Acridone Derivate Simultaneously Featuring Multiple Functions and Its Applications

Article abstract of DOI:10.1021/acs.analchem.9b01289

Compared with plenty of single-functional molecules, multifunctional molecules are scarce and have high demand in further research. In this work, a multifunctional molecule called 10-methyl-2-amino-acridone (MAA) is presented. Interestingly, MAA simultaneously features electrochemistry, two-photon fluorescence, visible-light-induced oxidase mimic, and photoelectrochemistry (PEC) activity, and the related properties are studied in detailed. Multiple functions integrated into one molecule allow MAA to become a versatile signal probe. Therefore, the MAA acted as an electrochemical indicator to detect exosomal total protein with high sensitivity at first. In addition, MAA is used for one- or two-photon fluorescence imaging in vitro and in vivo, including cells, three-dimensional (3D) tumor spheroids, zebrafish, and exosomes. The results suggest that MAA not only possesses favorable photostability, but it is also suitable for imaging in deep tissue. Furthermore, the visible-light-induced oxidase mimic and photoelectrochemical activities of MAA are selectively inhibited by Cu²⁺, and the relevant mechanism is carefully analyzed. On the basis of this phenomenon, we develop a dual-modal detection strategy for detection of Cu²⁺ in river water. Compared with a single signal readout model, this strategy is able to avoid false positive and negative detection through two series of data mutually validating each other. Therefore, our study shows that the "multiple-in-one" MAA provides a blueprint for the investigation and application of a multifunctional organic molecule.

Full text of DOI:10.1021/acs.analchem.9b01289

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Article
An Acridone Derivate Simultaneously Featuring
Multiple Functions and Its Applications
Yaokun Xia, Wenhui He, Juan Li, Lupeng Zeng, Tingting Chen, Yijuan Liao, Wei-Ming
Sun, Jianming Lan, Shuangmu Zhuo, Jing Zhang, HuangHao Yang, and Jing-Hua Chen
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01289 • Publication Date (Web): 28 May 2019
Downloaded from http://pubs.acs.org on May 28, 2019
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Analytical Chemistry
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An Acridone Derivate Simultaneously Featuring Multiple Functions
and Its Applications
Yaokun Xia†, Wenhui He†, Juan Li‡, Lupeng Zeng†, Tingting Chen†, Yijuan Liao§, Weiming Sun†, Jianming Lan†, Shuangmu Zhuo∥, Jing
Zhang*§, Huanghao Yang*‡, Jinghua Chen*†
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†Department of Pharmaceutical Analysis, The School of Pharmacy, Fujian Medical University, Fuzhou, Fujian Province 350122 (P. R.
China) E-mail: cjh_huaxue@126.com
‡MOE Key Laboratory for Analytical Science of Food Safety and Biology, State Key Laboratory of Photocatalysis on Energy and
Environment, College of Chemistry, Fuzhou University, Fuzhou, Fujian Province 350108 (P. R. China) E-mail: hhyang@fzu.edu.cn
§Department of Chemical Biology, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian Province 350002
(P. R. China) E-mail: 1601225385@qq.com
∥
Key Laboratory of Optoelectronic Science and Technology for Medicine of Ministry of Education, Fujian Normal University, Fuzhou,
Fujian Province 350007 (P. R. China)
ABSTRACT: Compared with plenty of single-functional molecules, multifunctional molecules are scare and have high demand in further
research. In this work, a multifunctional molecule called 10-methyl-2-amino-acridone (MAA) is presented. Interestingly, MAA
simultaneously featuring electrochemistry, two-photon fluorescence, visible-light-induced oxidase mimic, and photoelectrochemistry (PEC)
activity, and the related properties are studied in detailed. Multiple functions integrated into one molecule allows MAA to become a
versatile signal probe. Therefore, the MAA is acted as electrochemical indicator to detect exosomal total protein with high sensitivity at
first. In addition, MAA is used to one- or two-photon fluorescence imaging in vitro and in vivo, including cells, 3D tumor spheroids,
zebrafish, and exosomes. The results suggest that MAA not only possesses favorable photostability, but it is also suitable for imaging in
deep tissue. Furthermore, the visible-light-induced oxidase mimic and photoelectrochemical activities of MAA is selectively inhibited by
Cu2+, and the relevant mechanism is carefully analyzed. Based on this phenomenon, we develop a dual-modal detection strategy for
detection of Cu2+ in river water. Compared with single signal readout model, this strategy is able to avoid false positive and negative
detection through two series of data mutually validating each other. Therefore, our study shows that the “multiple-in-one” MAA provides a
blueprint for the investigation and application of multifunctional organic molecule.
Introduction
structure modification flexibility,9,10 it is possible to introduce
appropriate groups in acridone, which not only has potential to
optimize its electrochemical and fluorescent performance but also
may integrate different functions in single compound to enrich
multifunctional organic molecule. Therefore, significant effort
should be further invested in improving performance and
exploring more functions of ADs.
