Simple Colorimetric and Fluorescence Chemosensing Probe for Selective Detection of Sn2+Ions in an Aqueous Solution: Evaluation of the Novel Sensing Mechanism and Its Bioimaging Applications

of the Novel Sensing Mechanism and Its Bioimaging Applications

Article

Simple Colorimetric and Fluorescence Chemosensing Probe for

Selective Detection of Sn Ions in an Aqueous Solution: Evaluation

Palanisamy Ravichandiran, Vignesh Krishnamoorthi Kaliannagounder, Antony Paulraj Bella,

Anna Boguszewska-Czubara, Maciej Mas ł yk, Cheol Sang Kim, Chan Hee Park, Princy Merlin Johnson,

Byung-Hyun Park, Myung-Kwan Han, Ae Rhan Kim, and Dong Jin Yoo *

Cite This: https://dx.doi.org/10.1021/acs.analchem.0c03196

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sı Supporting Information

ABSTRACT: An easily accessible colorimetric and ﬂuorescence

probe 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-

benzenesulfonamide (4CBS) was successfully developed for the

selective and sensitive detection of Sn²in an aqueous solution.

The sensing mechanism involves reduction of −CO into −C−

OH groups in 4CBS upon the addition of Sn , which initiates the

ﬂuorescence turn-on mode. A better linear relationship was

achieved between ﬂuorescence intensity and Sn²⁺concentration

in the range of 0−62.5 μM, with a detection limit (LOD) of 0.115

μM. The binding mechanism of 4CBS for Sn²was conﬁrmed by

Fourier transform infrared analysis, NMR titrations, and mass

(

electrospray ionization) spectral analysis. Likewise, the proposed

sensing mechanism was supported by quantum chemical

calculations. Moreover, bioimaging studies demonstrated that the chemosensing probe 4CBS is an eﬀective ﬂuorescent marker

for the detection of Sn in living cells and zebraﬁsh. Signiﬁcantly, 4CBS was able to discriminate between Sn in human cancer cells

and Sn in normal live cells.

ndustrialization has resulted in the release of a large amount

Nevertheless, investigations on the physiological mechanisms

associated with Sn(II) ions are limited because of the lack of

eﬀective Sn(II)-sensing methods. In the environmental

perspective, Sn(II) can remain in the environment for a

longer time and fairly remain non-biodegradable, while the

accumulation of Sn(II) for several years on water soils

distresses the microorganisms. Generally, Sn(II) can pollute

the water system which induces the toxicity to algae,

of heavy metals into the environment, seriously threatening

−3

human life.

In the human body, small concentrations of

inorganic cations take part in signiﬁcant biological, chemical,

and enzymatic activities at the cellular level. However, excess

consumption of these metal ions may lead to numerous neural

disorders such as Wilson’s disease, Alzheimer’s disease, and

Menkes syndrome. Among the various transition metals, tin

(

Sn) is an important element in human health. At trace levels,

phytoplankton, and fungi. Phytoplankton is an important

microorganism which delivers oxygen to the other micro-

organisms in water. This is a signiﬁcant measure of the food

chain in the aquatic system. The surface of the water can be

mainly polluted in contact with Sn(II) that inhibits the

penetration of sunlight which pave the way of harmful eﬀects

Sn(II) could help in the development of body muscles and act

as an eﬀective catalyst in the synthesis of nucleic acids and

proteins. Tin also supports hair growth, maintains body

homeostasis, and prohibits spread of cancer cells in humans. A

lack of Sn(II) can cause serious issues in humans such as loss

of hearing, breathing trouble, and shortage of hemoglobin

to the aquatics life. Besides, Sn(II) is the most dangerous

production. However, accumulation of large amounts of

Sn(II) can damage live cells, which would lead to inhibited

zinc metabolism. This in turn can cause severe illness in both

divalent metal ion which induce several serious eﬀects to the

Received: July 28, 2020

Accepted: November 26, 2020

the lung and gastric systems. Given the dangerous impacts of

Sn(II) to humans, consumption of Sn(II) must be closely

monitored. According to the World Health Organization’s

(

WHO) guiding principles for metals, the maximum levels of

allowed Sn(II) in drinking water and canned foods are 8.4 ×

−

−3

−6

to 8.4 × 10 and 2.105 × 10 M, respectively.

XXXX American Chemical Society

Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Article

humans. In recent times, Sn(II) is utilized in many ﬁelds such

as paint/plastic industries and agricultural ﬁeld via pesticides.

The utilization of Sn(II) continuously increases, which is one

of the major reasons for its poisoning. The Sn(II) has

comparatively short hydrogen bonds, whereas humans can

intake Sn(II) by breathing, by the consumption of food, and by

the skin. The accumulation of Sn(II) can induce acute and

long-term eﬀects in the human body. The acute eﬀects include

eye irritations, heavy sweating, breathing trouble, and urination

complications. The long-term eﬀects are damage in liver

functioning, disorders in the immune system, chromosomal

antibacterial properties. It was established that nitrogen

and sulfur atoms play a vital role to enhance the biological

activities showed by the compounds. Also, naphthoquinone

derivatives possess excellent ﬂuorescent behavior, a good molar

absorption coeﬃcient, high quantum yield, and relatively low

toxicity to normal human cells. However, their utilization as

chemical sensors for metal ions or biomolecule recognition is

very limited. Hence, naphthoquinone backbones are suitable

candidates for further development of selective chemosensing

probes in the ﬁeld of optical sensors.

