Fabrication of a water-soluble near-infrared fluorescent probe for selective detection and imaging of dipeptidyl peptidase IV in biological systems

Article abstract of DOI:10.1039/c9tb02301a

Dipeptidyl peptidase IV (DPP-IV) is a transmembrane glycoprotein known to regulate T cell activation, which is related to various pathological processes and has become a potential target to treat type 2 diabetes mellitus. Therefore, it is significant for the evaluation of endogenous DPP-IV activity in various biological systems. Herein, a water-soluble near-infrared (NIR) fluorescent probe HCA-D based on cyanine dyes as the fluorophore and glycyl-prolyl peptide as the specific recognition sequence was developed for the assay of dipeptidyl peptidase IV (DPP-IV) activity. Upon addition of DPP-IV, HCA-D can emit a significant turn-on NIR fluorescence signal under physiological conditions and exhibit high selectivity toward DPP-IV. This feature was available for quantifying DPP-IV in the range from 0.62 to 10 ng mL^-1 with a detection limit of 0.19 ng mL^-1. Furthermore, the present probe was successfully employed for monitoring DPP-IV in serum samples from diabetic and healthy people, and imaging of DPP-IV in living cells and tumor mice models. These results demonstrate that the designed probe provides a promising tool to explore the relationship between DPP-IV and diabetes mellitus or other diseases. Perhaps, it may become a prospective image-guided tumor resection indicator based on the abnormal expression of DPP-IV activity in the future.

Full text of DOI:10.1039/c9tb02301a

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Page 1 of 22
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Fabrication of a water-soluble near-infrared fluorescent
probe for selective detection and imaging of dipeptidyl
peptidase IV in biological system
a
a
a
b
a
a
Xiumei Guo , Shuai Mu , Jian Li , Yintang Zhang , Xiaoyan Liu , Haixia Zhang ,
Hong Gaoa*
aState Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metals
Chemistry and Resources Utilization of Gansu Province and College of Chemistry and
Chemical Engineering, Lanzhou University, Lanzhou 730000, China.
bHenan Key Laboratory of Biomolecular Recognition and Sensing, College of Chemistry and
Chemical Engineering, Shangqiu Normal University, Shangqiu 476000, China.

Corresponding author Tel: +86-931-8912510; fax: +86-931-8912582;
E-mail: liuxiaoy@lzu.edu.cn
1

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Abstract
Dipeptidyl peptidase IV (DPP-IV) is known as a transmembrane glycoprotein to
regulate T cell activation, which is related to various pathological processes and has
become a potential target to treat type 2 diabetes mellitus. Therefore, it is significant
for the evaluation of endogenous DPP-IV activity in various biological system. Herein,
a water-soluble near-infrared (NIR) fluorescent probe HCA-D based on Cyanine dyes
as the fluorophore and glycyl-prolyl peptide as the specific recognition sequence was
developed for assay of dipeptidyl peptidase IV (DPP-IV) activity. Upon addition of
DPP-IV, HCA-D can emit significant turn-on NIR fluorescent signal under
physiological conditions and exhibit high selectivity toward DPP-IV. This feature was
available for quantifying DPP-IV in the range from 0.62 to 10 ng/mL with detection
limit of 0.19 ng/mL. Furthermore, the present probe was successfully employed for the
monitoring DPP-IV in serum samples from the diabetics and healthy people, imaging
of DPP-IV in living cells and tumor mice models. These results demonstrate that the
designed probe provides a promising tool to explore the relationship between the DPP-
IV and the diabetes mellitus or other diseases. Perhaps, it may become a prospective
image-guided tumor resection indicator based on the abnormal expression of DPP-IV
activity in the future.
Keywords: Near Infrared, Fluorescent Probe, Dipeptidyl Peptidase IV, Bioimaging
1
. Introduction
Studies have shown that the clinical measurement of tumor-related biomarkers is
1
of great significance for the diagnosis, treatment and management of tumors . Among
many potential biomarkers, enzymes are gaining increased attention as they have
important roles in many physiological, pathological, and pharmacological processes2.
3
4
For example, caspase (Cas) , leucine aminopeptidase (LAP) , dipeptidyl peptidase
5
6
7, 8
(
DPP) , aminopeptidase N (APN) and nitroreductase have been consider as potential
indicators of various specific diseases including cancer. Among them, Dipeptidyl
2

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peptidase IV (DPP-IV, CD 26), a transmembrane glycoprotein with proteolytic activity,
is capable of cleaving N-terminal dipeptides from polypeptides with proline or alanine
in the penultimate position9, 10 and plays a considerable biomedical interest due to an
association between the upregulated of DPP-IV with some diseases11, 12. In recent years,
with the diversification of people’s dietary rules, the number of people with diabetes is
increasing. Owing to its popular expression and proteolytic activity, DPP-IV is also
1
3
involved in the regulation of glucose metabolism processes . Furthermore, some
evidences show that DPP-IV inhibition, as an effective therapy for type 2 diabetes,
exerts positive effects by degrading incretins, and control of blood glucose levels 14, 15
.