Development of multifunctional probes is of great significance for
sensors, biolabeling and other applications.1 For example,
multifunctional probes are often employed to construct multiple
modal sensing assay, and produce diverse signal to realize more
sensitive and efficient analysis.2 But most of multifunctional
probes are constructed by combining different components
together, which suffers the complicated preparation process with
high cost.3,4 Although emergence of some nanomaterials and
organic molecules realizes different functions integrate into one
component, their tedious structure and synthesis route as well as
low thermal and chemical stability restrict their applications.5-7 In
recent years, synthesis of acridone derivates (ADs) is expected to
develop a multifunctional molecule, because of its structure
simple, modification flexibility and specificity that is extended
conjugated plane and electron donor-acceptor system.8-10 Despite
various ADs have been synthesized that supported by previous
our works and other groups,11-13 several key issues still need to be
addressed to synthesize ADs with excellent properties. (1) As
electrochemical indictor, the related researches about ADs are
restricted by poor solubility and electrode types.14-16 (2) The short
excitation wavelength of a proportion of ADs may lead to
photodamage, biological autofluorescence, photobleaching and
shallow penetration depth.17,18 (3) The most important is that up
till now the multifunctions in the same ADs is still rarely reported.
Considering acridone structure is very special, containing an
electron-accepting group (C=O) along with an electron-donating
nitrogen atom bearing a lone electron pair in acridone moiety and
Herein, we present the synthesis of an acridone derivate with
methyl and amino modification called MAA. With a simple
synthetic method, the modified MAA obtained the following
fascinating properties, some of which in ADs were even barely
reported. (1) MAA exhibits good water solubility and low toxicity.
(2) The electrochemical activity of MAA is measured on different
electrodes, including gold electrode (GE), glass carbon electrode
(GCE), and indium tin oxide (ITO) electrode. Meanwhile, the
reaction between MAA and biomacromolecule such as protein is
validated, which is potential to develop a convenient sensing
approach with label-free pattern. (3) Based on large π-conjugated
system and electron donors (-CH3 and -NH2), the relatively high
fluorescence quantum yield, long lifetimes, high photostability,
long emission wavelength, and large stokes shift prevent self-
quenching effect and reduce the interference from excitation.19 In
addition, the small molecular weight of MAA, enables easy
penetration of cell membranes for live cell staining processes.20 (4)
Conjugating electron donating (-CH3 and -NH2) and accepting
(C=O) substituents through π-bridges (-π-), the bright two-photon
fluorescence of MAA is observed at 810 nm excitation that so
called biological optical window, which is able to eliminate the
biological autofluorescence, reduce photodamage, and increase
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tissue penetration depth.21,22 As far as we know, the two-photon
procedures: (1) The 0.1 g compound 2, 2.5 mL Na2S (30 wt%)
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fluorescence property of ADs is rarely explored. (5) It is
interesting that MAA is found to possess effective visible-light-
induced oxidase mimic activity, catalyzing 3,3',5,5'-
tetramethylbenzydine (TMB) from colorless to blue under xenon
lamp, which discards relative harsh reaction conditions because of
absence of destructive hydrogen peroxide. Taken together, the
vast potential of MAA as multifunctional signal probe is explored
in this work.
and 5 mL ethanol were mixed and added into a round bottom
flask, and add into water to 50 mL. The mixture was heated at
130 ℃ under N2 for 4 h. (2) The obtained solution was poured
into ice water, and the solid precipitate was acquired through
centrifugation. Then the precipitate was dried in vacuum drying
box. (3) Finally, the precipitate was separated and purified by
preparative HPLC method with gradient elution to obtain high
pure MAA. The conditions of preparative HPLC method were as
follows: chromatographic column was C18, mobile phases were
acetonitrile and double distilled water, and the flow rate was 2
mL/min, and the column temperature was 35℃. FAB-MS: m/z
Experimental Section
9
Reagents and apparatus. All chemicals were obtained from
commercial suppliers and used without further purification.
Acridone (AR, 98%) was obtained from MACKLIN Reagent
Company. CD63 antibody, AGO2 antibody, TSG 101 antibody,
and EpCAM antibody were provided from Abcam. The HeLa and
HepG2 cells were purchased from China Center for Type Culture
Collection in Shanghai. Pierce bicinchoninic acid (BCA) Protein
Assay Kit and enhanced chemiluminescence reagent were
purchased from Thermo Fisher Scientific. 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from
Aladdin Reagent Co. Ltd. (Shanghai, China). Phosphate buffered
saline (PBS), fetal bovine serum (FBS) and roswell park
memorial institute (RPMI) were purchased from Hyclone.
Hochest 33342 was purchased from Nanjing Kaiji Biological
Technology Development Co. Ltd. 1,1'-Dioctadecyl-3,3,3',3'-
Tetramethylindocarbocyanine (DiI) was purchased from
Beyotime. Zebrafish, culture fluid, and melanin inhibitor were
provided from Nanjing one tree pear flower Biotechnology Co.
Ltd.. 3,3',5,5'-tetramethylbenzidine (TMB) was purchased from
Tokyo Chemical Industry (TCI, Shanghai). All other chemicals
were analytical grade. All aqueous solutions were prepared using
ultra-pure water (18.2 MΩ, Milli-Q, Millipore). Electrochemical
signals were measured by Electrochemical analyzer (CHI 660E,
Shanghai Chen Hua Instrument Co. Ltd., China). The
ultracentrifuge was Beckman optima XPN-100 (U.S.A.). The
transmission electron microscopy (TEM) images were carried out
on a JEM-2010 microscope (JEOL Ltd., Japan). The fluorescence
spectra were carried out using a fluorescence spectrometer (F-
4600, HITACHI, Japan). The UV-Vis absorption spectra were
recorded on UV-Vis spectrometer (2450, SHIMADZU, Japan).