Recently, some chemosensing probes were reported for the

detection of Sn ions. For example, Jonaghani et al. reported

destruction, damage in the brain, and lack of red blood cells.

Hence, Sn(II) is the most relevant and signiﬁcant metal ion to

study about its biological and environmental impacts. Besides,

there is a signiﬁcant need to develop simple and eﬃcient

methods for the detection of Sn(II) in the environment and

biological bodies. This should provide precise, rapid, and

highly sensitive responses to speciﬁc analytes.

the synthesis of quinoline-based naphthothiazole as a

ﬂuorescent and colorimetric chemosensing probe for Zn

and Sn ions. Qu et al. described the preparation of

carbazole-containing diarylethene units for the selective

detection of Sn and Cu ions. Very recently, Singh et al.

reported the synthesis of N-heterocyclic unsymmetrical

Several analytical techniques have been reported for the

detection and quantitative analysis of Sn ions. The

determination of Sn²was reported using the atomic

absorption spectroscopy in the aqueous solution with an

organosilatranes for the ﬂuorescence detection of Sn ions.

Salem et al. prepared citrate-stabilized silver nanoparticles for

in situ sensing of Sn ions. Gao et al. described the synthesis

of chiral carbon dot-based nanosensors for Sn and lysine

enantiomer recognition. Recently, Gul et al. described the

LOD of 8.4 × 10⁻M. Inductively coupled plasma mass

spectroscopy was used for the sensing of Sn and Sn ions.

synthesis of phenolphthalein and BODIPY-based colorimetric/

−

The LOD for this study was achieved as 1.1 × 10 and 7.0 ×

ﬂuorescence dual-channel sensors for Sn and Al recognition

−

M, respectively. Recently, UV/vis spectroscopy was

and evaluated their bioimaging applications. Most of these

employed in the identiﬁcation of Sn and the probe exhibits

reported chemosensing probes have several practical disadvan-

tages such as interference of coexisting metal ions, poor

performance in aqueous solutions, poor selectivity, weak

emission properties, and diminished biocompatibility which

means that they induce the toxicity or immune response upon

residing in the human/animal living cells. Thus, in the

treatment of medical implants to the humans, the biocompat-

ibility of the material is very important to avoid the denial of

an LOD of 7.5 × 10⁻M. X-ray ﬂuorescence spectroscopy

was used in the determination of Sn in canned foods with an

LOD of 4.2 × 10⁻M. Also, a polymeric membrane-based

ionophore was used to detect Sn in real samples with an

LOD of 4.0 × 10 M via potentiometric membrane detection

−

techniques. These methods have several practical disadvan-

tages such as the need for expensive instruments/sophisticated

maintenance, uncertain sample preparation, and the need for

skilled operators. In contrast, optical recognition of analytes

speciﬁcally for toxic materials is attracting wide attention

among researchers in chemical, biological, and environmental

implantation by the body cells and tissues.

The development of dual-channel ﬂuorescence and colori-

metric sensing probes for the detection of metal ions in the

aqueous solutions is needed because of their color-based easy

detection and need for inexpensive instruments. Speciﬁcally,

dual-channel recognition of single-metal ion detection attracts

wide attention because of the simple naked-eye detection, less

time-consuming process, and highly eﬃcient ﬂuorescence

analyses. These optical sensors provide simple operation and

have several practical advantages such as high selectivity/

sensitivity, rapid response, inexpensive cost, real-time detec-

tion, and no need for sophisticated instruments. Among

various optical sensors available today, ﬂuorescence chemo-

sensing probes have attracted wide attention and are

recognized as a powerful tool for the detection of speciﬁc

targets because of their simple operation, rapid response, and

analysis. However, the maximum of the available dual-

channel sensing probes are highly active in the organic

medium, although these probes have shown weak sensing

performance in the aqueous medium that restricts the

applications in the environmental analysis. Besides, these

probes are interfered with the same family of alkaline earth

ability to bioimage live human or animal cells. Colorimetric

chemosensing probes have received much attention for metal

ion detection because of their simple “naked-eye” recognition

that can be easily monitored using simple spectroscopic

instruments or even by an untrained technician. These

combined colorimetric and ﬂuorescence dual-channel chemo-

sensing probes provide deﬁned data with simple operative

metal ions. Hence, inspired by the requirement of active

colorimetric/ﬂuorescence chemosensing probes in the aqueous

medium, we report the synthesis and sensing behavior of a new

ﬂuorescence and colorimetric chemosensing probe 4-((3-

chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-

benzenesulfonamide (4CBS) that exhibited excellent selectiv-

modes. Typically, chemosensing probes consist of two major

units, a receptor and a signal unit. Receptors such as amides,

thiophenols, ureas, and pyrrole moieties are often used to bind

with suitable metal ions, and molecules such as naphthoqui-

nones function as a reporter for the detection of speciﬁc metal

ity and sensitivity toward Sn in water (20 mM HEPES, pH =

7.5). The chemosensing probe displayed higher selectivity

toward Sn , whereas the interference of the coexisting metal

ions was strictly inhibited. Moreover, the chemosensing probe

4CBS presented a good limit of detection, complex stability

ions. Naphthoquinone cores are widely present in many

natural compounds such as lawsone, menadione, and

constant (K ), and rapid response (less than 10 s) in the

plumbagin. In our previous reports, we synthesized a new

library of 1,4-naphthoquinones grafted with nitrogen and sulfur

aqueous medium than the formerly stated chemosensing

probes. Also, the sensor probe 4CBS functioned through a

novel sensing mechanism called reduction-enabled ﬂuores-

atoms and explored their anticancer, cytotoxic, and

Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Article

Scheme 1. Green Synthesis of the Chemosensing Probe 4CBS in Water

cence turn-on response which was thoroughly explored by

Fourier transform infrared (FT-IR), NMR, and mass [electro-

spray ionization−mass spectrometry (ESI−MS)] spectral

analyses. The detection of Sn²was accomplished in human

live cells and zebraﬁsh. Moreover, discriminative identiﬁcation

3 h. Later, the optical density was recorded at 450 nm

(Microplate reader, Tecan, Austria). The cells without sample

treatment were used as a control, and the cytotoxicity test was

performed in triplicate.

