments were
tested in buffered human serum (HS)/PBS solution (v/v = 1 : 9,
pH 7.2). The response time and selectivity of CARA toward
lipase were tested in the same way as the test in PBS. The
concentration dependence of lipase in human serum was
detected by adding a serious of concentrations of lipase to
the CARA in buffered HS/PBS solution (v/v = 1 : 9, pH 7.2). Then,
their fluorescence spectra were recorded after incubation at
37 1C for 1 h.
2.6. Detection of hydrolysis of CARA by lipase
RA, CA, and CARA were dissolved with DMSO as a stock
solution (1 mM). Then the solution was diluted with PBS to a
final concentration of 100 mM. CARA solution in PBS (100 mM,
2 mL) was incubated with lipase (5 mg mLÀ1) at 37 1C for 1 h.
3. Results and discussion
3.1. Design and detection mechanism of the CARA probe for
lipase
The solutions of CA, RA, CARA, and CARA + Lipase were analyzed Lipase is an interface catalytic hydrolysis enzyme that can
using an Agilent 1260 Infinity II. The chromatographic conditions hydrolyze the ester bonds of fatty acid esters. According to
were as follows: Kromasil 100-5-C18 (4.6 mm  250 mm) column; the function of lipase, we designed and synthesized a ratio-
column temperature: 30 1C; mobile phase: 0.1% trifluoroacetic metric fluorescent probe (CARA) based on FRET for detecting
acid–water and acetonitrile, gradient elute; flow rate: 1 mL minÀ1
detection wavelength: 254 nm.
;
lipase activity. The coumarin fluorophore (CA) and rhodamine
fluorophore (RA), the classic partners of the FRET platform,
were employed as the energy donor and acceptor of CARA,
respectively. The two fluorophores are linked through the fatty
2.7. Lipase activity assay in PBS
CARA was dissolved in DMSO as a stock solution (1 mM). In all acid ester bond that is easily broken by lipase. In addition, CA is
the experiments, the final concentration of CARA in PBS (pH a water-insoluble dye and RA is a water-soluble dye, so they
7.2, 0.01M) was 10 mM and the mixture was incubated at 37 1C. were linked to construct amphipathic molecules. These
The effect of pH was conducted as follows. CARA was added to amphipathic molecules not only enhanced the water-solubility
PBS (2 mL) with different pH values ranging from 5.5 to 8 with of the CARA probe, but also easily formed a micro-interface to
or without lipase (5 mg mLÀ1). After incubation for 1 h, the meet the requirement of the interface catalysis of lipase. CA
fluorescence spectra of the mixture were tested. The response shows strong blue fluorescence from 450 nm to 560 nm (Fig. 1A,
time of CARA towards lipase was tested as follows. CARA in PBS blue solid line) and RA has obvious UV-vis absorbance from
(pH 7.2, 2 mL) was incubated with lipase (0.5 mg mLÀ1) at 37 1C 490 nm to 600 nm (Fig. 1A, red solid line), so there is a great
for different times, and then the fluorescence spectra at a overlap between the fluorescence spectra of CA and the UV-vis
specific time were measured respectively. The selectivity of absorption spectra of RA. The fluorescence of CA is absorbed
CARA toward lipase was tested by adding lipase and other well by RA to emit the red fluorescence of RA. Thus, the free
biological substances. Each of the following biological sub- probe CARA shows strong red fluorescence at 585 nm and weak
stances including Mg2+, Zn2+, BrÀ, Cu2+, Fe2+, NO2À, Fe3+, blue fluorescence at 470 nm upon excitation at 405 nm, which
+
Ca2+, NH4 , S2O32À, IÀ, Phe, Pro, Glu, a-amylase, tryptone, yeast can be attributed to the FRET process between CA and RA. The
extract, BSA and lipase was separately incubated with CARA apparent FRET efficiency of the CARA probe was calculated to be
solution (2 mL), where the concentration of lipase was 88.8% according to a reported method (Fig. S1, ESI†).36 As lipase
5 mg mLÀ1, and the levels of some ions and amino were was added into the CARA solution, the ester bond of CARA was
100 mM, and the concentrations of a-amylase, tryptone, yeast hydrolyzed by lipase to generate CA and RA, which increased the
extract, and BSA were 1 mg mLÀ1. The fluorescence spectra of distance between CA and RA and weakened the FRET effect
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Fig. 1 (A) Normalized fluorescence spectra of a donor (CA) (blue solid line), normalized absorption spectra of an acceptor (RA) (red solid line), and the
spectra overlapping domain (purple zone). (B) The proposed mechanism of CARA for detecting lipase activity.