The separation and purification of MAA was carried out using
preparative high performance liquid chromatography (LC-20AP,
SHIMADZU, Japan). The one- and two-photon fluorescence
imaging of HeLa cells, zebrafish and exosomes were conducted
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225 ([M+H]+); H-NMR (400 MHz, CDCl3) δ: 8.572 (d, ArH),
7.841 (s, ArH), 7.718 (t, ArH), 7.514 (d, ArH), 7.443 (d, ArH),
7.261 (CDCl3), 7.202 (d, ArH), 3.887 (s, NH2), 1.595 (s, CH3);
13C-NMR (400 MHz, DMSO-d6): δ: 177.65 (-C=O), 142.21 (=C-
N), 140.89 (=C-NH2), 136.37 (=C-N), 133.40 (=CH-), 127.86
(=CH-), 123.59 (=C-C=O), 123.43 (=C-C=O), 121.73 (=CH-),
120.54 (=CH-), 116.05 (=CH-), 114.46 (=CH-), 110.67 (=CH-),
33.55 (-N-CH3).
Cells culture and exosomes isolation. HepG2 cells were cultured
in DMEM containing 10 % FBS and 1 % penicillin-streptomycin,
and maintained in a humidified atmosphere of 5 % CO2 at 37 ℃.
Before exosomes isolation, HepG2 cells (1×108 cells) were
cultured in DMEM containing 5 % FBS (exosomes depleted) for
48 h. Subsequently, the culture supernatant was collected and
centrifuged according to previous reported protocol.24 Briefly, the
obtained cell culture supernatant was centrifuged at 300 g for 10
min, 2,000 g for 20 min, and 11,000 g for 45 min to deplete intact
cells, cell debris, and protein, respectively. The supernatant then
was centrifuged at 110,000 g for 70 min, and the transparent
sediment was exosomes. Finally, the exosomes were resuspended
in sterile PBS and stored at -80 ℃. Additionally, the HeLa cells
were cultured under the same conditions.
One- and two-photon cellular imaging. The HeLa cells were
seeded on glass coverslips and incubated at 37 ℃ for 24 h. Then
the HeLa cells first were stained with Hochest 33342 (1 μM) for
15 min. Next, the cells were incubated with MAA (20 μM) for 4 h.
After every staining, the media was removed, and the cells were
washed with PBS (pH 7.4) for three times to remove the unbound
dye. After that, the cells were imaged on a Zeiss LSM880
confocal microscope. The fluorescence of Hoechst 33342 and
MAA were excited at 405 nm (blue channel) and 488 nm (green
channel), respectively. The excitation wavelength of the laser was
810 nm for two-photon imaging. The three-dimensional
multicellular spheroid (3DMTS) was constructed according to
previous literatures with some modifications.25,26 The 6.0 mL
RPMI containing 90 mg agarose was microwaved until agarose
dissolution. The 50 μL of the hot agarose solution was added into
each well rapidly and allowed to cool for 30 min. Next, the HeLa
cells were seeded in above 96-well plates at 6000 cells per well
and were incubated under 5 % CO2 at 37 ℃ for 72 h. After that,
the resulting 3DMTS were incubated with MAA for certain time.
Then, the 3DMTS was washed by PBS for three times. Finally,
confocal microscopy was used for the spheroids imaging.
on
a confocal fluorescence microscopy (Zeiss, LSM880,
Germany).
Preparation of MAA. The synthesis route of MAA was
displayed in Figure S1 following previous published procedure
with some modifications.23 Firstly, the compound 1 (10-methyl-
acridone) was synthesized through substitute reaction. An amount
of 0.4811 g 9(10H)-acridone was put into ethanol and potassium
hydroxide mixture, and the temperature was risen to 110-140 ℃.
Then, 20 mL N,N-Dimethylformamide and 10 g iodomethane
were added into above system, and heated at 165 ℃ for 40 min.
The solution was poured into ice water and the yellowish green
solid precipitate appeared. The solid was filtered off, washed with
10 mL water and further purified by recrystallization in absolute
alcohol. The solid was oven dried at 70 ℃, and the compound 1
was obtained. MS (EI) ([M+H]+)=210.
One- and two-photon zebrafish imaging. The zebrafishes were
maintained in culture solution containing 2 % melanin inhibitor at
room temperature for 2 days. 10 μM MAA was incubated with
zebrafishes at room temperature for 12 h. Finally, the MAA
labeled zebrafishes were imaged by Zeiss LSM880 confocal
fluorescence microscope for one-photon and two-photon
fluorescence imaging after washing by zebrafishes culture fluid
for three times to remove the free MAA.
After that, 0.2105 g compound 1 was dissolved in 6 mL acetic
acid, and then added into 2 mL concentrated nitric acid and 2 mL
acetic acid, and heated at 58-62 ℃ for 2 h. The solution was
poured into ice water, and the golden solid was appeared. The
solid was filtered off, washed, and oven dried. The compound 2
One- and two-photon exosomes imaging. The HepG2 cells
derived exosomes were treated with 10 μM MAA in PBS (pH 6.8)
for 30 min at room temperature. After washing three times by
(10-methyl-2-nitro-acridone)
was
obtained.