Thus, the accurate measurement of DPP-IV activity in complex biological samples,
especially in plasma, will be a great significance for disease diagnosis, drug discovery
and clinical practice.
Up to date, the activity of DPP-IV has been assayed using chromogenic substrates,
1
6
17
such as glycyl-proline b-naphthylamide , glycyl-proline p-nitroanilide
and
nonpeptide-based small-molecule fluorescent probes18-20. Although these probes are
commonly available, the most of them have poor aqueous solubility and usually require
organic solvents such as acetonitrile 21, 22 and DMSO as solubilizer. However, the use
of organic co-solvents in enzymatic assays for DPP-IV may lead to unreliable results.
For example, the DPP-IV activity has been reported to be reduced to 50% by modulated
in 1% DMSO 18, 19. Obviously, it is an unneglectable limiting factor for the practical
application in biological system. Therefore, it is imminent to develop a near-infrared
fluorescent probe with favorable water solubility for real-time tracking DPP-IV activity
in biological sample or vivo.
In the present study, we aimed at developing a water-soluble small-molecular
probe (HCA-D) for near-infrared fluorescence (NIRF) specific detection and imaging
of DPP-IV in biological system. Herein, in order to endow the probe with both water
solubility and availability, a cyanine skeleton containing a sulfonic acid group (HCA)
3

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was constructed and synthesized via multi-step reactions. Subsequently, the fit-for-
2
3
purpose probe for specific recognition of DPP-IV , HCA-D is prepared by covalently
linking the amino group of HCA with DPP-IV-responsive unit (a dipeptide consisting
of proline and alanine, glycylprolyl (Gly-Pro) group) (Scheme 1). Finally, based on the
specific interaction between DPP-IV and the responsive unit of HCA-D, the proposed
probe was applied to detect DPP-IV activity in living cells (Raw264.7 macrophage) and
living animals, respectively. To the best of our knowledge, this is the first report of a
water-soluble small-molecular probe for bioimaging of DPP-IV in vitro/vivo.
Scheme 1. Proposed response mechanism of HCA-D for DPP-IV
2
2
. Experimental Section
.1 Reagents and Materials
2
,3,3-trimethyl-3H-indole and Phosphorous oxychloride were obtained from J&K
Chemical Ltd. (Beijing, China). 1,3-propane sultone, Potassium tert-butoxide, Sodium
acetate, Acetic anhydride, (S)-1-(2-(tert-butoxycarbonylamino)acetyl)pyrrolidine-2-
carboxylic acid (Boc-Gly-Pro-OH), O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-
tetramethyluronium hexafluorophosphate (HATU), N,N-Diisopropylethylamine
(
DIPEA), 3-Nitrophenol, SnCl -2H O and CF COOH were obtained from Saan
2 2 3
Chemical Technology (Shanghai) Co., Ltd. DPP-IV was obtained from Kaiwei
Yicheng Technology (Beijing) Co., Ltd. Sitagliptin was purchased from Shanghai
Yuanye Bio-Technology (Shanghai, China).
2
.2 Instruments
4

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1
13
H NMR and C NMR spectra were recorded using a JEOL400 MHz instruments
(
Japan). Mass spectra were measured using Brukermicr OTOF II with ESI mode
America). Absorption spectra were measured on a UV-visible spectrophotometer (TU-
(
1
5
810, China). Fluorescence spectra were recorded on a Fluorescence Spectrometer (RF-
301pc, Japan) with a Xenon lamp. Cell imaging were performed by Confocal
fluorescence microscopy (FV1000, Olympus, Japan) and Living animal imaging were
carried out with IVIS Spectrum (Canada) equipped with molecular imaging software
5
2
2
.0.6.20.
.3 Synthesis of HCA-D
.3.1 Synthesis of compound 1
1
,3-propane sultone (2.3 g, 18.8 mmol) and 2,3,3-trimethyl-3H-indole (3.0 g, 18.8
mmol) were dissolved in 100 mL of toluene and refluxed for 24 h. The resulting
production was filtrated and washed with petroleum ether and a purple-red powder
(Compound 1) was obtained.
2
.3.2 Synthesis of compound 2
To a chilled solution of DMF (20 mL, 273 mmol, 5.5 eq) in 20 mL CH Cl under
2
2
N atmosphere, 20 mL of POCl (17.5 mL, 115 mmol, 2.3 eq) in DCM were dropwise
2
3
added under an ice bath. After 30 min, cyclohexanone was added (5 g, 50 mmol, 1 eq),
and the resulting mixture was refluxed with vigorous stirring at 80 °C for 3 h, poured
into ice-cold water, and kept it overnight to obtain compound 2 as a yellow solid (8.0
g, 90%).
