A novel “OFF–ON–OFF” fluorescence chemosensor for hypersensitive detection and bioimaging of Al(Ⅲ) in living organisms and natural water environment

Article abstract of DOI:10.1016/j.jphotochem.2020.112618

High-selectivity detection of trace Al³⁺ ions in water and biological systems by a fluorescence imaging method is not yet mature. Herein, we synthesized a novel reversible Al³⁺-specific fluorescent probe named RBLF by linking Rhodami

Full text of DOI:10.1016/j.jphotochem.2020.112618

Journal Pre-proof
A novel “OFF–ON–OFF” fluorescence chemosensor for hypersensitive
detection and bioimaging of Al(null) in living organisms and natural water
environment
Chun Kan (Supervision), Xiaotao Shao (Conceptualization)
(Methodology) (Software) (Data
curation)<ce:contributor-role>Writing- original
draft)<ce:contributor-role>Writing- review and editing), Linyun Wu
(Visualization) (Investigation), Yao Zhang (Software), Xiaofeng Bao
(Validation), Jing Zhu (Supervision)
PII:
S1010-6030(20)30417-2
DOI:
https://doi.org/10.1016/j.jphotochem.2020.112618
JPC 112618
Reference:
To appear in:
Journal of Photochemistry & Photobiology, A: Chemistry
Received Date:
Revised Date:
Accepted Date:
10 February 2020
22 April 2020
6 May 2020
Please cite this article as: Kan C, Shao X, Wu L, Zhang Y, Bao X, Zhu J, A novel
“OFF–ON–OFF” fluorescence chemosensor for hypersensitive detection and bioimaging of
Al(x2162;) in living organisms and natural water environment, Journal of Photochemistry &
amp; Photobiology, A: Chemistry (2020),
doi: https://doi.org/10.1016/j.jphotochem.2020.112618

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A novel “OFF–ON–OFF” fluorescence chemosensor for hypersensitive detection
and bioimaging of Al(Ⅲ) in living organisms and natural water environment
Chun Kan a, 1, *, Xiaotao Shao a, 1, Linyun Wu a, Yao Zhang a, Xiaofeng Bao b, Jing
Zhu c, *
a
College of Science, Department of Chemistry and Material Science, Nanjing Forestry
University, 159 LongpanRoad, Nanjing 210037, PR China
b School of Environmental and Biological Engineering, Jiangsu Key Laboratory of Chemical
Pollution Control and Resources Reuse, Nanjing University of Science and Technology,
Nanjing 210094, China
c
Department of Pharmacy, Jiangsu Key Laboratory for Pharmacology and Safety
Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, 138
XianlinDadao, Nanjing 210023, China
*Corresponding author, Email address: kanchun@njfu.edu.cn (Chun Kan).
1These authors contributed equally to this work.
Highlights
 A new hypersensitive Al3+ probe based on rhodamine B was developed.
 The probe can be considered as a reversible fluorescent switch to detect Al3+ with
1:1 binding stoichiometry.
 The probe can respond immediately to Al3+, which is reflected in the instantaneous
change of solution color and fluorescence.
 The detection limit of this probe is as low as 14.23nM.
 The probe has been successfully applied to image Al3+ in living cells, animal and
plant tissues.
 The probe has been proved to work properly in natural water.
Abstract
High-selectivity detection of trace Al3+ ions in water and biological systems by a
fluorescence imaging method is not yet mature. Herein, we synthesized a novel
reversible Al3+-specific fluorescent probe named RBLF by linking Rhodamine B and
pyridine-3-sulfonyl chloride through o-phenylenediamine in several reactions. RBLF
exhibited an ultra-high sensitive and excellent selective response towards Al3+ by
fluorescence and UV-visible spectra in aqueous solutions. What is more worth
mentioning is that its response is fast, dual changes in color and fluorescence of the