(Fig. 1B). The fluorescence intensity increases at 470 nm and values (Fig. S2, ESI†). With the variation of pH from 5.5 to 8,
decreases at 585 nm upon excitation at 405 nm. Hence, CARA the fluorescence intensity showed little changes at 585 nm in
could be employed to quantify lipase activity through measuring the presence or absence of lipase, which indicated that the
the fluorescence intensity at 470 nm and 585 nm.
fluorophore RA is stable at various pH values. Under the same
conditions, the fluorescence intensity at 470 nm decreased,
which may be attributed to the protonation of the amino group
of CA. Although the fluorescence ratio (I470 nm/I585 nm) decreased
with the increase in pH, the different values of the fluorescence
3.2. Effect of pH and time
Firstly, the fluorescence intensities of the CARA probe were
measured in the presence or absence of lipase at different pH
Fig. 2 (A) The ratio of the fluorescence intensity (I470 nm/I585nm) of CARA (10 mM) in the presence or absence of lipase in PBS at different pH values from 5.5
to 8.0. (B) The difference value of the fluorescence ratio (I470 nm/I585 nm) between CARA and CARA + lipase in PBS at various pH values from 5.5 to 8.0. (C) The
ratios of the fluorescence intensity (I470 nm/I585 nm) of CARA (10 mM) in the presence or absence of lipase in PBS (pH 7.2) at 37 1C for 0–90 min, which were
obtained at 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, and 90 minutes. (D) The I470 nm/I585 nm ratio of CARA (10 mM) in the presence of lipase and other species
including Mg2+, Zn2+, BrÀ, Cu2+, Fe2+, NO2À, Fe3+, Ca2+, NH4+, S2O32À, IÀ, Phe, Pro, Glu, a-amylase, tryptone, yeast extract, and BSA in PBS (pH 7.2).
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ratio (I470 nm/I585 nm) between CARA and CARA with lipase was not influenced by other species (Fig. 2D, the red column).
change little (Fig. 2B). The results indicated that the CARA These above results indicated that CARA possessed high
probe can respond to lipase activity in a wide pH range. selectivity to lipase and excellent anti-interference capacity to
Subsequently, the kinetics of the CARA probe to lipase was other species.
investigated through recording the fluorescence intensities of
CARA incubated with lipase (3500 U LÀ1) at pH 7.2 for different
3.4. Concentration-dependent detection of lipase
times (Fig. S3, ESI†). As shown in Fig. 2C, the fluorescence ratio
(I470 nm/I585 nm) of the free probe CARA changed little within
90 minutes, indicating that the CARA probe has good stability in
aqueous solution. With the addition of lipase, the fluorescence
ratio (I470 nm/I585 nm) increased with time and changed little after
60 minutes (the red line, Fig. 2C), demonstrating that the
hydrolysis of CARA by lipase reached equilibrium at 60 minutes.
Thus, in the next experiments, the incubation time of CARA is
60 minutes.
To evaluate the sensitivity and linearity of the CARA probe to
lipase activity, the titration experiments were implemented in
PBS solution containing 1% DMSO at pH 7.2. Various units of
lipase were added into the 10 mM CARA solution, and the
fluorescence spectra were recorded upon excitation at 405 nm
after incubation for 60 min. As shown in Fig. 3A, the free probe
CARA showed two fluorescence emission peaks at about
470 nm and 585 nm. Along with lipase concentration increased,
the fluorescence emission of the donor group (CA) increased
gradually at 470 nm and the fluorescence emission of the
receptor group (RA) decreased at 585 nm. The fluorescence
ratio (I470 nm/I585 nm) increased gradually from 1.77 to 9.10 with
the change of lipase from 0 to 35 000 U LÀ1 (0–5 mg mLÀ1). The
CARA probe showed a good linearity between the fluorescence
intensity ratio and lipase concentration, which fitted to the
equation y = 3.30 Â 10À4 Â [lipase] + 2.3521 with R2 = 0.999
(Fig. 3B). The limit of detection (LOD) is estimated to be 48.5 U LÀ1
based on 3d/k (d is the standard deviation of blank measurements,
n = 10, and k is the slope of the linear equation). Thus, CARA
can be used to detect lipase activity in PBS solution quantitatively.
Both UV absorbances at 420 nm and 566 nm of CARA changed
slightly in the presence of lipase as compared to that of free CARA,
which indicated that neither the CA conjugated system nor the RA
conjugated system has changed before and after adding lipase
(Fig. S5, ESI†). Thus, the UV absorbance further suggested that the
fluorescence change was attributed to ester bond cleavage rather
than structure changes of the CA or RA moiety, which was further
confirmed by HPLC and HRMS spectra (Fig. S6, ESI†).