2
.3.3 Synthesis of compound CySO Cl
3
Compound 1 (281 mg, 1 mmol) and compound 2 (86 mg, 0.5 mmol) were
dissolved in 7 mL of acetic anhydride, then added sodium acetate (82 mg, 1 mmol).
After stirring at room temperature for 1-2 h, the product was processed by filtration and
washing with petroleum ether to give a green solid, which was purified on silica gel
1
(
CH Cl / MeOH) to obtain compound CySO Cl. H NMR (400 MHz, DMSO-d6 ) δ =
2
2
3
5

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8
.26 (d, J = 14.1 Hz, 2H), 7.61 (d, J = 7.1 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 7.46 - 7.38
(m, 2H), 7.27 (t, J = 7.6 Hz, 2H), 6.52 (d, J = 14.2 Hz, 2H), 4.42 - 4.33 (m, 4H), 2.74
(t, J = 5.7 Hz, 4H), 2.57 (t, J = 6.7 Hz, 4H), 2.09 - 1.97 (m, 4H), 1.87 - 1.78 (m, 2H),
1
3
1
1
2
2
.67 (s, 12H). C NMR (101 MHz, DMSO-d ) δ =172.24, 148.02, 143.24, 142.13,
6
41.16, 128.65, 126.60, 125.09, 122.48, 111.58, 101.99, 48.97, 47.81, 42.89, 27.54,
6.02, 23.49, 20.58.
.3.4 Synthesis of compound HCA
3
-Nitrophenol (200 mg, 1.438 mmol), and potassium tert-butoxide (161 mg, 1.438
mmol) were dissolved in anhydrous DMF (5 mL). The mixture was stirred at 25 °C for
3
0
0 min under a nitrogen atmosphere. Then, a solution of compound CySO Cl (345 mg,
3
.48 mmol) in anhydrous DMF (3 mL) was added to the mixture via handheld syringe,
which was further stirred for 6 h. The solvent was removed under reduced pressure,
then the crude product was purified on silica gel (CH Cl /MeOH) to give compound
2
2
1
HCA. H NMR (400 MHz, DMSO-d ) δ = 8.43 (d, J = 14.1 Hz, 1H), 7.69-7.61 (m, 2H),
6
7
.55 (d, J = 8.0 Hz, 1H), 7.47-7.36 (m, 2H), 7.30 (t, J = 7.4 Hz, 1H), 6.88 (s, 2H), 6.73
dd, J = 8.6, 1.9 Hz, 1H), 6.67 (s, 1H), 6.47 (d, J = 14.2 Hz, 1H), 4.39 (t, J = 7.4 Hz,
H), 2.69 (s, 4H), 2.59 (t, J = 6.6 Hz, 2H), 2.09-1.98 (m, 2H), 1.85-1.75 (m, 2H), 1.70
(
2
1
3
(
s, 6H). C NMR (101 MHz, DMSO-D ) δ = 173.99, 162.85, 156.15, 155.49, 142.51,
6
1
1
2
2
42.34, 141.50, 138.73, 130.29, 130.18, 129.19, 125.68, 123.04, 114.96, 114.88,
13.29, 112.19, 101.25, 97.83, 52.92, 49.61, 49.12, 48.31, 43.40, 28.46, 24.27, 23.97,
0.84.
.3.5 Synthesis of compound HCA-D
Boc-Gly-Pro-OH (218 mg, 0.8 mmol), HATU (228 mg, 0.6 mmol) and DIPEA
(129 mg, 165 μL, 1 mmol) were dissolved in 3 mL of anhydrous DMF, and the solution
was stirred at 0 °C under nitrogen atmosphere for 60 min. Then, a solution of compound
HCA (100 mg, 0.2 mmol) in anhydrous DMF (3 mL) was added dropwise into the
mixture, and the reaction mixture was stirred overnight at 0 °C to room temperature.
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The solvent was removed by reduced pressure, and the crude product was purified by
silica gel chromatography (CH Cl /MeOH from 30:1 to 5:1) to get the product HCA-
2
2
D-Boc.
HCA-D-Boc (64.4 mg, 0.1 mmol) was dissolved in DCM (3 mL) and TFA (1 mL).