Live Cell Imaging Study. L929, HaCaT, HeLa, and MCF-

7 cells were cultured in 24-well plates for 24 h for ﬂuorescence

bioimaging studies. Next, the culture media was removed and

washed twice with 1× phosphate-buﬀered saline (PBS, pH =

of Sn in human cancer cells was demonstrated to prove the

utility of the sensor probe 4CBS. Moreover, very few sensing

probes could discriminatively detect Sn in human cancer cells

from normal cells, and our reported chemosensing probe

7.4). The cells were treated with 6.25 μM Sn for 10 min.

After incubation, the cells were washed twice with PBS and

treated with 6.25 μM 4CBS for 10 min. Subsequently, the cells

were washed thrice using PBS and ﬁxed with paraformaldehyde

CBS shows the best performances in terms of quick response

(

within 10 min) and the lower concentration of the ligand

used.

(

4%, Biosesang, Korea) solution for 5 min. Fluorescence

images of the cells were captured using a ﬂuorescence

microscope (ECLIPSE Ts2-FL, Nikon) with 50 μm magniﬁ-

cation under red and green ﬁlters. DIC images were captured

under the white LED source. Fluorescence bioimaging studies

were performed under a green ﬁlter (excitation = 488 nm;

emission = 548 nm) and a red ﬁlter (excitation = 594 nm;

emission = 654 nm). The mean ﬂuorescence intensity of

EXPERIMENTAL SECTION

■

Synthesis of the Ligand 4CBS. The ligand 4CBS was

synthesized according to our previously reported procedure

with slight modiﬁcations (Scheme 1). The procedure for the

preparation of the ligand and its structural characterizations are

brieﬂy deliberated in the Supporting Information (Figures S1,

S2).

bioimages was determined using ImageJ software.

Preparation of the Stock Solution. The stock solution of

Zebraﬁsh Imaging Study. Hatched zebraﬁsh babies were

−

.0 × 10 M 4CBS was prepared in 10 mL of dimethyl

maintained in an E3 embryo medium (28 °C). At ﬁrst, 6.25

−

sulfoxide (DMSO) (0.0072 g). 1.0 × 10 M alkaline metal

μM Sn (predissolved in water) was incubated with the

ions of Na , K , Cs , Li , Sr , Ba , Cd , Zn , Sn , Ni ,

zebraﬁsh for 20 min. After the treatment period, the unreacted

Co , Mg , Ca , Mn , Hg , Cu , Fe , Fe , Al , La , and

Sn was removed by washing with PBS (pH = 7.4). Next, the

Sn were prepared using double-distilled water. From the

metal-treated zebraﬁsh were further incubated with 6.25 μM

CBS for 30 min. Finally, the unreacted traces of 4CBS were

−

prepared stock solutions, 10 μL (2.50 × 10₅M) of a metal ion

solution and 25 μL of 4CBS (1.25 × 10⁻M, predissolved in

DMSO) were added into 3965 μL of the aqueous solution (20

mM HEPES, pH = 7.5). The proportion of the ﬁnal 4000 μL

of the prepared test solution contains 3975 μL of water and 25

μL of DMSO. The prepared solutions were used for metal ion-

binding studies. Spectroscopic studies were carried out at room

temperature (RT).

removed by washing with PBS, and ﬂuorescence bioimaging of

zebraﬁsh was conducted using a confocal laser scanning

microscope (Carl Zeiss, Germany, LSM 510 META) under a

green ﬁlter (excitation = 550 nm; emission = 580 nm) and red

ﬁlter (excitation = 630 nm; emission = 660 nm) installed at the

Center for University-Wide Research Facilities (CURF) at

Jeonbuk National University (JBNU, Jeonju, South Korea).

CBS−Sn Complex Preparation for Mass (ESI-MS)

and FT-IR Analyses. An acetonitrile solution (CH CN, 15

RESULTS AND DISCUSSION

mL) of the sensor probe 4CBS (0.054 g, 0.01 M) and SnCl₂

0.028 g, 0.01 M) was constantly stirred in the presence of

trimethylamine (0.5 equiv) for 3 h at RT. The obtained solid

■

(

Design and Synthesis of the Ligand 4CBS. The

reducing and electrophilic properties are the most signiﬁcant

complex was ﬁltered and washed with anhydrous CH CN (40

characters of the Sn ion. From our previous report, it was

mL). The isolated ﬁnal product was characterized by FT-IR

and ESI-MS analyses.