MS
(EI)
([M+H]+)=255. Next, the MAA was prepared as following
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Analytical Chemistry
PBS, the MAA labeled exosomes were obtained. The exosomes
comparing with BCA protein assay that is a classic protein
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fluorescence imaging procedure as following: (1) the HepG2 cells
were stained by Hochest 33342 (1 μM) for 15 min and washed by
PBS for three times. (2) The aforementioned cells were incubated
with MAA labeled exosomes for 1 h, followed by washing with
PBS for three times. (3) The one-photon fluorescence imaging
was measured by Zeiss LSM880 confocal fluorescence
microscope. The fluorescence of Hoechst 33342 and MAA were
excited at 405 nm (blue channel) and 488 nm (green channel),
respectively. As for two-photon fluorescence imaging, the images
were obtained by Zeiss LSM880 confocal fluorescence
microscope with excitation of 810 nm. For fluorescence co-
localization analysis, the MAA-labeled exosomes were incubated
with DiI for 30 min to acquire MAA and DiI dual-labeled
exosomes. The DiI was excited by using laser at 561 nm.
analysis method. Figure S9A illustrates that the linear relationship
is obtained between ΔIp and the concentration of BSA. As a
demonstration, our method is applied to the determination of the
total protein in exosomes solution. Exosomes are membrane-
enclosed vesicles secreted by various cells, and they carry
abundant molecular information of their parent cells such as
protein.27 The exosomal protein concentration is higher in
advanced-stage disease, so they have been revealed as a
promising biomarker for liquid biopsy.28 Before analysis content
of exosomal protein, the exosomes are characterized according to
International Society for Extracellular Vesicles (ISEV)
guidelines.29 At first, a mean diameter of exosomes measured by
dynamic light scattering (DLS) is about 130 nm (Figure 1A). The
morphologies of exosomes are characterized by transmission
electron microscope (TEM) (Figure 1A, inset), exhibiting saucer
like, and the average diameter shows ~100 nm that smaller than
that detected by DLS, which is ascribed to shrink of exosomes
during the preparation process for TEM.30 The western blot (WB)
images for different categories proteins validate the efficiency of
the separation of exosomes (Figure 1B). Figure S9B shows the
exosomal total protein concentration that acquired by our method
is closed to that of the traditional BCA method. The t-test is
utilized to quantitatively analyze if two series of data from
electrochemistry and BCA are significantly different from each
other. The P-value is 0.517, much larger than 0.05, indicating that
there is no significant difference between two results, so this
strategy is reliable in practical application. Taken together, this
approach is prospective in generating label-free pattern for
detecting other proteins selectively coupling with aptamer
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Cu2+ detection by use of MAA as oxidase mimicking. The Cu2+
detection was based on color change of TMB. In a typical
experiment, 10 μM of MAA was incubated with different
concentration Cu2+ for a certain time in pH 4.0 HAc-NaAc buffer
solution (10 mM) at room temperature. Following that, 500 μM
TMB was added in the above solution and illuminated for 5 min
under visible light (λ≥400 nm). Finally, 2 μL of 2 M H2SO4 was
added, and the absorbance at 450 nm was measured by a UV-Vis
spectrophotometer.
Cu2+ detection by photoelectrochemistry. Detection of Cu2+ was
carried out as follows: the ITO electrode was modified with MAA
before detection firstly. Subsequently, the ITO electrode was
immersed in a NaAc-HAc buffer solution (10 mM, pH 4.0) with
different concentration Cu2+ to measure the photocurrent signal.
Electron spin resonance (ESR). 10 μM MAA and 100 mM spin
trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in anhydrous
methanol solution were photoirradiated with visible light for 0, 5
or 10 min, respectively. Subsequently, the prepared samples were
transferred to a quartz capillary tube and placed in the ESR cavity,
and obtained the ESR spectra using a JES FA 200 ESR
spectrometer (JEOL, Japan).
technology that is similar to previous work.31
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Results and discussion
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Synthesis and characterization of MAA. The synthesis of MAA
is accomplished as described in Figure S1 with substituent and
reduction reaction: Firstly, link a methyl and a nitro to the 2, 10-
positions of acridone, respectively.14 Subsequently, the nitro is
reduced to amino by Na2S, and the crude product is purified by
preparative HPLC to obtain pure MAA. The relevant
characterization data are provided in Figure S2~Figure S4.
Moreover, the color of MAA and water solubility exhibit obvious
change comparing with acridone and other intermediates (Figure
S5). Above results suggest that MAA is synthesized successfully.
Electrochemical properties and detection of exosomal total
protein. The electrochemical activity of MAA is studied on
different electrodes firstly. A couple of redox peaks of MAA are
acquired on different electrodes (Figure S6A) compared to
acridone (Figure S6B). The peak current of MAA is proportional
to the square root of scan rate, indicating a diffusion-controlled
process (Figure S7A and Figure S7B). The effect of pH on formal
potential and peak current of MAA suggests protons participate in
the electrode process (Figure S7C and Figure S7D), and the
electron transfer number is equal to proton number. The relevant
details are exhibited in section 1 (Supporting Information).