The resulting mixture was stirred at room temperature for 6 h. The solvent was
evaporated, and then the residue was purified by silica gel chromatography with CH Cl2
2
1
/
MeOH (1:1) to get the final product HCA-D. H NMR (400 MHz, DMSO-d ) δ=10.69
6
(s, 1H), 8.49 (d, J = 14.9 Hz, 1H), 7.90 (s, 1H), 7.79 – 7.71 (m, 2H), 7.53 – 7.47 (m,
3
4
6
1
1
4
2
H), 7.45 – 7.38 (m, 2H), 6.78 (d, J = 14.9 Hz, 1H), 4.55 (s, 2H), 3.85 (s, 2H), 2.68 (s,
H), 2.59 (t, J = 6.4 Hz, 2H), 2.07 (s, 2H), 2.00 – 1.88 (m, 3H), 1.78 (s, 2H), 1.71 (s,
1
3
H), 1.19 (s, 6H). C NMR (101 MHz, DMSO-D ) δ =178.13, 171.42, 165.21, 160.56,
6
58.73, 153.45, 145.64, 142.75, 142.59, 141.93, 129.43, 128.62, 127.64, 123.37,
19.30, 117.72, 117.01, 116.31, 114.91, 113.87, 113.33, 105.80, 105.53, 61.10, 50.93,
8.04, 46.65, 44.53, 30.10, 29.09, 27.86, 27.81, 24.88, 24.46, 20.47.
.4 General procedures for DPP-IV activity assay
All spectrum measurements were performed in PBS buffer (10 mM, pH 7.4) at 37
°
C. Various physiologically important species (150 mM KCl; 150 mM NaCl; 100 μM
ZnCl ; 2.5 mM MgCl ; 100 μM CuSO ; 1.0 mM Vitamin C; 5.0 mM glutathione; 1.0
2
2
4
mM cysteine; 500 μg/mL human serum albumin (HSA); 10 μg/mL trypsin; 10 μg/mL
pepsase; 10 μg/mL lysozyme; 30 μg/mL Tyrosinase; 10 μg/mL Alkaline phosphatase;
1
0 μg/mL Chymotrypsin; 10 μg/mL Compound proteinase; 10 μg/mL Alkaline protease;
0 μg/mL Glucose Oxidase; 10 μg/mL catalase lipase; 800 ng/mL Dipeptidyl
1
peptidases 8 (DPP 8); 1000 ng/mL fibroblast activation protein (FAP) and 40 ng/mL
DPP-IV) were prepared in distilled water. The test solution was prepared according to
the following procedure: 20 μL of stock solution of probe HCA-D (1 mM),
corresponding volume of PBS buffer solution, and appropriate volume of the solution
of DPP-IV or other substances were added and mixed in a 2 mL test tube. Subsequently,
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the volume of the mixture was adjusted to 1.0 mL by PBS buffer solution. After
incubation at 37 °C for 30 min, the UV-vis absorption spectra data was recorded from
4
6
2
00 to 800 nm, and the fluorescence measurements were operated in the range from
50 to 800 nm (λ = 640 nm, λ = 690 nm, slit width: 5/5 or 10/5 nm).
ex
em
.5 Selectivity of HCA-D for DPP-IV
The probe HCA-D (20 μM) were mixed with DPP-IV (40 ng/mL) and a series of
physiologically important species (KCl, NaCl, ZnCl , MgCl , CuSO , vitamin C,
2
2
4
glutathione, cysteine, HSA, trypsin, pepsase, lysozyme, Tyrosinase, Alkaline
phosphatase, Chymotrypsin, Compound proteinase, Alkaline protease, Glucose
Oxidase, catalase, lipase, DPP 8 and FAP), respectively. Then, the volume of the mixed
solution was adjusted to 1.0 mL with PBS buffer. Afterwards, the fluorescence
measurements were measured as described above procedure.
2
.6 Quantitative detection of DPP-IV
The probe HCA-D (20 μM) were mixed with various concentrations of DPP-IV
(0.0, 0.625, 1.25, 2.5, 5.0,10.0, 20.0, 30.0, 40.0, 50.0, 60.0, 80.0, 100.0 ng/mL) and
incubated at 37 °C in a water bath. After 30 min, the fluorescence intensity was
determined at 690 nm with an excitation at 640 nm.
2
.7 Assay of DPP-IV activity in human serum samples
Human serum samples from diabetes patients and health people were collected by
the clinical laboratory in Gansu Provincial People’s Hospital laboratory and were not
further processed. The assay was directly performed by 1.0 mL of mixture containing
2
0 μL of 1.0 mM probe, 10 μL of blank or spiked (at DPP-IV level of 2.0, 5.0, 8.0
ng/mL) serum sample and 970 μL of 10 mM PBS buffer solution.
2
.8 Inhibition of DPP-IV
DPP-IV (5.0 ng/mL) and the different concentrations of inhibitor sitagliptin (0, 1.0,
.0, 10.0, 20.0, 50.0, 100.0, 150.0, 200 and 300 nM) were mixed and incubated in a
5
water bath thermostatically regulated at 37 °C for 30 min. Then the mixture was excited
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at a wavelength of 640 nm, and fluorescence emission intensity was recorded at 690
nm.