In Vitro Cytotoxicity Assay of 4CBS. Mouse ﬁbroblast

cells (L929), human keratinocyte cells (HaCaT), human

cervical cancer cells (HeLa), and human breast cancer cells

found that the Sn ion binds with the host molecule, the

strong electronegative −OH or −NH groups preferably

participating in the coordination mechanism. Nevertheless,

if the host molecule does not have −OH groups, the available

redox centers (−CO) in the host molecule can be reduced

(

MCF-7) were cultured in Dulbecco’s modiﬁed Eagle medium

to −C−OH groups by the metal Sn that enables the metal to

Hyclone, USA) at 37 °C with 5% CO . For cytotoxicity

bind. With respect to these features of Sn , we rationally

designed and synthesized the chemosensing probe 4CBS

which consists of strong electronegative groups (−NH) and

analysis, both cells were seeded with a density of 30,000 cells/

well. After monolayer conﬂuency, a cell culture media with 11

various concentrations of 4CBS (0.0975−100 μM, predis-

solved in DMSO) was added to the respective wells. After 24 h

treatment of 4CBS with cells, a 10% CCK-8 solution (Dojindo

Laboratories, Japan) was added to each well and incubated for

reactive redox centers (−CO) for the detection of the Sn

ion. The synthesis of the chemosensing probe 4CBS was

achieved by a simple, green approach, as presented in Scheme

1. The reaction of 2,3-dichloro-1,4-naphthoquinone (1) with

Anal. Chem. XXXX, XXX, XXX−XXX

Article

Figure 1. Visual recognition of metal ions (1.25 × 10⁻³M) with the sensor probe 4CBS (1.25 × 10⁻⁵M) in the aqueous medium (20 mM HEPES,

pH = 7.5).

p-aminobenzenesulfonamide (2) was carried out via a Michael-

like addition reaction. The reaction was completed by a green

method in which water was employed as a solvent medium.

The nucleophilic substitution product of the ligand 4CBS was

obtained under reﬂux conditions for 3 h with an excellent yield.

Colorimetric Responses of 4CBS toward Metal Ions.

In the beginning of the investigation, the colorimetric behavior

of the sensor probe 4CBS was evaluated upon the addition of

00 equiv of various alkaline earth metals in the aqueous

medium (20 mM HEPES, pH = 7.5). As depicted in Figure 1,

the light maroon color of the probe turned to milky white with

the addition of Sn . The prepared chemosensing probe 4CBS

did not show any notable color changes with the addition of

other metal ions. Poor yellow color conversions of Fe and

Fe ions were noticed, but this was due to the original color of

the iron ions. The speciﬁc color change of 4CBS with Sn²⁺

was attributed to the several electron transitions in the 4CBS−

Figure 2. Fluorescence selectivity study of 4CBS in the presence and

absence of various M in the aqueous solution; λ_e_x= 280 nm.

Sn complex such as π to π*, d−d, ligand−metal charge-

transfer, and metal−ligand charge-transfer eﬀects.

particles with Sn²⁺in water. To characterize this aggregation

phenomenon, initially, the dynamic light scattering (DLS)

Emission Properties of 4CBS with Metal Ions. A

solvatochromic study was carried out to determine the

absorption behavior of the sensor probe 4CBS in diﬀerent

solvents with 20 mM HEPES, pH = 7.5. The obtained

absorption spectrum showed three strong peaks for 4CBS at

analysis was carried out to conﬁrm the intriguing 4CBS−Sn

aggregation. The DLS analysis of the 4CBS−Sn complex

indicates the maximum particle size distribution obtained at

−

33, 280, and 467 nm. The examined absorption behavior of

CBS in diﬀerent solvents and water displayed a smooth UV/

190.50 ± 18.52 nm with 5.0 × 10 M Sn . However, the

population of aggregated particles was reduced upon the

addition of higher concentration of 1.25 × 10⁻M Sn

vis spectrum with high ligand solubility (Figure S3 ). Water

with 20 mM HEPES, pH = 7.5 was selected for metal ion-

binding studies because of the excellent performance of the

ligand 4CBS in the aqueous medium. Also, a wavelength of

(164.22 ± 12.30 nm, Figures S5, S6). To get a deep insight

into the formation of aggregated particles, the morphology of

the 4CBS−Sn complex was studied by atomic force

microscopy. The obtained results indicate that the particle

size distribution is more consistent to the DLS analysis of the

4CBS−Sn complex (Figures S7, S8). Besides, the eﬀect of

aggregates/precipitates in the ﬂuorescence emission property

80 nm was carefully chosen as the ﬂuorescence excitation

wavelength based on the ﬂuorescence study of 4CBS at

diﬀerent excitation wavelengths (Figure S4). In contrast, using

a lower excitation wavelength for highly diluted solutions

shows a ﬁne emission spectrum. However, employing such a

lower excitation wavelength associated with other practical

challenges is likely harmful to the skin/eyes to the humans and

besides, it leads to the DNA damage in tissues of the biological

system. Thus, extensive care must be taken while employing

such a lower excitation system for the environmental and

of the 4CBS−Sn complex was explored. Upon the addition

of increased concentration of Sn (0−100 equiv) to the

aqueous 4CBS solution, without any intervention, the

ﬂuorescence emission intensity was progressively intensiﬁed

up to 60 equiv of Sn , although small/acceptable emission

decrement was noticed with the addition of 70−100 equiv of

biological sample analysis. Initially, selectivity of the sensor

Sn to the aqueous 4CBS that was due to the formation of

−

probe was determined with 20 various metal ions (2.50 × 10

larger aggregated particles. These combined results indicate

that the formed aggregates/precipitates induced negligible

M) of Na , K , Cs , Li , Sr , Ba , Cd , Zn , Ni , Co ,

Mg , Ca , Mn , Hg , Cu , Fe , Fe , Al , La , and Sn .