Moreover, the MAA also can bind with biomacromolecule, such
as protein, to induce electrochemical signal change. The result
indicates the forming of a 1:1 association between MAA and BSA
(Figure S8), and the interaction mechanism and binding ratio are
showed in section 2 (Supporting Information). Based on the
described properties, we thereupon envisage that MAA is acted as
an electrochemical indicator for protein concentration analysis
because of the inherent high sensitivity of electrochemistry
Diameter (nm)
Figure 1. (A) The size distribution of exosomes analyzed by DLS,
inset: The TEM image of exosomes. (B) WB images in exosomes
for CD 63, TSG 101, AGO2 and EpCAM.
Optical properties and fluorescence imaging in cells, 3D
tumor spheroids, zebrafish, and exosomes. The optical
properties are explored in detailed. Because of electron-donating
effect of methyl and amino, UV-Vis absorbance peaks of MAA
are red-shifted from ~250 nm to ~260 nm and ~390 nm to ~420
nm, respectively (Figure S10A), and optimal fluorescence
emission wavelength is located at 540 nm (Figure S10B),
exhibiting strong green fluorescence (Figure S10B, inset).
Compared with acridone (Δλ=40 nm, Figure 2A), MAA has a
three-fold increase of Stoke shift (Δλ=120 nm, Figure 2B), which
is promising to achieve precise imaging and accurate sensing. The
absolute fluorescence quantum yields and lifetime are 12.3 % and
461.82 ns, respectively, which is superior to acridone (Table S1).
Especially, the long fluorescence lifetime of MAA may provide a
neat opportunity for fluorescence lifetime imaging in future.32
Surprisingly, two-photon fluorescence of MAA is found in our
work (Figure 2C). The slop of double logarithm plot between
fluorescence intensity and laser power remains at 2 (Figure 2D),
indicative of quadratic dependence, illustrating it is a two-photon
excitation process.33 Thus, MAA is potential to act as probe for
fluorescence imaging.
The low toxicity is precondition for bioimaging. As shown in
Figure S11, high viability is obtained after the cells being
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Figure 2. (A) The excitation (a) and emission (b) spectrum of acridone. (B) The excitation (a) and emission (b) spectrum of MAA. (C)
Two-photon fluorescence spectrum of MAA with femtosecond pulsed laser. (D) Double logarithmic plots of between two-photon
fluorescence intensity and laser power. (E) Two-photon microscopy images of HeLa cells incubated with MAA (a), bright field image of
HeLa cells (b), overlay image of a and b (c). (F) Two-photon confocal images of MAA for photobleaching in HeLa cells. (G) Two-photon
microscopy images of 3D tumor spheroids (a), the two-photon 3D Z-stack images of an intact spheroid (b), the two-photon Z-stack images
were taken from the top to bottom (c). (H) Two-photon microscopy images of living zebrafish incubated with MAA (a), bright field image
of zebrafish (b), overlay image of a and b (c).
incubated with 20 μM MAA even for 20 h, which indicates that
MAA presents hypotoxicity under the cell imaging conditions. To
investigate the fluorescence imaging feasibility of MAA in vitro,
the uptake of MAA by HeLa cells is conducted. One- (Figure S12)
and two-photon (Figure 2E) fluorescence microscopy images
illustrate that MAA penetrates cell membrane efficiently. To
understand the distribution of MAA in cell, HeLa cells are
incubated with nuclear dye Hoechest 33342 and MAA. The nuclei
of HeLa cells stained Hoechest 33342 shows a blue color (Figure
S12B). The green emission from MAA full of whole cell (Figure
S12C and Figure S12D), indicating the MAA not only gets into
the cytoplasm, but also enters the nuclei. Besides, photobleaching
experiment is conducted in HeLa cells due to photostability being
an important parameter for long-term biological imaging. Figure
S13 and Figure 2F display the fluorescence intensities with one-
or two-photon excitation for 1500 s are still changed minimally,
and quantitative photobleaching results (Figure S14) show MAA
possesses superior photostability than other fluorescent probes
that reported by previous literatures.25,34-38 Furthermore, the
imaging depth is the other important parameter for fluorescence
imaging because it is conducive to noninvasive biological
imaging. A tissue penetration depth experiment using 3DMTSs as
model is investigated. The fluorescence intensity is obtained from
3DMTS only 58.8 μm for the one-photon excitation (Figure S15).
Compared with one-photon excitation, signal is detected even to
104.9 μm for the two-photon excitation (Figure 2G). Additionally,
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from the quantification graphs of one-photon (Figure S16A) and
two-photon (Figure S16B) fluorescence images for 3DMTS, the
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deeper penetration of two-photon excitation is observed
intuitively, which is ascribed to the low absorption of near-
infrared (NIR) light by the biological environment,39 and enables
MAA as an effective two-photon probe for fluorescence imaging
to observe physiological condition in deep of tissue.
Currently, fluorescence imaging in vivo become a hotspot
because of it being an effective avenue to understand the
pathological process and drug adsorption, distribution,
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metabolism, and excretion (ADME).40 To investigate the
application of MAA imaging in vivo, the zebrafish is acted as
study subject because of its small size, transparent body, rapid
maturation, and highly genome homologous with human.41,42
Before fluorescence imaging, the zebrafish that is incubated with
MAA is washed by culture fluid for several times to eliminate
non-specific adsorption. From Figure 2H and Figure S17, the
zebrafish emits green fluorescence after cultured in MAA solution
for 12 h, which illustrates the MAA is introduced into the body.