2
.9 Cell culture and cytotoxicity Assay
Raw 264.7 cells provided by Cell Bank of Shanghai Institute of Biochemistry and
Cell Biology, Chinese Academy of Sciences (Shanghai, China), were grown in RPMI
640 medium supplemented with 10% fetal bovine serum at 37 °C in a 5% CO /95%
1
2
air incubator. The cytotoxicity of the probe to Hela cells was evaluated through the
methyl thiazolyl tetrazolium (MTT) assays. The cells were initially seeded into 96-well
4
plate at a density of 1×10 cells/well, and various concentrations (0.0, 2.5, 5.0, 10.0,15.0,
2
0.0, 25.0, 30.0 μM) of the probe in 200 μL DMEM mediums were added into each
well. After incubation for 24 h, 0.02 mL of the MTT (0.5 mg/mL) were diluted in the
plates and followed by culture for another 4.0 h. Then the formed formazan was
dissolved after placing 100 μL DMSO in the wells. Afterwards, the cell viability rate
was calculated by measuring the absorbance values at 490 nm with ELISA reader
(
Spectra Max Plus 384, Molecular Devices, Sunnyvale, CA).
.10 Fluorescence Imaging of DPP-IV in the living cells and tumor-bearing
mice model
Raw 246.7 cells were grown in RPMI 1640 medium, supplemented with 10% fetal
bovine serum at 37 °C in a 5% CO / 95% air incubator. Cells were treated with HCA-D
2
2
(10 μM) for 60 min, then images were performed under a confocal microscope (Leica
SP8, Germany). The excitation wavelength was set at 635 nm, and fluorescence
emission windows of 650-730 nm were used.
Kunming mice were feed by the medical center of Lanzhou University. All in vivo
experiments are approved by the Laboratory Animal Health Association. The hepatic
tumor-bearing mice models were constructed by subcutaneously injecting H22
6
hepatoma cells (2×10 cells) on their right flanks. Simultaneously, the health mice and
tumor-bearing mice models were feed in the same feeding environment. After about
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3
two weeks, the tumor grew to about 100 mm . Subsequently, the fur in the tumor region
was removed by using 8% Na S aqueous solution and the mice were anaesthetized by
2
intraperitoneal injection with 3.5% chloral hydrate (45 mg/kg). Then 200 μL of HCA-
D (10 μM) PBS solution (pH=7.4) was intratumorally injected into the tumor-bearing
mice. Meanwhile, the control groups were also treated by the probe HCA-D in the same
region. Furthermore, the fluorescence signal of the health and tumor-bearing mice were
observed at 0-2 h post injection on Carestream In-Vivo FX Professional Imaging
System (New Haven, CT) equipped with fluorescent filter sets (excitation/emission,
6
40/700 nm) Professional Imaging System (Canada, CT) equipped with fluorescent
filter sets (excitation/emission, 635/700 nm).
3
3
Results and discussion
.1 Synthesis and Spectral Characterization of HCA-D
All synthesis steps of probe are provided in Figure.S1 (in the Electronic
Supplementary Information, ESI), and the characterization results of HCA-D was
1
13
identified by H-NMR, C-NMR and HRMS. (Figure.S2-S9).
3
.2 Spectral properties of probe HCA-D
In general, the idealized probe should be highlighted on the trends in spectral
changes induced by analytes. Figure 1 shows that the fluorescence and UV-vis spectra
of HCA-D are obviously different before and after reaction with DPP-IV. As shown in
Figure.1A, a desirable emission band centered at 690 nm emerges in the fluorescence
spectrum of HCA-D in the presence of DPP-IV. Meanwhile, the absorbance bands of
HCA-D induced by DPP-IV also undergo a distinct hyperchromic shift along with a
visible change in the color of the solution from bluish violet to light blue (see the inset
of Figure 1B). Notably, the spectral profiles of both HCA and HCA-D caused by DPP-
IV have no obvious difference and the final color of the HCA-D induced by DPP-IV is
similar with that of HCA. These observations imply the probable formation of HCA in
the reaction system, thereby supporting its feasibility for the detection of DPP-IV
1
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activity.
Figure 1. The fluorescence emission spectra (A) and UV/Vis absorption spectra (B) of HCA
20 μΜ, black line), HCA-D (20 μΜ) before (red line) and after (blue line) reaction with DPP-IV
(
(40 ng/mL) in PBS buffer of pH 7.4 at 37 °C for 30 min. Inset of photos are HCA-D (left), HCA
and HCA-D after (right) reaction with DPP-IV under natural light.
3
.3 The selectivity of HCA-D for DPP-IV detection
While DPP-IV detection could be plagued by the physiological substances, the
selectivity of HCA-D was further investigated. As shown in Figure 2, the slight
difference in response intensity of fluorescence can be observed upon the addition of
Human serum albumin (HSA) and fibroblast activation protein (FAP), respectively.