eﬀects in the emission intensity for the 4CBS−Sn complex in

No obvious ﬂuorescence improvement was obtained in the

the aqueous solution (Figure S9 ). The ﬂuorescence changes of

−

emission spectrum of 4CBS (1.25 × 10 M). Upon the

the 4CBS−Sn complex in an aqueous environment were

−4

addition of Sn (2.50 × 10 M) to the 4CBS solution, the

intensity of the emission peak was signiﬁcantly increased via

the ﬂuorescence turn-on mode. Adding Sn²to the aqueous

solution of 4CBS resulted in a 43-fold higher emission

intensity than that of the ligand 4CBS alone because of

exciplex complex formation (Figure 2). According to the

explored by the addition of diﬀerent amounts of CH CN. The

ﬂuorescence intensity of the 4CBS −Sn complex increased in

80−100% aqueous medium (Figure S10). Moreover, the

concentration of the HEPES, pH = 7.5 buﬀer for the 4CBS−

Sn complex was investigated, and a 20 mM HEPES, pH = 7.5

buﬀer solution was suﬃcient to show the higher ﬂuorescence

intensity (Figure S11). The solvent eﬀect was also studied for

colorimetric responses of the ligand 4CBS toward Sn , it is

clearly shown that the ligand was formed the aggregated

the 4CBS−Sn complex, and none of the organic solvents

Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

solution (20 mM HEPES, pH = 7.5). As in Figure 3 , an

Article

increased the emission intensity. Nevertheless, higher

ﬂuorescence intensity was observed in water than in other

solvents (Figure S12). Therefore, water with 20 mM HEPES,

pH = 7.5 (which contains 99.375:0.625 v/v of H O/DMSO)

buﬀer was used as an optimized solvent medium for 4CBS to

recognize Sn .

After we determined the selectivity of 4CBS for Sn ,

quantitative analysis of Sn²was carried out in the aqueous

−

Figure 4. Complexometric titration of 4CBS (1.25 × 10 M) and

−4

Sn (2.50 × 10 M) with the coexistence of other metal ions (2.50

10⁻M) in the aqueous solution (20 mM HEPES, pH = 7.5); λ =

80 nm; λ_e_m= 397 nm.

stoichiometric ratio attained in the 4CBS−Sn complex

Figure S16 ). Furthermore, the 1:1 binding ratio was

(

con ﬁ rmed by mass (ESI) analysis of the 4CBS−Sn complex

(

Figure S17). The peak for the sensor probe 4CBS was

−

Figure 3. Emission titration analysis of 4CBS (1.25 × 10 M) with

obtained at a m/z of 363.00000 [M] (calcd m/z 362.78400),

−5

the increased concentration of Sn (0−6.25 × 10 M) in the

and the peak for the 4CBS−Sn complex was noted at a m/z

of 484.21024 [M + Na]⁺(calcd m/z 483.50890). This

observation conﬁrmed that the 1:1 binding ratio was obtained

^a^q^u^e^o^u^s^s^o^l^u^tⁱ^oⁿ⁽²⁰^m^M^H^E^P^E^S^,^p^H⁼⁷^.⁵⁾^;^λex = 280 nm.

increase in the concentration of Sn²⁺in the 4CBS solution

resulted in a gradual increase in the ﬂuorescence emission band

centered at 397 nm. A good linear relationship between

in the 4CBS−Sn complex. Next, the complex stability

constant (K ) was determined from the Benesi−Hildebrand

analysis by employing the ﬂuorescence titration data from

−1

ﬂuorescence emission intensity and Sn concentration was

Figure 3. The calculated K value was 5.0 × 10 M (Figure

S18 ). The detection limit (LOD) was calculated according to

the formula 3σ/k. The calculated LOD value for Sn

obtained in the range of 0−62.5 μM (R = 0.9979, Figure S13).

Thus, the ﬂuorescence titration study indicates that the

chemosensing probe 4CBS has good detectability and could

be used to quantitatively recognize Sn²in the aqueous

medium. Next, we examined the eﬀect of interference of other

detection was 0.115 μM, which is much lower compared to

the WHO limitations.

Reversibility Analysis of the Ligand 4CBS. Reversibility

is a requirement in the development of novel chemical sensors

in real-time applications. Thus, the reversibility behavior of

metal ions with the sensor probe 4CBS and Sn . In this study,

various metal ions were added to the aqueous 4CBS−Sn

complex (20 mM HEPES, pH = 7.5), and their ﬂuorescence

changes were recorded. The obtained results reveal that the

coexistence of metal ions has no or a negligible inﬂuence on

the sensing of Sn²by the probe 4CBS (Figure 4). Moreover,

the selectivity of 4CBS was examined as the ratio of the slope

of the calibration plot for Sn²to the slope of the plot for a

given metal cation interference (Figure S14 ). Besides, the

metal ion interference was examined by UV/vis complexo-

metric titration analysis (Figure S15). These cumulative UV/

vis and ﬂuorescence studies recommend that the sensing

4CBS was investigated with the added amount of Sn . The

ﬂuorescence intensity increased after adding a certain amount

of Sn to the aqueous 4CBS (20 mM HEPES, pH = 7.5). This

ﬂuorescence intensity diminished after the addition of a

chelating agent ethylenediaminetetraacetic acid (EDTA) (140

equiv) into the 4CBS−Sn complex. Furthermore, the

reversibility competence of the chemosensing probe 4CBS

was analyzed. The obtained results show that the chemo-

sensing probe 4CBS could be reversed by a maximum of 3

cycles (Figure S19). These ﬁndings reveal that the chemo-

sensing probe 4CBS was regenerated, supporting the high-

grade reversibility of 4CBS (Figure 5).

property of the probe 4CBS for Sn was not inﬂuenced by the

coexisting metal ions except Hg , Fe , and Fe ions. The

binding and the interference of these three metals were

reduced to a signiﬁcant amount of absorbance and

ﬂuorescence intensities; nevertheless, the obtained signals are

Evaluation of the Binding Mechanism. FT-IR Analysis.