probe solution that can occur instantaneously after adding with Al3+. Its detection limit
for Al3+ ions in aqueous system solutions is 14.23 nM, which is far below the maximum
allowable amount of Al3+ in drinking water. Moreover, the probe provided an effective
method for detecting low concentrations of Al3+ in natural water, living cells, zebrafish
and plant tissues.
Keywords: Hypersensitive fluorescent probe; Aluminum (Ⅲ) detection; Reversibility;
Natural water; Biological application
1. Introduction
As the third most abundant element in the earth's crust and the first most abundant
in metallic elements, aluminum is widely found in nature and the biosphere in the form
of silicate, bauxite and ion. At present, the progress and development of various
industries seem to be inseparable from aluminum, because of its excellent physical and
chemical properties, these also laid its status as the second category of metals.
Aluminum is often used in industrial production and daily life, such as water
purification, food additives, cosmetics and medicine, etc. [1]. This makes it easier for
aluminum to contact directly with living organisms, which may lead to environmental
degradation or accumulation of aluminum in the body [2]. However, aluminum is not
an essential metal element for human body, nor is it a trace element. When the content
of Al3+ in human body accumulates to a certain degree, it will produce toxicity harmful
to human. In 2017, aluminum was included in the list of carcinogens by the World
Health Organization's International Agency for Research on Cancer. Because human
brain tissue has affinity for Al3+, a large amount of Al3+ can destroy the human central
nervous system and immune system, cause erythrocyte hypochromic anemia, inhibit
the activity of a variety of enzymes in the body and accelerate human aging. In terms
of disease, Alzheimer disease [3-5]and ParKinson Diseautomotive service engineers
[6-9] can be induced; a large amount of Al3+ can also affect the ingestion and utilization
of essential microelements needed by human body, thereby increasing the risk of breast
cancer in women [10]. For animals and plants, when the content of Al3+ reaches a
certain level, the soil will become acidified, which is not conducive to the growth of
plant roots and seeds [11, 12]. When lakes, rivers and oceans contain excessive amounts
of Al3+, the growth and development of various aquatic fish will be inhibited, causing
fish die due to lack of essential nutrients [13]. The World Health Organization (WHO)
states that the weekly intake of Al3+ by human should be less than 7 mg per kilogram
[14]. Therefore, it is crucial to design a highly selective and sensitive method for
detecting the content of Al3+ in order to reduce its damage to the body and ecology.
Prior to the advent of new analytical methods, there have been several traditional
methods to detect aluminium ion. The widely used detection technology mainly
includes Inductively coupled plasma optical emission spectroscopy (ICP-OES),
Inductively coupled plasma mass spectrometry (ICP-MS), Graphite furnace atomic
absorption spectrometry (GF-AAS), Atomic fluorescence spectrometry (AFS) and
chromatography [15-19]. Although these analysis methods have the advantages of high

accuracy and good stability, the shortcomings such as high use and maintenance costs,
complicated equipment debugging, and poor convenience have greatly limited their
wide application. In recent years, fluorescence analysis has developed rapidly and
become a new research hotspot [20, 21]. Compared with traditional high sensitivity
methods, the fluorescence analysis method has the advantages of low cost, easy
operation, repeatability and diversity of detection materials. Fluorescence sensor is the
most typical method of fluorescence analysis. Moreover, fluorescent sensors have
obvious advantages over other methods in ion monitoring of living cells and biological
tissues, because of their nondestructive properties, instantaneous responsiveness and
indicator dye diversity [22-25]. Fluorescent sensors are mostly a class of compounds
derived from fluorescent groups such as Rhodamine[26-29], coumarin [30-32], pyrene
[33, 34] and naphthalene [35, 36]. Among them, Rhodamine is favored by researchers
for its excellent photostability, high fluorescence quantum yield, high molar extinction
coefficient, unique switching mechanism [17], low toxicity and other outstanding
photophysical properties, and has gradually developed into a popular fluorescent group
[37]. Most rhodamine-based fluorescent sensors work by opening and closing the spiral
ring. At first, the helix ring was in a closed loop state without fluorescence generation;
under the action of ion complexation, the rhodamine lactam spiral ring structure is
broken, and the ring opens and emits fluorescence [38, 39].
In this paper, we designed and synthesized an Al3+ hypersensitive fluorescence
sensor called RBLF with rhodamine B as the fluorophore. Firstly, rhodamine B reacts
with phosphorus oxychloride to get its acyl chloride, which makes it easier to react with
o-phenylenediamine later. We modified the probe (N-TC) synthesized in our previous
work and replaced the original linking group with o-phenylenediamine that has a rigid
structure and a strong planar conjugate effect [40]. The amino group is an electron-
donor group, which will greatly enhance the fluorescence effect and sensitivity of probe.
However, pyridine-3-sulfonyl chloride with good ion coordination ability was still
retained as the recognition group, and the final probe RBLF was obtained (scheme 1).