Meanwhile, a negligible change in fluorescence signal responses upon addition of other
common substances. In contrast to the prominent fluorescence enhancement caused by
low concentration DPP-IV (40 ng/mL), the results illuminate that HCA-D has a high
selectivity for detection of DPP-IV activity.
1
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Figure 2. Fluorescence spectra and fluorescence intensity changes of HCA-D (20 μΜ) upon
interaction with DPP-IV or other substances in PBS buffer (10 mM, pH = 7.4). (a) 40 ng/mL DPP-
IV; (b) 150 mM KCl; (c) 150 mM NaCl; (d) 100 μM ZnCl ; (e) 2.5 mM MgCl ; (f) 100 μM CuSO ;
2
2
4
(g) 1.0 mM Vitamin C; (h) 5.0 mM Glutathione; (i) 1.0 mM Cysteine; (j) 500 μg/mL HSA; (k) 10
μg/mL Trypsin; (l) 10 μg/mL Pepsase; (m) 10 μg/mL Lysozyme; (n) 30 μg/mL Tyrosinase; (o)10
μg/mL Alkaline phosphatase; (p) 10 μg/mL Chymotrypsin; (q) Compound proteinase; (r) 10 μg/mL
Alkaline protease; (s) 10 μg/mL Glucose Oxidase; (t) 10 μg/mL Catalase lipase; (u) 10 μg/mL
Lipase. (v) 800 ng/mL Dipeptidyl peptidases 8 (DPP 8); (w) 1000 ng/mL fibroblast activation
protein (FAP). The fluorescence excitation spectra were detected in PBS buffer (10 mM, pH = 7.4)
at 37 °C for 30 min. λ = 640 nm, λ = 690 nm. Error bars represent standard deviation (n = 3).
ex
em
3
.3 Response Mechanism
From a theoretical design perspective, the reaction product of HCA-D and DPP-
IV is a pivotal evidence for the insight into sensing mechanism. Figure S9 and Figure
S10 illustrate the HRMS spectral of the HCA-D and the corresponding products of
HCA-D (20 μM) and DPP-IV (with low (5 ng/mL) and high (40 ng/mL) concentration),
respectively. As expected, the peak at m/z 491.1977 derives from HCA (Figure.S5)
appears in HRMS spectrum of the reaction mixture and its relative abundance increased
with the incremental addition of DPP-IV. More importantly, a concomitant decreasing
and even disappearing the probe HCA-D peak at m/z 645.2729 is also observed. These
data validate the product of HCA-D and DPP-IV is HCA. In this context, the spectral
properties of HCA-D and HCA may account for the response mechanism (Figure1).
Herein, the enhancement fluorescence intensity of HCA-D induced by DPP-IV should
be ascribed to the strong donor ability of amino group (HCA) compared with that of
glycylprolyl (Gly-Pro) group, illuminating that intramolecular charge transfer (ICT) of
HCA is mainly luminescent mechanism. Furthermore, we conducted a preliminary
theoretical calculation of HCA-D and the resultant product HCA. On the basis of the
respective optimal ground state and excited state geometry of the HCA-D and HCA,
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Figure S11 displays the lower HOMO-LUMO orbital energy gap of HCA compared
with that of HCA-D. Additionally, relative to HCA-D, a bathochromic shift of HCA is
evidently observed in the fluorescence emission and UV/Vis absorption spectra
2
4
(
Figure1). These lines of evidence reflect the ICT luminescent mechanism of HCA .
3
.5 Effect of solvent on the activity of DPP-IV
As mentioned above, the activity of enzyme may be affected in the organic co-
solvent system. Figure S12 reveals that a clear decline in fluorescence intensity of
HCA-D induced by DPP-IV in 2% DMSO-reacting system compared to that in PBS
buffer solution, indicating that the use of DMSO is a significant factor associated with
adverse effect on DPP-IV activity. It is to say, DPP-IV activity is sensitive to the DMSO
or other organic solvent. As shown in Figure S12, a critical threshold for complete
deactivation of DPP-IV is caused by about 20% DMSO. These data further confirm that
the main line of research in synthesis of a water-soluble probe is a crucial point on the
way to accurate assay of DPP-IV activity.
3
.6 Optimization of experimental conditions
In any case, it is essential to optimize the response conditions of the probe before
performing the following experiments. First, the response time of the probe to DPP-IV
was investigated. Figure 3A shows that the response signal reaches equilibrium at 30
min. Therefore, 30 min was selected as the reactive time in the subsequent experiments.
Next, Figure 3B can be observed that the probe has the best response to DPP-IV in the
pH range from 7.0 to 10.0, which is beneficial to the practical application of HCA-D
under physiological conditions and pH 7.4 is selected for the further experiments.
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Figure 3. The effect of response time (A) and pH (B) on fluorescence intensity of HCA-D
induced by DPP-IV. Error bars represent standard deviation (n = 3).