To explore the obtained sensing mechanism, we performed

FT-IR analysis of the 4CBS−Sn complex. As given in Figure

6, the vibrational frequencies of the functional groups in the

sensor probe 4CBS were identiﬁed. Very strong and sharp

still discernible to identify the presence of Sn .

Evaluation of Coordination Stoichiometry and Com-

−

plex Stability Constant. To evaluate the coordination ratio

signals were obtained for −NH (3346 cm ) and −NH (3216

−1

of the 4CBS−Sn system, we constructed a Job’s plot. As

cm ) groups. After establishing coordination bonds of 4CBS

Sn was added to the 4CBS solution, the ﬂuorescence

intensity steadily increased. The highest emission intensity was

obtained at a 0.5 mol fraction, which indicates a 1:1 binding

with Sn , a red-shifted IR signal appeared for the −NH group

−

(3349 cm ), whereas no peak shift was noticed for the −NH

−

group (3216 cm ). Moreover, a strong new stretching

Anal. Chem. XXXX, XXX, XXX−XXX

the aqueous solution (20 mM HEPES, pH = 7.5); λ _e _x = 280 nm.

Article

NMR Titration Analysis. After receiving the basic

information about the coordination mechanism in the

CBS−Sn complex, NMR titration studies were performed

to acquire direct evidence for functional group participation in

the coordination mechanism. H NMR analysis of 4CBS was

carried out with an increasing amount of Sn . As shown in

Figure 7, the peak at 9.4799 ppm belongs to the −NH group in

CBS. As the concentration of Sn in the 4CBS solution

increased, all the proton signals started to migrate in large

upﬁeld shifts. New signals for two −OH and one −NH groups

appeared at 8.0077, 9.1782, and 9.3266 ppm, respectively. The

intensity of −OH (9.1782 ppm) and −NH (9.3266 ppm)

signals progressively decreased with increasing quantity of

Sn . All the proton signals were shifted (upﬁeld) with the

addition of 2 equiv of Sn , and no further peak shifting was

observed with excess Sn , indicating that the sensor probe

CBS reached its maximum saturation level at the solution

state.

−

Figure 5. Ligand regenerative study of 4CBS (1.25 × 10 M) with

the addition of Sn²(6.25 × 10 M) and EDTA (1.75 × 10 M) in

−5

−3

_T_h_e_n_,13C NMR analysis was investigated to conﬁrm the

reduction reaction-enabled sensing mechanism of the probe

CBS. Strong signals for −CO groups in the naphthoqui-

none moiety were obtained at 176.9433 and 179.9464 ppm.

The highest upﬁeld shifts of all the carbon signals were

observed upon adding 2 equiv of Sn . Speciﬁcally, the carbon

signals for −C−OH groups in the reduced form of 4CBS were

found at 141.7613 and 143.5276 ppm (Figure 8). After

NMR titration analysis, distortionless enhancement by polar-

ization transfer (DEPT) NMR analysis was carried out to

determine if any of the aromatic rings that participated in the

reduction reaction was induced by Sn . Eight tertiary carbons

are present in the chemosensing probe 4CBS. When DEPT 90

and DEPT 135 NMR titration experiments were executed with

equiv of Sn in the 4CBS solution, signals for eight tertiary

carbons with upﬁeld shifting were found in both experiments.

These combined results indicate that the aromatic rings were

not inﬂuenced by the addition of Sn , and the reduction

reaction is more speciﬁc at −CO centers (Figure 9).

Consequently, the achieved cumulative NMR titration analyses

revealed that a reduction-promoted sensing mechanism takes

place between 4CBS and Sn . In addition, −NH and −OH

functional groups in 4CBS helped to establish the coordination

bonding with Sn .

Electrochemical Investigation of the 4CBS−Sn Com-

plex. To gain a deeper insight into the sensing behavior of

CBS toward Sn , an electrochemical investigation was

Figure 6. FT-IR analysis of SnCl , 4CBS, and 4CBS−Sn complex.

accomplished using the cyclic voltammetry technique. First,

the electrochemical properties of 4CBS were measured in a

−

vibration signal appeared for the −C−OH group at 1035 cm

CH CN solution of 0.1 M tetrabutylammonium tetraﬂuor-

−

because of the reduction of −CO groups in the

naphthoquinone moiety. IR signals showed negligible or no

changes for the remaining aromatic functional groups in 4CBS.