Scheme 1. Synthesis of RBLF
2. Experimental
2.1. Reagents and instrumentation
Unless specifically stated, none of the solvents and chemical reagents purchased
from Aladdin (Shanghai, China) require further purification. The water used in the
whole experiment was ultrapure water. The silica gel GF254 plate used for TLC
analysis and the silica gel used for chromatography column were all purchased from
Haiyang Chemical Co., Ltd. (Qingdao, China). Metal ion stock solution was prepared
in advance with nitrate or chloride corresponding to the metal.
An Avance III HD 600MHz fully digital superconducting nuclear magnetic
resonance spectrometer was used to record NMR spectra with CDCl3 as solvent and
tetramethylsilane (TMS) as the internal standard. The fluorescence spectra and the UV–
Vis absorption spectra were performed by an Edinburgh FLS920 fluorescence
spectrophotometer and a Shimadzu UV-3600 UV–vis-NIR spectrophotometer
respectively. Electrospray ionization mass spectra (ESI-MS) were acquired with a
Thermo-Fisher Scientific dual-focus high resolution instrument under electron
ionization conditions. Bioimaging were implemented with a laser confocal microscopy
(ZEISS, LSM710) and inverted fluorescence microscope (OLYMPUS, IX81).
2.2. Synthesis of compound 1
Rhodamine B (960 mg, 2 mmol) was stirred and dissolved in anhydrous
dichloroethane at a round bottom flask of 50 mL. POCl3 (1 mL, 11 mmol) was slowly
added under the atmosphere of N2, and then the system was reflux for 6h. After the
reaction, the solvent and excess POCl3 were removed by decompression to obtain the
purple oily acyl chloride. The purple oil obtained previously was redissolved in 40 mL
acetonitrile, o-phenylenediamine (432 mg, 2 mmol) dissolved in 10 mL acetonitrile and
triethylamine (1 mL, 7.2 mmol) was added in drops. The reaction process was
monitored by TLC after overnight stirring at room temperature. When the reaction was
completed, decompress to remove the solvent. The crude products were extracted three
times with dichloromethane and saturated NaCl solution, and then extracted once with
reversed-phase. The organic phase was dried, filtered, and concentrated by vacuum
1
distillation to obtain compound 1(787.8 mg, 74%). H NMR (600 MHz, CDCl3), δ
8.03(d, J=7.26 Hz, 1H), 7.56(m, 2H), 7.25(dd, J1=6.18 Hz, J2=1.5 Hz, 1H), 6.95(m, 1H),
6.64(s, 2H), 6.65(d, J=7.92 Hz 1H), 6.40(t, J=7.5 Hz, 1H), 6.27(t, J=33.72 Hz 4H),
13
6.08(d, J=7.14 Hz, 1H), 3.32(s, 8H), 1.63(m, 1H), 1.14(t, J=6.9 Hz, 12H) ppm. C
NMR(126 MHz, CDCl3), δ 166.50, 154.01, 152.32, 148.90, 144.54, 134.68,
132.67,131.97, 128.91, 128.73, 128.41, 124.33, 123.46, 122.12, 120.32, 118.23, 116.98,
116.81, 108.04, 98.10, 68.13,45.81, 45.20, 44.43, 12.60, 12.52, 8.62 ppm.
2.3. Synthesis of RBLF
Compound 1 (532 mg, 1 mmol) was dissolved in an appropriate amount of
dichloromethane, pyridine-3-sulfonyl chloride (179.04μL, 1.5 mmol) and triethylamine