We also observe that MAA is tissue-dependent affinity, and
mainly accumulated at the head, abdominal cavity and tail (Figure
2H). MAA is accumulated at eyes, but the eyeball is dark, which
demonstrates that the MAA not enters across the blood-ocular
barrier. The abdominal cavity is the brightest part of the body and
indicates that MAA enters into the digestive system, revealing
that swallowing is a primary avenue for MAA entering into
zebrafish. The brightness of the dorsal aorta illustrates that MAA
enters into the circulatory system, which is important for MAA
transportation in zebrafish body. In brief, MAA is a potential
fluorescent probe for in vivo imaging, and above results from
zebrafish possess a certain reference value for other animal
models owing to zebrafish having high homology with mammals.
Furthermore, recent evidence indicates that exosomes play an
important role in transfer of informative molecules between
neighboring or even distant cells.43 Notably, it is difficult to
understand the distribution of exosomes in cell due to lack of
effective imaging probe. Thus, the development of a proper probe
for clear visualization of exosomes is highly desirable. Before
exosomes imaging experiment, the recipient cell nuclei are stained
by Hoechest 33342 and display blue fluorescence (Figure S18B).
The MAA labeled exosomes exhibiting green fluorescence, and
located at cytoplasm (Figure S18C and D). To further validate
exosomes being imaged in cell, we utilized DiI, a fluorescence
dye commonly used for staining exosomes,44,45 to label exosomes
and study exosomes internalization by cells. The cell nuclei
showed blue fluorescence (Figure S19A). After the MAA and DiI
dual-labeled exosomes were cultured with HepG2 cells for a
certain time, the green and red fluorescence in cytoplasm are
observed (Figure S19B and S19C). In addition, the orange
fluorescence is emergence (Figure S19D), which suggests that the
MAA green fluorescence is co-localized with the red fluorescence
of DiI. All these results indicate that exosomes are uptake by cells
and imaged indeed, which was similar with previous reports.46,47
Equally, the MAA labeled exosomes are visualization under two-
photon fluorescence microscopy (Figure S18E, F and G). So we
have realized the one- and two-photon fluorescence imaging for
exosomes, and provide a new way for tracking of exosomes in
vitro. Compared to existing strategies for labelling exosomes, our
conjugation-free
labelling
strategies
without
complex
pretreatment, chemical modification or engineering for exosomes
is time- and labor- saving, and user- and bio-friendly.48-50
Visible-light-induced
oxidase
mimic
and
photoelectrochemistry activities. Besides above properties, the
visible-light-induced oxidase-mimicking activity of MAA is
0.6
A
found. As far as we know, the visible-light-induced oxidase
0.5
mimic is scarcely reported in acridone derivate. Under visible
0.4
light irradiation, MAA is able to catalyze the oxidation of
0.3
different substrates, including TMB (Figure S20A), 2,2'-Azinobis-
0.2
(3-ethylbenzthiazoline-6-sulphonate) (ABTS) (Figure S20B) and
0.1
o-Phenylenediamine (oPD) (Figure S20C). It is noted that MAA
0.0
A
k
I
2
3
A
A
T
D
O
n
N
P
N
shows catal
B
ytAic Oact
T
ivity
K
under mild reaction conditions without
I
a
A
a
l
T
D
S
C
N
b
E
the use of destructive H2O2 and the catalytic reaction is
controllable just regulating light irradiation time simply. In order
to elucidate the reactive species in this catalytic reaction induced
by visible light, different scavengers for related reactive species
0 min
5 min
10 min
B
318.1 318.2 318.3 318.4 318.5 318.6 318.7
Magnetic Field/mT
Figure. 3 (A) Effects of scavengers on the TMB-MAA
chromogenic reaction under visible light irradiation. (B) The ESR
spectra of MAA with spin trap DMPO after irradiation with
visible for different time. (C) Proposed mechanism for visible
light stimulated oxidase mimic activity of MAA.