.7 Quantitative detection of DPP-IV
3
Under the optimized conditions, the emission intensity of the probe HCA-D
exhibits a good linear fluorescence response to DPP-IV in the concentration range of
2
0
.62-10 ng/mL (see the inset of Figure 4B, R = 0.9998). From the slope of the
regression line, the detection limit (LOD) of the probe is determined to be 0.19 ng/mL
of DPP-IV. Evidently, the proposed probe exhibits a clear-cut advantage in
performance of LOD and water-solubility compared to the reported nonpeptide-based
small molecule fluorescent probes (Table S1 in the Electronic Supplementary
Information). In addition, it has been reported that the levels of DPP-IV in human serum
2
5
have been determined ranging from 200 ng/mL to 700 ng/mL . According to the result
of anti-interference experiments (see Section 3.3) and above linear range of the probe,
the proposed approach has enough sensitivity and selectivity for analysis of DPP-IV in
biological samples.
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Figure 4. (A) Fluorescence response of 20 μΜ HCA-D after incubation with various
concentrations of DPP-IV (0.625-100 ng/mL) at 37 °C in PBS buffer (10 mM, pH 7.4) for 30 min.
(B) Linear relationship between the fluorescence intensity of HCA-D and the concentration of
DPP-IV (0.625-10.0 ng/mL). Error bars represent standard deviation (n = 3).
3
.8 Inhibition assay of DPP-IV
To further verify the anti-interference ability of HCA-D for DPP-IV activity in
complicated bio-samples, inhibition assay was performed by using sitagliptin, a
selective inhibitor of DPP-IV. As shown in Figure 5, it does not interfere with the
fluorescence signal of the probe HCA-D itself and the fluorophore HCA upon addition
of the inhibitor. However, the fluorescence signal of HCA-D induced by DPP-IV is
gradually decreased relative to the increased concentration of the sitagliptin.
Simultaneously, the similar inhibition phenomenon is also observed in human serum
(Figure S13). Based on these data, the IC50 values of sitagliptin against recombinant
DPP-IV and the DPP-IV in human serum are calculated as 12.5 nM and 12.1 nM,
respectively, meaning that the probe HCA-D features excellent anti-interference ability
in a complex biological system and this approach can offer a robust platform for
screening of DPP-IV inhibitor. To this end, human serum can be served as an enzyme
source to replace the conventionally expensive recombinant DPP-IV for high-
throughput screening of DPP-IV inhibitor.
Figure 5. (A) HCA-D (20 μΜ) and HCA (20 μΜ) were treated with sitagliptin inhibitor (0-300
nM), respectively. (B) The inhibitory effect of sitagliptin on DPP-IV and the fluorescence
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intensity of the HCA-D (20 μΜ) after incubated with the DPP-IV (10 ng/mL) that was treated by
different concentration of Sitagliptin (0-50 nM).
3
.9 Determination of the Michaelis constant
It is well-known that the enzyme kinetic behavior is significant for the quantitation
of the activity-based fluorescent probes 21, 26. Figure S14 depicts the kinetic behavior of
the hydrolysis of HCA-D by recombinant DPP-IV and the Michaelis-Menten equation
is utilized to estimate kinetic parameters. As shown in Figure S14, the kinetic parameter
Michaelis constant (K ) and Maximum rate (Vmax) values for HCA-D hydrolysis in
m
-1
DPP-IV are calculated to be 8.76 μM and 70.88 μM min , respectively, which indicates
that the enzyme has a better affinity and hydrolytic efficiency of DPP-IV for the present
probe substrate than that of the probes in the reported literature25, 28. It also validates
that HCA-D can be viewed as a highly selective fluorescent probe substrate to quantify
trace DPP-IV in complex biological samples.
3
.10
Quantification of DPP-IV in human serum
In order to extend the practicality of the probe HCA-D, the proposed method was
applied for determining DPP-IV in human serum samples from diabetes patients and
health people. Furthermore, the recovery experiments of DPP-IV in the spiked samples
were carried out and recoveries were calculated. The results show satisfactory
recoveries as well as relative standard deviation (RSD) (Table S2), suggesting that
HCA-D can be used as a tool to qualify DPP-IV activity in complex biological samples.
Subsequently, HCA-D was employed for monitoring DPP-IV level in serum
samples from the type 2 diabetes patients and healthy people. To ensure a
representativeness, serum samples consisted of twenty-five patients with type 2
diabetes and twenty-four healthy volunteers were randomly selected from the clinical
laboratory in Gansu Provincial People’s Hospital laboratory to participate in the
assessment of DPP-IV level. As shown in Figure 6, the DPP-IV levels in serum samples
from diabetes patient are apparently higher than that from healthy group. This result
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further elucidates that there is a significant relationship between the DPP-IV level and
the type 2 diabetic.