(138.99 μL, 1 mmol) were added and stirred at room temperature for 6 h. The progress
of the reaction was monitored by chromatography. After the reaction, the solvent was
evaporated under reduced pressure. The compound was rapidly separated by silica gel
column with CH2Cl2/Ethyl Acetate (9:1, v/v) used as an eluent. Apale pink solid (342.4
mg, 51%) was obtained as the fluorescence sensor RBLF towards Al3+. 1H NMR (600
MHz, CDCl3), δ 8.99(d, J=1.74 Hz 1H), 8.76(dd, J1=4.74 Hz, J2=1.2 Hz, 1H), 7.99(t,
J=10.38 Hz 2H), 7.62(m, 2H), 7.38(m, 1H), 7.32(d, J=7.32 1H), 7.25(d, J=8.04 Hz, 1H),
7.10(m, 1H), 6.79(m, 1H), 6.62(t, J=8.88 3H), 6.44(d, J=7.56 2H), 6.27(s, 2H), 6.21(dd,
J1=7.92 Hz, J2=1.2 Hz, 1H), 3.36(m, 8H), 1.67(s, 1H), 1.18(t J=7.02 Hz 12H) ppm. 13C
NMR(126 MHz, CDCl3), δ 168.08, 153.95, 153.22, 151.49, 149.31, 148.07,
136.71,134.97, 134.82, 133.26, 131.47, 130.27, 129.01, 128.82, 128.07, 126.91, 124.80,
124.51, 123.79, 123.60, 120.43, 108.84, 105.93, 98.03, 68.85, 53.45, 44.49, 31.44,
30.21, 29.70, 12.48 ppm. ESI-MS m/z: calculated for C39H39N5O4S: 673.2723, [M+H]+
found, 674.2804; [M+Na]+ found, 696.2626.
2.4. Preparation for spectral experiments
The stock solutions of cationic (5mM) including Al3+, Ag+, Cr3+, Ca2+, Cd2+, Co2+,
Fe2+, Fe3+, Li+, K+, Mn2+, Mg2+, Ni+, Na+, Zn2+ and Pb2+ were respectively prepared.
−
−
−
2−
2−
Some common anions including H2PO4 , Br−, BrO3 , B4O72−, I−, NO3 , SO4 , S2O8
−
and IO3 are also configured as solutions in the same way as before. The stock solution
of RBLF was prepared at 5mM with the solution of EtOH/H2O (1:2, v/v). The excitation
wavelength for spectral experiments was selected at 520nm, and the emission
wavelength was selected from 530nm to 700nm. In addition, the reversibility of RBLF
was determined by fluorescence spectrometry and UV–Vis absorption spectrometry
with fluoride ion solution. To verify whether RBLF can respond in the real environment,
we examined its performance in natural water. In addition to that, we adjusted the pH
of the system to around 7.3 before testing, which simulated the physiological pH
environment, and completed the test as soon as possible.
2.5. Cytotoxicity and cell imaging studies
The incubation conditions and methods of the cells, as well as the specific
procedures of toxicity test and cell imaging, were described in the supporting materials.
2.6. Zebrafish and plant imaging
Detailed information about zebrafish and the processing of biometric imaging are
described in the supplementary materials.
3. Results and discussion
3.1. Spectral response of probe toward metal ions and anions