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such as hydroxyl radicals (∙OH), singlet oxygen (1O2), superoxide
anions (O2 ) and photo-genetrated holes (h+) are added to the
system. From Figure 3A, the absorbance of oxidized TMB
(oxTMB) has almost no change after reacting with ∙OH scavenger
isopropanol (IPA) or tert-butanol (TBA), which demonstrates that
∙OH is not mainly reactive species. Similarly, the photocatalytic
activity is not affected in the presence of NaN3, which is
•̶
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1
1
employed to scavenge O2. It shows that O2 is not the main
reactive oxygen species. As an efficient enzyme for decomposing
of H2O2 to H2O and O2, catalase (CAT) can not affect the
absorbance of oxTMB, which indicates that H2O2 is not produced
during the illumination process. In contrast, the absorbance
dramatically decreased after adding h+ scavengers such as EDTA,
ammonium oxalate (AO) or KI, demonstrating that h+ is one of a
reactive specie during the catalytic process. The absorbance of
oxTMB obviously decrease after the reaction system purged by
N2, showing the dissolved oxygen plays a key role in the
photocatalytic reaction. The catalytic activity is also suppressed in
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•̶
the presence of superoxide dismutase (SOD) that a O2 scavenger,
•̶
indicating O2 is a crucial specie. Electron spin resonance (ESR)
spectroscopy provides direct evidence to validate that MAA
•̶
generates O2 under visible light irradiation and the quantity of
•̶
O2 depends on the illumination time (Figure 3B). When MAA is
irradiated by visible light, it generates electrons in the lowest
unoccupied molecular orbital (LUMO) and leaves h+ in highest
occupied molecular orbital (HOMO) to react with TMB. The
produced electrons may be trapped by dissolved oxygen to form
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.5
0.4
0.3
0.2
0.1
0.0
C
a

h
0
2
4
6
8
10
Concentration
(
M
)
400
500
600
700
800
900
Wavelength (nm)
1.0
0.8
0.6
0.4
0.2
0.0
0.5
1.6
1.0
D
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
E
F
0.4
0.3
0.2
0.1
0.0
-0.1
0.8
0.6
0.4
0.2
0.0
a


0
2
4
6
8
10

h
Concentration (M)
+
g
A
+
+
+
3
+
2
+
2
2
a
l
o
n
u
N
A
Z
C
C
+
+
+
2
+
+
2
+
+
+
2
+
+
2
+
+
3
2
2
3
2
g
30 40 50 60 70 80 90 100
Time (s)
a
l
o
a
K
b
e
n
n
g
u
A
N
C
P
F
C
A
Z
C
M
M
Figure. 4 (A) Structural sketch map of MAA-Cu. (B) Transition orbitals involved in the crucial excited state. (C) UV-Vis response to
different concentration Cu2+ (from a to h: 0, 0.5, 2, 3, 5, 7, 9, 10 μM), the inset shows corresponding calibration curve (D) Selectivity of the
UV-Vis toward Cu2+ and different metal ions. (E) photocurrent response to different concentration Cu2+ (from a to h: 0, 0.5, 2, 3, 5, 7, 9, 10
μM), the inset shows corresponding calibration curve. (F) Selectivity of the PEC toward Cu2+ and different metal ions.
•̶
O2 radicals that also can oxidize TMB. Moreover, the PEC is
conducted to verify the photoinduced charge transfer of MAA in
this system. A reproducible photocurrent response to light
irradiation on/off cycles is observed (Figure S21), which indicates
there are charge generation, separation, and transfer process.
Based on above results, the mechanism of substrates oxidation by
photoactivated MAA is shown in Figure 3C. The Michaelis-
Menten (Km) constant obtained (Table S2) from Michaelis-
menten equation of MAA (Figure S22) is lower than that of
fluorescein and 2-aminoacridone (2-AA) as visible light induced
oxidase mimic, indicating that it has higher affinity to TMB. The
catalytic constant (Kcat) and catalytic efficiency (Kcat/Km) of
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MAA are superior to 2-AA (Table S2), which is ascribed to the
larger binding energy between MAA and TMB through molecular
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simulation (Figure S23). Although the Kcat and Kcat/Km of
MAA are lower than fluorescein to date (Table S2),51 which
maybe improved significantly through introducing appropriate
groups based on its structure modification flexibility.
Detection of Cu2+. Quite impressively, we find that the visible-
light-induced oxidase mimicking activity and photocurrent of
MAA is selectively inhibited by Cu2+. Molecular simulation is
used to understand the mechanism about this phenomenon. The
density functional theory (DFT) computation indicates that Cu2+
and TMB are both inclined to bind with carbonyl group of MAA
in solution (Figure 4A and Figure S23A). Note that the binding
energy between Cu2+ and MAA is as large as 6.21 eV, which is
greatly larger than that (0.20 eV) between TMB and MAA.
Considering the stronger interaction between Cu2+ and MAA, the
carbonyl group of MAA will be occupied by Cu2+ rather than
TMB, leading to the weakened catalytic efficiency. Besides, the
time-dependent DFT (TD-DFT) calculation reveals that an
obvious electron transfer from the π orbital of MAA to the 4s
orbital of Cu2+ occurs in the MAA-Cu compound (Figure 4B),
which is further confirmed by the natural bond orbital (NBO)
charge (0.956 |e|) on Cu atom (Figure 4A). Such an electron
transfer can justify the decrease of photocurrent (Figure S21) and
ESR (Figure S24) response in presence of Cu2+. Because Cu2+ is
harmful to liver and kidneys if short-time exposure to high-level
of Cu2+,52 an analysis platform based on MAA could be put
forward for Cu2+ detection. Before the detection of Cu2+, some
important conditions were investigated. As shown in Figure S25,
pH 4.0 was chosen for further study (Figure S25A), 5 min was the
optimal irradiation time (Figure S25B). Thus, under optimal
conditions, along with the increase of Cu2+ concentration, the UV-
Vis absorbance is decreased (Figure 4C). As expected, the
difference in color of solution is significant with the change of
Cu2+ concentration, which is observed directly by naked eyes
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(Figure S26).
A
highly linear relationship between Cu2+
concentration and absorbance at 450 nm is observed in the range
of 0.5 μM to 10 μM. The correlation equation could be expressed
as ΔA=0.042C+0.0263 (r=0.9912), where the ΔA is defined as A0-
A (A0 and A are the absorbance at 450 nm in the absence and
presence of Cu2+, respectively) (Figure 4C, inset). The detection
of limit (LOD) of 0.36 μM is based on 3σ method. In addition,
this method exhibits good ability to resist interference from
common metal ions (Figure 4D). It is worthy noting that the ΔA of
Ag+ and Fe3+ was negative, which indicates these metal ions could
oxidized TMB directly. This phenomenon is reported by previous
literature.53,54 However, the ΔA of Ag+ and Fe3+ is relatively low,
which is not able to influence the selectivity of this method.