3.3. Selectivity of CARA to lipase
Selectivity is a crucial parameter to evaluate the quality of a
fluorescent probe. To test the selectivity of CARA, we measured
the fluorescence of CARA in the presence of different species
including lipase (35 000 U LÀ1), Mg2+, Zn2+, BrÀ, Cu2+, Fe2+,
NO2À, Fe3+, Ca2+, NH4 , S2O32À, IÀ, Phe, Pro, Glu, a-amylase,
+
tryptone, yeast extract, and BSA (Fig. S4A, ESI†). As lipase was
added into CARA solution, the fluorescence intensity enhanced
2 fold at 470 nm and fell by half at 585 nm upon excitation at
405 nm. The fluorescence ratio (I470 nm/I585 nm) of CARA with
lipase increased 4 fold compared with that of free probe CARA
(Fig. 2D, the black column). Upon addition of other species,
both the fluorescence intensity of CARA at 470 nm and 585 nm
changed slightly, and the fluorescence ratio (I470 nm/I585 nm
)
floated barely. These results exhibit that CARA permits specific
response to lipase over other species. Subsequently, we evaluated
the anti-interference capacity of CARA through recording the
fluorescence intensity of CARA with the addition of lipase and
other different species. The fluorescence intensity of CARA still
increased at 470 nm and decreased at 585 nm in the presence of
3.5. Application in the detection of serum lipase activity
both lipase and other different species, which was consistent Serum lipase activity is a key biological indicator for diagnosing
with the fluorescence change of CARA with only lipase (Fig. S4B, acute pancreatitis in the early stage, so we evaluated the
ESI†). The fluorescence ratio (I470 nm/I585 nm) of CARA with lipase feasibility of the CARA probe for detecting lipase activity in
Fig. 3 (A) Fluorescence spectra of CARA (10 mM) in PBS buffer (pH 7.2, 0.01M) with different concentrations of lipase. (B) Relationship between I470 nm/I585 nm of
CARA and the concentrations of lipase from 0 to 35 000 U LÀ1 and a linear relationship between I470 nm/I585 nm and [lipase] from 0 to 8750 U LÀ1 (inset). The
results are presented as means Æ SD with three replicates (n = 3).
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Fig. 4 (A) Fluorescence spectra of CARA (10 mM) in buffered HS/PBS solution (v/v = 1 : 9, pH 7.2) with different concentrations of lipase. (B) Relationship
between I480 nm/I593 nm of CARA and the concentrations of lipase from 0 to 35 000 U LÀ1 and a linear relationship between I480 nm/I593 nm and [lipase] from
0 to 4375 U LÀ1 (inset). The results are presented as means Æ SD with three replicates (n = 3).
human serum samples. The fluorescence intensity of CARA was lipase at 37 1C for 1 h in HS/PBS solution, the fluorescence
measured in the mixture of human serum (HS) and phosphate intensity enhanced gradually at 480 nm and weakened
buffer solution (HS/PBS, containing 10% HS) in the absence or obviously at 593 nm with the concentration of lipase, and a
presence of exogenous lipase. As shown in Fig. 4A, when the good linear relationship was built between the fluorescence
CARA probe was incubated with various concentrations of intensity ratio (I480 nm/I593 nm) and lipase concentration ranging
Fig. 5 (A) Fluorescence spectra of CARA (10 mM) toward lipase and other biological substances including Mg2+, Zn2+, BrÀ, Cu2+, Fe2+, NO2À, Fe3+, Ca2+
,
NH4+, S2O32À, IÀ, Phe, Pro, Glu, a-amylase, tryptone, yeast extract, and BSA in HS/PBS (v/v = 1 : 9). (B) Fluorescence spectra of CARA (10 mM) toward the
coexistence of lipase and other biological substances including Mg2+, Zn2+, BrÀ, Cu2+, Fe2+, NO2À, Fe3+, Ca2+, NH4+, S2O32À, IÀ, Phe, Pro, Glu, a-amylase,
tryptone, yeast extract, and BSA in HS/PBS (v/v = 1 : 9). (C) The relative ratio of I480 nm/I593 nm of CARA (10 mM) in the presence of lipase and other species
including Mg2+, Zn2+, BrÀ, Cu2+, Fe2+, NO2À, Fe3+, Ca2+, NH4+, S2O32À, IÀ, Phe, Pro, Glu, a-amylase, tryptone, yeast extract, and BSA in HS/PBS (v/v = 1 : 9).