For the sake of determining which ions can be detected by RBLF, several common
metal ions including Ag+, Al3+, Ca2+, Cd2+, Co2+, Cr3+, Fe2+, Fe3+, K+, Li+, Mg2+, Mn2+,
Na+, Ni+, Pb2+ and Zn2+ were selected for fluorescence spectrum and UV-Vis absorption
spectrum test in EtOH/H2O (1:2, v/v) solution containing 10μM RBLF. The specificity
of probe is the primary factor to test whether the probe has good performance.
Fluorescence spectrum test results showed that the intensity of the solution increased
significantly only when added Al3+. Although the addition of Fe2+ produced a slight
fluorescence enhancement effect higher than other ions, it was negligible compared to
Al3+. The addition of other ions had little effect on the fluorescence intensity of the
solution originally containing only RBLF (Fig. 1). In addition, the solution added with
Al3+ became pink visible to the naked eye, while the other solutions remained colorless
and transparent. RBLF itself has no absorption peak at 500-600nm. Only after adding
Al3+, obvious absorption peak also appeared in the UV-Vis absorption spectrum (Fig.
S6).
Fig. 1. (A) Fluorescence spectra of RBLF (10 μM) in a EtOH/H2O (1:2, v/v) solution
with the existence of various metal ions. The inset in (A) represents the fluorescence
intensity (λex= 520nm, λem=582nm) of RBLF in the presence of various metal ions.
We also designed a set of competitive experiments to verify whether RBLF can
accurately detect Al3+ in this environment when other metal ions are present, so as to
evaluate the specificity of RBLF to Al3+. As shown in Fig. 2, the fluorescence intensity
of solutions containing RBLF and other measured metal ions increased sharply after
the addition of Al3+, which was close to or even more than that of the probe solution

in presence of only Al3+. This indicated that other metal ions have no negative impact
on the specificity of RBLF for detecting Al3+, which indirectly indicates that RBLF has
higher selectivity.
In the normal working environment of the probe, there must be not only metal ions,
but also other anions. Therefore, we designed an experiment to test the interference of
−
−
2−
−
several anions during RBLF operation. We added H2PO4 , Br−, BrO3 , B4O7 , I−, NO3 ,
SO4 , S2O8 and IO3 respectively to the solution containing Al3+ and RBLF.
Excitingly, even with the addition of the above anions, the fluorescence intensity of the
original solution did not decrease. This showed that even in complex ionic environment
RBLF can also efficiently detect Al3+, which provides the possibility to detect Al3+ in
natural water environment (Fig. S7).
2−
2−
−
Fig. 2. Comparison of fluorescence intensity of probe solutions containing different
metal ions (10 μM) before and after adding Al3+ in EtOH/H2O solution (1:2, v/v). Blue
bars: RBLF added with several common cations. Red bars: add Al3+ to above mixed
solutions.
3.2. Response of RBLF to pH value and time
The spiral ring of rhodamine compounds are sensitive to pH. When the pH value
of the environment is very low, spironolactone is destroyed to produce a strong
fluorescence, which may be a potential interference factor, so the pH titration
experiment is very necessary. As can be seen from Fig. S8, when pH < 6, RBLF had a
distinct fluorescent signal, but gradually decreased. When pH is between 6 and 12,
RBLF has almost no fluorescence. For RBLF-Al3+, when pH value was less than 8, the
fluorescence intensity was strong and stable. When pH > 8, fluorescence intensity
began to decline drastically. The above test results showed that the RBLF fluorescence
signal is weak at physiological pH, which provides a good fluorescence background for
biological imaging of RBLF. However, RBLF-Al3+ had a strong fluorescence intensity,

indicating that bioimaging is achievable.
The response time of the probe is also an important criterion to evaluate its
performance, so we explored the response time of RBLF. 60μL of Al3+ was added to
the probe system solution prepared in advance, and then the fluorescence intensity was
tested every 20s. As shown in Fig. 3, the fluorescence intensity increased significantly
after the addition of Al3+. The rising speed was obviously accelerated between 80s and
180s, but gradually slowed down after 180s, and the fluorescence intensity reached its
maximum and became stable after 240s. Therefore, RBLF can be viewed as a
hypersensitive probe for Al3+. All the above experiments were conducted in EtOH/H2O
(1:2, v/v) solution.
Fig. 3. Fluorescence spectra of RBLF (10 μM) with Al3+ in the EtOH/H2O (1:2, v/v)
solution. The excitation wavelengths were 520 nm and the wavelengths of emission
were 582 nm. Inset: Plot of the fluorescence intensities at 582 nm during a period of
280s.
3.3. Sensitive quantification of RBLF to Al3+
To further explore the sensing properties of the probe along with the interaction
between RBLF and Al3+, a fluorescence titration experiment were performed.
According to the fluorescence titration experiment results (Fig. 4), when Al3+ was
slowly added dropwise in EtOH/H2O (1:2, v/v) solution containing RBLF, the
fluorescence intensity of the mixed solution began to rise slowly, and the color of the
solution turned pink. When 1.2 equivalents of Al3+ were added, the fluorescence
intensity increased rapidly and the color of the solution deepened. After adding 10

