Water-dispersible rhodamine B hydrazide loaded TiO2 nanoparticles for "turn on" fluorimetric detection and imaging of orthosilicic acid accumulation in-vitro in nephrotoxic kidney cells

Article abstract of DOI:10.1166/jnn.2018.16338

Silica (SiO₂) is the inevitable form of silicon owing to its high affinity for oxygen, existing as a geogenic element perpetrating multifarious health problems when bioavailable via anthropogenic activities. The hydrated form of silica viz. orthosilicic acid (H₄SiO₄) excessively displays grave toxicity, attributed to prolonged exposure and incessant H⁺ ions generating capacity inflicting pulmonary toxicity and renal toxicity silica. The diverse deleterious potency of silica highlights the desirability of selective and sensitive detection of toxic species (mainly orthosilicic acid) bioaccumulation in affected living human cells. In this paper we have reported, the design of water-dispersible turn-on fluorimetric sensing material for the detection of orthosilicic acid in the aqueous phase and in live cells. The sensing material was prepared by adsorbing a suitable rhodamine derivative (i.e., Rhodamine B hydrazide (Rh1)) on water dispersible TiO₂ nanoparticles. The function of the sensing system, which is composed of Rh1 and TiO₂ (Rh1@TiO₂), is accredited to H⁺ ion (from orthosilicic acid) induced spirolactam ring-opening of the rhodamine derivative generating orange fluorescence and bright pink colouration. The sensing system was efficiently utilized for fluorimetric detection and imaging of orthosilicic acid accumulation in-vitro in human kidney cells (HK cells). To the best of our knowledge, this is the first time this sensing system (Rh1@TiO₂) is reported for detection of toxic silica species accumulation in-vitro in human kidney cells. The advantages, such as good water dispersibility, the absence of organic solvents during fluorimetric studies, quick turn-on type signal transduction, low-level imaging, which are offered by the synthesized sensing material (Rh1@TiO₂), make it a potential candidate to fabricate medical tool for early identification of silicainduced nephrotoxicity, which can help to reduce the burden and risk of chronic kidney disease development.

Full text of DOI:10.1166/jnn.2018.16338

Article
Journal of
Nanoscience and Nanotechnology
Vol. 18, 8142–8154, 2018
Copyright © 2018 American Scientific Publishers
All rights reserved
Printed in the United States of America
www.aspbs.com/jnn
Water-Dispersible Rhodamine B Hydrazide Loaded TiO2
Nanoparticles for “Turn On” Fluorimetric Detection and
Imaging of Orthosilicic Acid Accumulation In-Vitro in
Nephrotoxic Kidney Cells
1
ꢀ†
2ꢀ†
2
2
Starlaine C. Mascarenhas , Ram U. Gawas , Barun Kumar Ghosh , Mainak Banerjee ,
1
ꢀ∗
2ꢀ∗
2ꢀ∗
Anasuya Ganguly , Amrita Chatterjee , and Narendra Nath Ghosh
1
2
Department of Biological Sciences, and Department of Chemistry, Birla Institute of Technology and Science,
Pilani K K Birla Goa Campus, Zuarinagar, Goa 403726, India
Silica (SiO ꢀ is the inevitable form of silicon owing to its high affinity for oxygen, existing as a
2
geogenic element perpetrating multifarious health problems when bioavailable via anthropogenic
activities. The hydrated form of silica viz. orthosilicic acid (H SiO ꢀ excessively displays grave toxic-
4
4
+
ity, attributed to prolonged exposure and incessant H ions generating capacity inflicting pulmonary
toxicity and renal toxicity silica. The diverse deleterious potency of silica highlights the desirability
of selective and sensitive detection of toxic species (mainly orthosilicic acid) bioaccumulation in
IP: 185.251.14.213 On: Wed, 19 Sep 2018 01:37:21
affected living human cells C. I on pt yh ri si g ph at :p Ae rmw ee ri hc aa vne Sr ec pi eo nr t tei fdi c, t Ph eu bd l ei ss hi gen r so f water-dispersible turn-on