This binding mechanism can be rationally explained in the

following ways. It is known that the metal ion Sn²⁺is a strong

reducing agent, which can easily reduce ketonic group into

alcohols. Hence, it reduced −CO to −C−OH in the

naphthoquinone core. According to Pearson’s principle for

acid−base theory, strong electronegative functional groups

such as −OH and −NH in 4CBS were strongly attracted to

oborate (supporting electrolyte) at a scan rate of 0.1 V s . The

sensor probe 4CBS showed distinctive reduction peaks at

−1.517 and −1.121 V and oxidation peaks at −1.231 and

−1.651 V. As Sn was added to the 4CBS medium, the peak

current for the 4CBS−Sn complex signiﬁcantly increased

with large potential shifts at −0.944 and −1.310 V (Figure

S20 ). These results indicate that the chemosensing probe

4CBS has strong aﬃnity and sensitivity toward Sn .

Theoretical Calculations. Theoretical calculations were

executed for 4CBS and 4CBS−Sn complex systems using

the Gaussian 16 package. In the optimized structure of 4CBS,

the positions of naphthoquinone and sulfonamide units were

nearly in the same plane. The optimized structure of the

the strong electrophilic borderline base Sn . Therefore, the

obtained results indicate that coordination bonding of 4BCS

and Sn occurred by a reduction-enabled detection pathway in

which −OH and −NH groups were involved in the bonding

mechanism.

4CBS−Sn complex showed the formation of hydrogen bonds

of Sn with −OH and −NH groups in the naphthoquinone

Anal. Chem. XXXX, XXX, XXX−XXX

mL of DMSO-d (with 20 mM HEPES, pH = 7.5).

Article

Figure 7. H NMR titration study of 4CBS (3.0 × 10⁻²M, 0.0054 g) with (a) 0.0, (b) 0.5, (c) 1.0, (d) 1.5, (e) 2.0, and (f) 2.5 equiv of Sn²⁺in 0.5

Figure 8. ¹³C NMR titration study of 4CBS (9.0 × 10⁻²M, 0.0162 g) with (a) 0.0 and (b) 2.0 equiv of Sn²⁺in 0.5 mL of DMSO-d₆.

unit, which enhanced the stability of the system (Figure S21).

Next, the energy distributions of the highest occupied

molecular orbital (HOMO) and lowest unoccupied molecular

ICT eﬀect, which is consistent with the proposed binding

mechanism. The anticipated coordination mechanism of the

4CBS−Sn complex is given in Scheme 2.

orbital (LUMO) energy levels for 4CBS and its Sn complex

Fluorescence Bioimaging Studies. Identiﬁcation of

Sn²in Live Cells. Encouraged by the outstanding photo-

were examined. As shown in Figure 10, the energy of the

HOMO and LUMO orbital levels for the 4CBS−Sn complex

physical properties of the ligand 4CBS toward Sn , its

was lower than that of the ligand 4CBS. Also, the energy

biocompatibility was investigated to detect Sn in L929,

HaCaT, HeLa, and MCF-7 cells using ﬂuorescence bioimaging

studies. First, the toxicity of the ligand 4CBS was studied at 11

concentrations from 0.0975 to 100 μM. From the results,

about 95% of the cells were viable to the sensor probe 4CBS.

However, the viability of the cells was reduced when 4CBS was

overdosed at concentrations of 25−100 μM (Figure S22).

Therefore, the obtained results suggest that the ligand 4CBS is

biologically compatible and has low toxicity to live cells.

After the toxicity analysis of 4CBS, a bioimaging study was

carried out in L929, HaCaT, HeLa, and MCF-7 cells to

diﬀerences of 4CBS and 4CBS−Sn complex were calculated

with an energy diﬀerence of 1.86 eV. These outcomes indicate

that the eﬀective resonance attraction obtained in the 4CBS−

Sn complex. For the ligand 4CBS, the energy migrated from

the naphthoquinone unit to sulfonamide units because of the

weak intramolecular charge-transfer (ICT) eﬀect, whereas in

the 4CBS−Sn complex, the maximum energy was localized

at the naphthoquinone−Sn units via a coordination bond

caused by a strong ICT eﬀect. Hence, the obtained results

imply that a stable complex was formed through the strong

Anal. Chem. XXXX, XXX, XXX−XXX

Article

−

Figure 9. DEPT titration analysis of 4CBS (9.0 × 10 M, 0.0162 g) in 0.5 mL of DMSO-d (a) DEPT 90 analysis of 4CBS without Sn , (b)

DEPT 90 analysis of 4CBS with 2 equiv of Sn , (c) DEPT 135 analysis of 4CBS without Sn , and (d) DEPT 135 analysis of 4CBS with 2 equiv of

Sn .

CBS in L929 and HaCaT cells. Fascinatingly, robust

ﬂuorescence emission was induced by 4CBS in HeLa and

MCF-7 cells. Increased ﬂuorescence intensities were obtained

in HeLa and MCF-7 cells, compared to the ﬂuorescence

emission induced in L929 and HaCaT cells. The ﬂuorescence

intensity of HeLa and MCF-7 cells was increased in a dose-

dependent manner (Figures S27 −S30). In contrast, HeLa cells

exhibit better ﬂuorescence images than the MCF-7 cells

(

Figure S31). Furthermore, it can be noticed that the

subcellular localization of 4CBS made Sn to mostly distribute

in the cytoplasm of the cancer cells within 10 min. Thus, these

results indicate that the sensor probe 4CBS could easily

penetrate the cancer cells (HeLa and MCF-7) and enhance the

ﬂuorescence emission intensity compared to normal live cells

(

L929 and HaCaT). The comparison of recently reported

Figure 10. Theoretical calculations of the ligand 4CBS (A) and

chemosensing probes with 4CBS showing the K , LOD, and

other analytical parameters is given in Table S1 . In contrast,

4CBS−Sn (B) complex.

the sensing probe 4CBS showed better sensitivity and

selectivity toward Sn . The bioimaging incubation period

Scheme 2. Proposed Detection Mechanism of the 4CBS−

Sn Complex

was competitively less than the reported sensing probes.