equivalent of Al3+, the rising speed of fluorescence intensity slowed down, and the
fluorescence intensity tended to be stable after adding 20 equivalent of Al3+. Therefore,
RBLF responded to the increase concentration of Al3+ with fluorescence intensity and
color.
In order to further explore the binding mechanism of RBLF and Al3+, we
calculated the binding constant of RBLF, which is a key factor, according to the Benesi-
Hildebrand formula (F-F0=ΔF= [Al3+] (Fmax-F0)/(1/Ka+[Al3+])based on the titration
experiment results. The binding constant is calculated by linearly fitting the
independent variable 1/[Al3+] with the dependent variable 1/(Fmax-F0). Where F0 is the
initial fluorescence intensity of probe RBLF, F is the fluorescence intensity of RBLF
after adding a certain amount of Al3+, and Fmax is the fluorescence intensity when Al3+
is excessive. From this linear relationship (Fig. S9A), the concentration range of Al3+
in the unknown solution can be roughly determined. In addition, the binding constant
can be calculated as1.16×104 M-1.
The detection limit is also a key component of probe performance. In the
beginning, we measured the fluorescence spectroscopy of standard solution containing
RBLF only every 5 min for a total of 10 times, and calculated the standard deviation of
fluorescence intensity with the fluorescence value at 582 nm (Fig. S9B). Then,
according to the formula DL=3SD/S, the detection limit of RBLF for Al3+ was
calculated to be 14.23nM, which was far lower than the maximum of 7μM in drinking
water stipulated by the World Health Organization.
Fig. 4. Different concentrations of Al3+ (0–50 equiv.) was added in EtOH/H2O (1:2, v/v)
solutions containing RBLF (10 μM). Inset: Fluorescence intensities plot of mixed
solution at 582 nm with gradually increased addition of Al3+.

3.4. Stoichiometry and sensing mechanism of RBLF
In order to further study the stoichiometry of RBLF and Al3+, the Job's plot curve
was drawn to confirm the complexation ratio of ions and target compounds in the liquid
phase. In this work, the coordination ratio between RBLF and Al3+ was determined by
reference to the fluorescence spectrum. The total concentration of RBLF and Al3+
remained at 50 μM, and the Al3+ content ratio ranged from 0 to1. The concentration of
Al3+ is accounted for as a function of the fluorescence intensity of the solution at 582
nm. As can be seen from the Fig. 5, when the mole fraction of Al3+ was 0.5, the
fluorescence intensity was the maximum, indicating that RBLF and Al3+ are 1:1 ratio.
On the basis of above experimental results, a reasonable sensing mechanism of RBLF
was proposed (Scheme 2).
Fig. 5. Job's plot to determine the stoichiometry between RBLF and Al3+
([RBLF]+[Al3+]=50 μM) in EtOH/H2O (1:2, v/v) solution at 582 nm.
Reversibility is a common characteristic of rhodamine-based probes for practical
application, and the reversibility of RBLF was studied below. As shown in the Fig. 6, a
constant amount of Al3+ and F- were added alternately and repeatedly in the solution of
RBLF to make a switchable fluorescence intensity change at 582nm, and the operation
could be repeated indefinitely by adding Al3+and F- successively. Therefore, the original

RBLF had fluorescence enhanced response to Al3+, which could be reversed by adding
F-. In other words, the original RBLF is the specific probe of Al3+, while RBLF-Al3+
can work as a probe for F-. This is mainly because fluorine and aluminum ions are
anions and cations, and they will form chemical bonds. The difference in
electronegativity between aluminum and fluorine atoms is greater than 1.7, so they may
form ionic bonds. Or they form complexes that reduce the coordination between Al3+
and RBLF.
Fig. 6. Fluorescent intensity of RBLF (10μM) at 582 nm in the solution of EtOH/H2O
(1:2, v/v) upon the alternate addition of Al3+ (1 equiv.) and F- (2 equiv.). Images of
fluorescence reversibility under 365 nm UV lamp.
Scheme 2. Proposed sensing mechanism of RBLF towards Al3+