fluorimetric sensing material for the d eD t ee cl i tvi oe nr e od f bo ry t hI no sg i el i cn i ct aacid in the aqueous phase and in live
cells. The sensing material was prepared by adsorbing a suitable rhodamine derivative (i.e., Rho-
damine B hydrazide (Rh1)) on water dispersible TiO nanoparticles. The function of the sensing
2
+
system, which is composed of Rh1 and TiO (Rh1@TiO ꢀ, is accredited to H ion (from orthosilicic
2
2
acid) induced spirolactam ring-opening of the rhodamine derivative generating orange fluorescence
and bright pink colouration. The sensing system was efficiently utilized for fluorimetric detection
and imaging of orthosilicic acid accumulation in-vitro in human kidney cells (HK cells). To the best
of our knowledge, this is the first time this sensing system (Rh1@TiO ꢀ is reported for detec-
2
tion of toxic silica species accumulation in-vitro in human kidney cells. The advantages, such as
good water dispersibility, the absence of organic solvents during fluorimetric studies, quick turn-on
type signal transduction, low-level imaging, which are offered by the synthesized sensing material
(
Rh1@TiO ꢀ, make it a potential candidate to fabricate medical tool for early identification of silica-
2
induced nephrotoxicity, which can help to reduce the burden and risk of chronic kidney disease
development.
Keywords: TiO2 Nanoparticles, Rhodamine Dependent Fluorimetry, Nanomaterial-Based
Sensor, Nephrotoxic Silica, Bio-Imaging of Live Cells, Human Kidney (HK) Cells.
1
. INTRODUCTION
other elements and oxygen forming silicates, like asbestos,
quartz, feldspar etc. Silica is abundantly present in rocks,
1
Silicon is the second most abundant constituent con-
tributing to 28% of earth’s crust. Silicon being highly
reactive converts from its elemental form and combines
sand and soil. However, it is not readily bioavailable as
it remains combined with oxygen and hence is generally
2
either with oxygen forming free silica (SiO ꢀ or with
categorised as a trace geogenic element. The threat arises
2
when silica emanates in air and water during the course
of various anthropogenic activities, like granite mining,
sand mining, etc., manifesting serious health problems
∗
†
Authors to whom correspondence should be addressed.
These two authors contributed equally to this work.
8142
J. Nanosci. Nanotechnol. 2018, Vol. 18, No. 12
1533-4880/2018/18/8142/013
doi:10.1166/jnn.2018.16338

Mascarenhas et al.
Water-Dispersible Rhodamine B Hydrazide Loaded TiO Nanoparticles for “Turn On” Fluorimetric Detection
2
including silicosis,3ꢁ4 bronchitis,5 systemic autoimmune
organic dyes in the working solution. This is a serious
concern for biotechnological applications as organic sol-
vents can exaggerate severe toxicity to biological systems
restricting their application in live cells or any other
living species. One of the possible ways to overcome this
difficulty is the use of water-dispersible nanomaterials on
which a chemodosimeter can be grafted by physical or
chemical modifications.
Nanomaterials, many of which possess excellent water
dispersibility, enormous surface area/volume ratio, large
adsorption capacity, great mechanical strength and sur-
face reactivity, intrinsic optical/electrochemical/magnetic/
spectroscopic properties, have proved their efficacy in
recent years as suitable candidates for the development
of material based sensors by selective interactions of
6
diseases like rheumatoid arthritis (RA), systemic lupus
7
8
erythematosus (SLE) and systemic sclerosis (SSc),
9
lung cancer. Due to grave toxicity exhibited by sil-
ica, OSHA has set harsh regulations limits to 50 ꢂg/m
3
in air.10
In recent times, limited epidemiological reports have
highlighted the potential of silica to exhibit nephrotoxi-
city as well, resulting mainly in chronic kidney disease
1
1–13
development.