Figure 6. Activity levels of DPP-IV in serum samples from the control (healthy volunteers) and
patients with type 2 diabetes.
3
.11 In vitro imaging of DPP-IV in living Cell samples
With our original intent in mind, we proceeded to research the cell toxicity of the
customized probe with different concentration. Herein, MTT assay was used to evaluate
the cell viability inducted by HCA-D. Figure S15 illustrates that the cell viability
remains above 85% after incubation with 10 μM HCA-D for 24 h, which provides
support for living cell imaging at HCA-D concentration of 10 μM.
It has been reported that DPP-IV exists in a membrane-bound state in macrophages
29.
Hence, fluorescence imaging experiments were implemented in macrophage (Raw
2
64.7) cells to ascertain the reliability of HCA-D for DPP-IV imaging in biological
system. As expected, Figure 7(a) displays the brilliant fluorescence that accumulated at
the cell membrane regions of Raw 264.7 cells after incubation with HCA-D for 30 min,
which is in good agreement with the reported results. Furthermore, when the Raw 264.7
cells were pretreated with different concentration of DPP-IV inhibitor (sitagliptin) for
6
0 min and washed with PBS buffer three times, then dealt with HCA-D for 30 min.
Figure 7(b-d) exhibits that the fluorescence intensity of HCA-D in cells is subjected to
gradual decrease along with an increased concentration of inhibitor. In combination
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with the inhibition experiments of DPP-IV (see Section 3.8, Figure 5), the result
indicates the positive correlation between the fluorescent intensity of HCA-D and the
amount of DPP-IV protein in the living cell. These observations prove that it is reliable
and valid to utilize HCA-D in imaging of DPP-IV in live organisms.
Figure 7. Confocal laser scanning microscopy (CLSM) images of Raw 264.9 cells (A). (a−d)
stained with different concentration of Sitagliptin (0, 10, 100, 150 nM) for 60 min and then
incubation with 10 μM HCA-D for 30 min. excitation, 635 nm; emission, 650−730 nm.
3
.12 In vivo imaging of DPP-IV in tumor-bearing mice model
Studies have shown that near-infrared fluorescent probes offer great advantages in
in vivo imaging 30, 31. To further explore the gamut of HCA-D for practical application,
the ability of HCA-D for imaging the living tumor mice models derived from mouse
H22 hepatoma was evaluated. Figure 8 shows that an evident fluorescence appears in
the tumor region within 20 min after an intratumoral injection of HCA-D. Strikingly,
the fluorescence intensity represents discernible increase within 30 min and
accumulates mainly at the whole tumor region. In sharp contrast, the fluorescence
signal of the control group is found to original from the autofluorescence of biological
matrix. Practically, the previous reports have revealed that the DPP-IV activity is
related to the dysfunction-related diseases 19 and it has been found in some kinds of
cancer cells, especially in hepatoma HepG2 cells 22. Therefore, this result further
demonstrates the plausible relationship between DPP-IV level and liver cancer and this
platform might be capable of meeting an ever-increasing demand for image-guided
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tumor resection.
Figure 8. In vivo HCA-D fluorescence imaging of DPP-IV in tumor-bearing mice after
tumor injection.
Conclusions
4
In summary, a near-infrared fluorescent probe (HCA-D) was designed and
synthesized. Attributed to the introduction of the sulfonated functional group into the
fluorophore framework, the fabricated fluorescence probe has good water solubility,
which profit accurate assay and imaging of DPP-IV in the biological system. In light of
this feature, the proposed HCA-D was carried out the practical application in evaluation
of the DPP-IV activity level in serum samples from the type 2 diabetes patients and
healthy people. Furthermore, imaging of DPP-IV in living cells and animal models have
also been validated by the probe due to its good biocompatibility, low cytotoxicity and
specificity. Therefore, this probe may be applied to investigate the relationship between
disease and DPP-IV activity. It is also possible to serve as a promising image-guided
tumor resection indicator for surgical operation in the future.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(NSFC) Fund (No. 21575055, No. 21707059), the Fundamental Research Funds for the
Central Universities (lzujbky-2019-67) and the Henan Key Laboratory of Biomolecular
Recognition and Sensing (HKLBRSK1802).
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Conflicts of interest
There are no conflicts to declare.
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A water-soluble near-infrared (NIR) fluorescent probe HCA-D based on Cyanine dyes as the fluorophore and
glycyl-prolyl peptide as the specific recognition sequence was developed for assay of dipeptidyl peptidase IV
(
DPP-IV) activity. The proposed probe exhibited good selectivity, sensitivity and stability. It was successfully
employed to detect DPP-IV activity in serum, including the type 2 diabetes patients and healthy people.
Furthermore, imaging of DPP-IV in living cells and animal models.
3
38x190mm (96 x 96 DPI)