To further elucidate the proposed sensing mechanism or the property of the
complexation between RBLF and Al3+, 1H NMR titration of RBLF was performed in
the absence or existence of Al3+ in DMSO-d6 solution (Fig. 7). As seen in the 1H NMR
spectra of free RBLF, the aromatic proton peak is strong and sharp. After the addition
of Al3+, the chemical shift of several peaks have changed, especially the Hq peak on the
pyridine ring. Its chemical shift was originally around δ 7.85 ppm. After adding Al3+,
the peak became weaker and wider, and the proton signal upfield shifted to around δ
7.80 ppm. This indicated that the N on the pyridine ring also participated in the
coordination of Al3+. Additionally, upon the addition of Al3+, a new strong peak
appeared at δ 5.7-5.8ppm, which is the active hydrogen of N in rhodamine
spironolactone, indicating that spironolactone transforms into an open-loop amide and
complexed with Al3+.
Fig. 7. Partial spectra of 1H NMR titration of RBLF with Al3+ in DMSO-d6
3.5. Study on detection of Al3+ by RBLF in natural water
In order to explore whether the probe RBLF can detect trace Al3+ well in the
natural water environment which is much more complicated than the test environment,
we collected two local river and lake water samples (Xuanwu Lake and Zihu Creek)for
experiment. All the water samples were filtered for removing excess Al3+. After that,
different amount of Al3+ were spiked within the samples. As can be seen from the Table
1, the recovery of Al3+ in natural water ranges from 89.6% to 99.0%. It is obvious that
even in real water samples where impurities and ions may interfere with the
complexation of RBLF with Al3+ and affect the fluorescence intensity, the recovery is
still good. Thus, the above experiments showed that RBLF could be used to detect Al3+
in the real environment.

3.6. Cytotoxicity and detection of intracellular Al3+
For further evaluating the bioavailability and value in intracellular Al3+
bioimaging of the synthesized probe RBLF, we conducted a living cell fluorescence
imaging experiment. Firstly, MTT assay was used to study the cytotoxicity, and the
possibility of its application in cells was preliminarily verified. As can be seen From
the Fig. S10, even when the concentration of RBLF in the environment reaches 20 μM,
the survival rate of MCF-7 cells can be maintained more than 85%. As expected, RBLF
has lower toxicity and is fully capable of working well in cells. Next, we designed
fluorescence experiments to observe the intracellular imaging of RBLF. We used blue
channels to track living cells, and divided the cells into three groups, which were
incubated with Al3+, RBLF, and the both. We used blue channels to mark cells and
observed cell imaging through bright fields, blue channels, fluorescent channels, and
superimposed channels. As shown in the Fig. 8, cells incubated with Al3+ alone did not
produce fluorescence. When the cells were incubated with RBLF alone for a few hours,
weak fluorescence began to appear in the cells. After adding 10μM of Al3+ for co-
incubation for a period of time, the cells maintained normal activity and morphology,
and a wide range of intense fluorescence appeared in the cells. The above results
indicated that RBLF had low biotoxicity and good biocompatibility. Ensure that it can
detect Al3+ normally in cells without affecting cell survival and reproduction.
Fig. 8. LSCM (laser scanning confocal microscopy) images of RBLF in living MCF-7
cells. (A-D): Cells with only treatment; (E-F): Cells treated with only RBLF (10 μM);
(I-L): Cells co-treated with RBLF and Al3+ (10 μM). (A, E, I) Bright field; (B, F, J) Blue
channel (λex = 340 nm, monitored at 420-480 nm); (C, G, K) Fluorescent channel (λex=
550 nm, λem = 560-680 nm); (D, H, L) Overlay channel. Conditions: 5% CO2/95% air
atmosphere at 37 ℃.