The hydrated form of silica (orthosili-
cic acid, H SiO ꢀ at a concentration range of 100–
4
4
1
20 mg/L induces severe nephrotoxicity on prolonged
1
4ꢁ15
exposure, accredited to its bioaccumulative propensity.
However, in spite of silica’s diverse toxicological poten-
tial, studies analysing cellular penetration and accumu-
lation of silica in targeted mammalian cells (specifically
human cells) on prolonged exposure, an essential prereq-
uisite for toxicity, are unavailable till date. Few reports
utilised labour intensive and expensive instrumentation like
their surface (pre-functionalized with the signal transduc-
29ꢁ30
tion unit) with the analytes.
Often nanomaterial-based
chemosensors outshine traditional detection techniques
with decreased sampling time, quicker analytical response,
1
6
SEM coupled with X-ray analysis, X-ray fluorescence
spectrometer (P-XRF) for silica deposit visualisation in
grasses17 and diatoms. However, these techniques suf-
fer from major limitations of longer data acquisition time,
necessity of sophisticated and expensive instrumentation,
requirement of trained personnel, tedious sample prepara-
tion (such as solubilisation or in-situ charring of cells to
estimate the silica content), thereby negating the possibil-
31ꢁ32
high sensitivity and low cytotoxicity.
Some of the
nanomaterials comprehensively used for biological and
environmental assessment of lethal analytes include metal
nanoparticles (e.g., Au, and Ag nanoparticles), carbon
materials (carbon nanotubes, and graphene), magnetic
nanoparticles (e.g., Fe, Ni, Co), quantum dots (such as
ZnS, CdTe, and CdSe) and metal-oxide nanomaterials
18
3
3ꢁ34
IP: 185.251.14.213 On: We d( v, i1z 9. ST ieOp ,2 0C 1u 8O 0, 1 Z: 3n 7O : ,2 1e tc.).
Of our interest, TiO2
nanoparticles are good candidates to be used as matrix for
Delivered by Ingenta
2
ity of its application in live human cell imaging of silica
Copyright: American Scientific Publishers
species. Owing to its trace amounts, influences of coexist-
ing substances in real samples, rising and severe deleteri-
ous effects induced by bioaccumulated silica species and
limitations of the analytical techniques, the development
of sensitive, rapid on-site detection of toxic silica species
sensing purpose owing to its unique properties including
enhanced adsorptive surface area, resistance to photo and
chemical erosion, chemical and biological inertness,
low-cost synthesis, monodispersity, and nontoxicity.
3
5
3
6
37
3
8
39
4
0
Moreover, excellent biocompatibility and easy cellular
(
especially orthosilicic acid) and imaging of silica contam-
4
1
uptake on dispersion in aqueous media further extends its
potential application for live-cell imaging of intracellular
toxic silica species deposits.
inated live mammalian cells are necessary.
Fluorimetric techniques involving small organic
molecules as chemo-reactants or chemo-dosimeters are
well-validated methods for the detection of toxic ana-
lytes or even imaging of biological species due to high
selectivity and sensitivity, instant signal transduction, low-
Interactions of Nanomaterials with the biological sys-
4
2–43
tems have been investigated by several researchers.
As a part of our continuous efforts on the development
of molecular probes for biological and environmentally
1
9ꢁ20
cost instrumentation etc.
with high quantum yield is a vastly used fluorophore for
In this context, rhodamine
4
4–51
noxious analytes
we report, herein, a water-dispersible
2
1ꢁ22
“turn-on” fluorimetric sensing material, which is com-
prised of rhodamine-B based chemodosimeter (rhodamine
molecular sensing of various analytes,
which often
sense the analytes by the opening of its non-fluorescent
spirolactam ring to a highly fluorescent and coloured
form.23 Some rhodamine derivatives have successfully
utilized this principle in developing fluorimetric and
B hydrazide (Rh1)) and TiO nanoparticles (Rh1@TiO ꢀ,
2
2
for the detection and imaging of toxic silica species
viz. orthosilicic acid) bioaccumulation in live cells.