Meanwhile, the photocurrent is also decreased with Cu2+
concentration change (Figure 4E), and ΔI is proportional to Cu2+
concentration from 0.5 μM to 10 μM. The correlation equation is
ΔI=0.0852C+0.0803 (r=0.9961), where the ΔI was defined as I0-I
(I0 and I were the photocurrent intensities in the absence and
presence of Cu2+, respectively) (Figure 4E, inset). The LOD is
0.17 μM (Figure 4E, inset). Similarly, PEC also shows good
selectivity (Figure 4F). The feasibility of this method
Table 1. Detection of Cu2+ in river water
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sample
Add (μM)
Detection by UV-Vis (μM)
Recovery %
96.0
Detection by PEC (μM)
2.01±0.042
Recovery %
100.5
1
River water 1
2
1.92±0.034
2
3
4
5
6
7
8
9
River water 2
River water 3
River water 4
River water 5
3
5
7
9
3.02±0.053
5.03±0.045
6.98±0.067
9.09±0.093
100.7
100.6
99.7
2.98±0.061
4.96±0.058
7.09±0.074
8.99±0.096
99.3
99.2
101.3
99.9
101.0
for Cu2+ detection in river water is validated using standard
addition method. From Table 1, the recovery rate ranged from
96.0-101.0
% and 99.2-101.3 % for UV-Vis and PEC,
respectively. The P-value from t-test is 0.999, much larger than
0.05, indicating that there is no significant difference between
data from colorimetry and PEC. In addition, set the concentration
that detected by PEC and UV-Vis as the x axis and y axis,
respectively, a good linear relationship is obtained (Figure 5),
which indicates the practicability of this strategy in the spiked
river water.55 Compared with conventional single signal readout
model, this work with dual-modal signal readout exhibits a more
reliable and accurate result, benefiting from the different
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powder and solution; the CVs of MAA and acridone; the
electrochemical properties of MAA; the interaction mechanism
between MAA and BSA; detection of exosomal total protein; UV-
Vis and fluorescence spectra; cytotoxicity of MAA; one-photon
fluorescence images of HeLa cells; photobleaching of MAA;
Quantitative photobleaching results for one- and two-photon
imaging; one-photon fluorescence images of tumor speroids;
quantification graph of one- and two-photon fluorescence images
for 3DMTS; one-photon fluorescence images of zebrafish;
fluorescence images of exosomes; one-photon fluorescence
images of MAA and DiI labelled exosomes; visible-light-induced
oxidase mimic of MAA for different substrate; photocurrent of
MAA and MAA-Cu; steady-state kinetic analysis; the optimized
structures of MAA-TMB and 2-AA-TMB; the ESR spectra of
MAA and MAA-Cu; optimization of experimental conditions;
photographs of the colorimetric method at different concentrations
of Cu2+ (Figure S1-S26).
mechanism and relatively independent signal transduction.56
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y=0.988x+0.057
R2=0.9987
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2
AUTHOR INFORMATION
2
4
6
8
10
Concentration (M, PEC)
Figure 5. Correlation of the detection results of Cu2+ in the
recovery test between the developed colorimetric and
photoelectrochemical methods.
Corresponding Authors
*E-mail: 1601225385@qq.com (J. Zhang).
*E-mail: hhyang@fzu.edu.cn (H. Yang).
*E-mail: cjh_huaxue@126.com (J. Chen).
Conclusion
Notes
In summary, we synthesized a smart probe MAA by a facile
method and study its multiple properties in details, including
electrochemistry, one- and two-photon fluorescence, visible-light-
induced oxidase-mimicking, and PEC characteristic. Based on
these properties, MAA was employed to act as an electrochemical
indicator for exosomal total protein analysis firstly. Then, one-
and two-photon fluorescence images in cell, zebrafish, and
exosomes were conducted successfully based on the tempting
fluorescence property of MAA. Subsequently, we studied the
related mechanism about Cu2+ inhibiting visible-light-induced
oxidase mimic and PEC activities of MAA, and develop a dual-
modal strategy for Cu2+ detection. Continued research on
introducing different groups to further improve the performance
of MAA based on its structure modification flexibility.
Additionally, we envision building lipsome to encapsulate
MAA,57 it is not only a promising content-release and signal
amplification system when bind with target, but also it can realize
multiple-modal signal readout in one platform.58 Consequently,
we believe that the multifunctional MAA can be used as an ideal
candidate for many chemical and biochemical applications.
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors gratefully acknowledge the National Science
Foundation of China (Nos. 21874019, 21635002, 21622502,
U1505221, 81771881), the Natural Science Foundation of Fujian
Province (Nos. 2017J07001, 2016J05035, 2017J06004,
2018J07004), United Fujian Provincial Health and Education
Project for Tacking the Key Research P.R. China (WKJ2016-2-
30), the Fujian Science and Technology Innovation Joint Found
Project (2016Y9050), Startup Fund for Scientific Research, Fujian
Medical University (2017XQ2015).
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