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from 0 to 4500 U LÀ1 with R2 = 0.995 (Fig. 4B). The LOD based
3d/k is estimated to be 69.88 U LÀ1. The CARA probe with a low
LOD has potential for distinguishing normal and acute
pancreatitis patient serum, because the serum lipase activity
of acute pancreatitis patients is three times higher than that
(190 U LÀ1) of normal people. Then, we tested the selectivity of
CARA toward lipase in serum samples. As shown in Fig. 5A and
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the black column of Fig. 5C, the fluorescence ratio (I480 nm
/
I593 nm) of CARA with lipase increased about 2 fold compared
with that of free CARA or CARA with other species. In addition,
the results of the interference experiments (Fig. 5B and the red
column of Fig. 5C) showed that the fluorescence ratio of CARA
with lipase was hardly influenced by the co-existent species.
These results indicate that CARA also permits a good selectivity
to lipase and show a high anti-interference capacity in the
sample containing serum. Compared with other fluorometric
methods for lipase activity (Table S1, ESI†), the novel probe
CARA that is a ratiometric fluorescent probe can effectively
alleviate the interference from the variations in environments
such as probe concentration and excitation intensity. These
results suggest that probe CARA can be used to detect serum
lipase activity and may have potential usage for clinical
diagnosis of pancreatitis.
12 Y. Choi, S. H. Shin, H. Jung, O. Kwon, J. K. Seo and J. M. Kee,
ACS Sens., 2019, 4, 1055–1062.
13 L. Dong, S. Shen, H. Lu, S. Jin and J. Zhang, ACS Sens., 2019,
4, 1222–1229.
14 L. Feng, Y. Yang, X. Huo, X. Tian, Y. Feng, H. Yuan, L. Zhao,
C. Wang, P. Chu, F. Long, W. Wang and X. Ma, ACS Sens.,
2018, 3, 1727–1734.
4. Conclusions
15 S. Y. Liu, H. Xiong, J. Q. Yang, S. H. Yang, Y. Li, W. C. Yang
and G. F. Yang, ACS Sens., 2018, 3, 2118–2128.
16 J. Ou-Yang, Y. F. Li, P. Wu, W. L. Jiang, H. W. Liu and
C. Y. Li, ACS Sens., 2018, 3, 1354–1361.
17 Z. Qiao, H. Zhang, Y. Zhang and K. Wang, iScience, 2020,
23, 101294.
In this study, we developed a novel FRET probe CARA for the
detection of lipase activity based on the coumarin–rhodamine
platform. CARA that works in an almost aqueous solution is an
environmentally friendly approach for the detection of lipase
activity, and it can be directly used to measure human serum
lipase activity without further processing. CARA exhibits an
18 Z. Shen, J. Xia, Q. Ma, W. Zhu, Z. Gao, S. Han, Y. Liang,
J. Cao and Y. Sun, Theranostics, 2020, 10, 9132–9152.
19 S. Ma, G. Chen, J. Xu, Y. Liu, G. Li, T. Chen, Y. Li and
T. D. James, Coord. Chem. Rev., 2021, 427, 213553.
20 L. Zhu, L. Shan, J. Zhu, L. Li, S. Li, L. Wang, J. Wang,
S. Zhang, H. Zhou, W. Zhang and H. Li, Sci. China: Life Sci.,
2020, 63, 1016–1025.
excellent linearity between the fluorescence ratio (I480 nm/I593 nm
)
and lipase concentration, and shows higher selectivity to lipase
than other species. Because CARA also can detect serum lipase
activity, it has potential for the clinical diagnosis of pancreatitis.
Conflicts of interest
21 A. Jiang, Y. Liu, G. Chen, Y. Li and B. Tang, Chem. Sci., 2020,
11, 1964–1974.
The authors declare no competing financial interest.
22 B. An, H. Zhang, J. Peng, W. Zhu, N. Wei and Y. Zhang, New
J. Chem., 2020, 44, 20253–20258.
23 J. Shi, Q. Deng, C. Wan, M. Zheng, F. Huang and B. Tang,
Chem. Sci., 2017, 8, 6188–6195.
24 J. Shi, S. Zhang, M. M. Zheng, Q. C. Deng, C. Zheng, J. Li and
F. H. Huang, Sens. Actuators, B, 2017, 238, 765–771.
25 X. M. Hou and W. Cheng, Biomed. Opt. Express, 2012, 3, 340–353.
26 O. I. Kolenc and K. P. Quinn, Antioxid. Redox Signaling, 2017,
30(6), 875–889.
Acknowledgements
The project is supported by the research grants awarded to W. Z.
from the National Key Research and Development Program
(2018YFA019500) and to N. N. Wei from the National Natural
Science Foundation of China (81703579) and the Shandong
Provincial Natural Science Foundation of China (ZR2017BH082).
27 O. N. Shilova, E. S. Shilov and S. M. Deyev, Cytometry, Part A,
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