Moreover, the ligand 4CBS is well-performing in the aqueous

medium through the colorimetric/ﬂuorometric responses. In

summary, the sensor probe 4CBS may be capable of

diﬀerentiating human cancer cells from the normal ones with

high sensitivity/selectivity, and it has the potential to be

utilized as an active bioimaging agent in biological analyses.

Bioimaging Studies in Zebraﬁsh. Zebraﬁsh larvae (5-day-

old) were grown under optimal conditions in the E3 medium

at 28 °C. Before adding the metal and ligand, the baby

zebraﬁsh were anaesthetized (50 mg/L tricaine). First, 6.125

conﬁrm the detection of Sn²⁺in mouse ﬁbroblast cells, human

keratinocyte cells, human cervical cancer cells, and human

breast cancer cells, respectively. Based on the toxicity study,

two major concentrations of 4CBS such as 6.125 and 12.50

μM were chosen for bioimaging analysis. Initially, 6.125 μM

μM Sn was incubated alone with the zebraﬁsh, and no or

negligible ﬂuorescence was emitted. Similar emission responses

were perceived for incubation of the ligand 4CBS (Figure

(

2a−f). Under a combined incubation of Sn and 4CBS

Sn and 4CBS were separately incubated with L929, HaCaT,

HeLa, and MCF-7 cells for 10 min. A very weak/negligible

level of ﬂ uorescence emission was observed in all the cells

6.125 μM each), respectively, with the zebraﬁsh, remarkable

ﬂuorescence intensity was detected around the eyes/yolk

extension in both green and red ﬁlters (Figure 12g−i). The

obtained data indicate that the chemosensing probe 4CBS has

(

Figures S23 −S26). Next, 6.125 μM Sn and 4CBS were

incubated in a combined manner with L929, HaCaT, HeLa,

and MCF-7 cells for 10 min. As depicted in Figure 11,

insigniﬁcant ﬂuorescence emission was obtained for the ligand

tissue-penetrable capacity and is able to detect Sn in small

animals such as zebraﬁsh.

Anal. Chem. XXXX, XXX, XXX−XXX

The Supporting Information is available free of charge at

Article

Figure 11. Fluorescence bioimaging detection of Sn²⁺in L929, HaCaT, HeLa, and MCF-7 cells that are treated with 6.125 μM Sn²⁺and 4CBS,

respectively, where images (a −d) = L929 cells; images (e −h) = HaCaT cells; images (i −l) = HeLa cells; and images (m−p) = MCF-7 cells.

Figure 12. Confocal bioimaging of Sn²⁺in zebraﬁsh, where images (a−c) = zebraﬁsh treated with 6.125 μM Sn ; images (d−f) = zebraﬁsh treated

with 6.125 μM 4CBS; and images (g−i) = zebraﬁsh treated with 6.125 μM Sn and 4CBS, respectively.

CONCLUSIONS

that 4CBS was utilized as a chemosensing probe to detect trace

■

amounts of Sn in human living cells and zebraﬁsh. In

In conclusion, a new naphthoquinone-based colorimetric and

ﬂuorescence probe 4CBS was successfully fabricated for

recognition of Sn²in the aqueous medium under physio-

logical pH. The structure of the synthesized chemosensing

contrast, the chemosensing probe 4CBS detects Sn in live

cells, showing distinct ﬂuorescence emission, which helps to

diﬀerentiate human cancer cells from normal live cells.

probe 4CBS was analyzed by FT-IR, H NMR, C NMR,

mass (ESI−MS), and elemental analyses. The sensing

mechanism was triggered by the reduction of −CO groups

ASSOCIATED CONTENT

■

sı

Supporting Information

by Sn , which was conﬁrmed by spectroscopic investigations.

Theoretical calculations were performed to justify the binding

mechanism and optical behavior of the sensor probe. The

chemosensing probe 4CBS showed high selectivity and

https://pubs.acs.org/doi/10.1021/acs.analchem.0c03196.

_s_e_n_s_i_t_i_v_i_t_y_f_o_r_S_n2 even in the presence of other metal ions.

Synthetic procedures, spectral data, solvatochromic

studies of the ligand 4CBS, Job’s plot, proposed

The LOD of the chemosensing probe for Sn was calculated

optimized geometries of 4CBS and its Sn complex,

and cell viability assay and bioimaging of 4CBS (PDF)

to be 0.115 μM, which is less than the WHO permissible

amount of Sn in drinking water. We further demonstrated

Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Jeonju-si, Jeollabuk-do 54896, Republic of Korea;

Article

AUTHOR INFORMATION

funded by Korea Institute of Energy Technology Evaluation

and Planning (KETEP) and the Ministry of Trade, Industry &

Energy (MOTIE) of the Republic of Korea (no.

■

Corresponding Author

Dong Jin Yoo − Department of Life Sciences, College of

Natural Sciences and Department of Energy Storage/

Conversion Engineering of Graduate School, and Hydrogen

and Fuel Cell Research Center, Jeonbuk National University,

orcid.org/0000-0002-5707-3361; Email: djyoo@

jbnu.ac.kr

20184030202210).

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