3.7. Fluorescence microscope images of living organisms
In addition to the above mentioned cell imaging, we also attempted to apply RBLF
to detect trace aluminum ions in living organisms. We selected zebrafish with high
transparency, small size and soybean sprout tissue with high water content, good
imaging effect for experiment. As shown in Fig. 9, almost no fluorescence was observed
in the zebrafish incubated in an aqueous solution containing only RBLF (A-C). After
30 min of incubation with Al3+, the fish began to show fluorescence in the body.
Especially in the fish's internal organs and tissue fluids, the fluorescence was more
pronounced, forming the outline of the entire zebrafish (D-F). Similarly, soybean
sprouts grown in an environment that contained Al3+ showed no fluorescence,
compared to a group of beans grown in an Al3+ solution that were immersed in a probe
solution for another 30 min and produced an incredibly bright red fluorescence
(Fig. 10). The above results indicate that RBLF, with its high sensitivity and
biocompatibility, can effectively detect trace amounts of Al3+ in living organisms,
showing great development prospects in biological detection.
Fig. 9. LSCM images of zebrafish (λex=543nm). Zebrafish were incubated for 30 min
in a solution of only RBLF (10μM) (A-C). Zebrafish firstly cultured for 30 min with
RBLF and then fed with Al3+ (10 μM) (D-F). (A, D) Bright field, (B, E) Mixed images,
(C, F) Fluorescent images. Conditions: 100% air atmosphere at 25 ℃.

Fig. 10. LSCM images of soybean sprouts root tissues (λex=543nm). Soybean raised in
the environment with only RBLF (10μM) (A-C). Soybean germinated in the
environment with only Al3+ (10μM) and also treated with RBLF (10μM) for 30 min (D-
F). (A, D) Bright field, (B, E) Fluorescent images, (C, F) Mixed images. Conditions:
100% air atmosphere at 25 ℃.
4. Conclusion
In summary, an Al3+ hypersensitive fluorescent probe RBLF based on rhodamine
B was synthesized and characterized. It can be regarded as a naked-eye probe, which
produces fluorescence effect and color change immediately after adding Al3+. RBLF
exhibits high selectivity and high sensitivity for Al3+, it has a lower detection limit of
14.23nM and a shorter response time of only 280s. In addition, RBLF-Al3+ can realize
an “OFF-ON” fluorescence response through adding F-, the solution also become
colorless gradually. RBLF-Al3+ has a good selectivity to F-, which can be thought as a
fluorescence probe. Furthermore, RBLF has been successfully used to detect trace
amounts of Al3+ in natural water, living cells, zebrafish and plant tissues on the basis of
its good water solubility and low toxicity.

Declaration of Interest Statement
We declare that we have no financial and personal relationships with other
people or organizations that can inappropriately influence our work, there is no
professional or other personal interest of any nature or kind in any product, service
and/or company that could be construed as influencing the position presented in, or
the review of, the manuscript entitled.
Author statement
Chun Kan and Jing Zhu: Supervision.
Xiaotao Shao: Conceptualization, Methodology, Software, Data curation, Writing-
Original draft preparation, Writing- Reviewing and Editing.
Linyun Wu: Visualization, Investigation.
Yao Zhang: Software
Xiaofeng Bao: Validation.
Acknowledgements
The authors express their gratitude to Jiangsu Six Talent Peak Award
(2015SWYY013) and The Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD), which provided financial supports. We also gratefully
and sincerely acknowledge the technical along with equipment supports from
Advanced analysis and testing center of Nanjing Forestry University.
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Table 1. Results for the determination of Al3+ in water sample
Sample
Al3+ added (μM)
Detected (μM)
2.69 ± 0.03
5.94 ± 0.09
8.67 ± 0.17
11.65 ± 0.09
14.01 ± 0.22
16.61 ± 0.18
2.78 ± 0.04
5.69 ± 0.09
8.86 ± 0.07
11.70 ± 0.11
14.37 ± 0.13
16.24 ± 0.06
Recovery (%)
89.6
3
6
9
12
15
18
3
6
9
12
15
18
Xuanwu Lake
99.0
96.3
97.1
93.7
92.3
92.5
94.9
98.4
Zihu Creek
97.5
95.8
90.2