(
We chose rhodamine B hydrazide (Rh1) as the chemod-
colourimetric probes for the determination of pH of a
solution;2
4–26
the present method is based on the same
52
osimeter unit, which was first reported by Dujols et al.
principle. Relevant to this, a couple of rhodamine deriva-
tives have also been used as simple dyes for the detection
of silica deposition in diatoms based on the formation of
orthosilicic acid via dissolution of silica in the aqueous
as a sensor for Cu(II) ions in water and later used
5
3–62
by others
for various sensing and imaging studies.
It was presumed that the acidic environment inside the
cell would induce spirolactam ring-opening and trans-
2
7ꢁ28
23ꢁ57
intra-cellular environment.
However, use of some
duce fluorescence.
To demonstrate our concept silica
amount of organic solvents is inevitable to dissolve these
affected nephrotoxic kidney cells were chosen.
J. Nanosci. Nanotechnol. 18, 8142–8154, 2018
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Water-Dispersible Rhodamine B Hydrazide Loaded TiO Nanoparticles for “Turn On” Fluorimetric Detection
Mascarenhas et al.
2
2
. EXPERIMENTAL DETAILS
2.3.2. Preparation of TiO Nanoparticle
2
2
.1. Chemicals
TiO nanoparticles were prepared using an EDTA pre-
2
Rhodamine B HCl and Hydrazine hydrate were pur-
chased from Sigma Aldrich (India) and used as received.
cursor based method, which was developed by Ghosh
6
4
and coworkers. In a 250 mL glass beaker, 21.1 mL
of TiCl (192.6 mM) was taken, and then an aqueous
solution of HNO was added to it dropwise to oxidized
TiCl was purchased from Spectrochem Pvt. Ltd. Mumbai
3
3
(
India). Ethylene diamine tetra acetic acid (EDTA) was
3
3
+
4+
Ti to Ti . Separately, in a beaker aqueous solution of
EDTA (10% w/v) was prepared by dissolving EDTA in
purchased from Merck, India. All solvents and other chem-
icals were of AR grade and were procured from different
commercial suppliers and used without further purifica-
tion. Millipore water (18 Mꢃꢀ was used for all spectro-
scopic studies.
hot water with dropwise addition of dilute NH OH solu-
4
tion. After complete dissolution of EDTA, the solution
was boiled to remove excess NH . This EDTA solution
3
4
+
was then added to a solution containing Ti ions (keep-
ing Ti :EDTA molar ratio 1:1) with constant stirring. The
4
+
2
.2. Instruments and Measurements
reaction mixture was evaporated to dryness over a hot plate
NMR spectra were recorded on Bruker Avance (400 MHz)
NMR spectrometer. Mass spectra were obtained from Agi-
lent technologies 6460 triple quard LC-MS (ESI). Flu-
orescence spectra were measured on a JASCO FP-6300
spectrofluorometer, the slit width was 2.5 nm for both
excitation and emission. Absorption spectra were recorded
on a JASCO V570 UV/Vis/NIR spectrophotometer. Infra-
Red spectra were taken on IR Affinity-1 FTIR spec-
trophotometer, Shimadzu. Elemental analysis was carried
out on Vario elementar CHNS analyzer. Thermogravimet-
ric analysis (TGA) data were recorded using a DTG-60,
Shimadzu. Field Emission Scanning Electron Microscopy
ꢀ
at 125 C to obtain the precursor powder for TiO . Finally,
2
ꢀ
the precursor was calcined at 550 C for 3 h to obtain TiO
2
nanoparticles with an average diameter of ∼35 nm.
2
.3.3. Preparation of the Sensing Material, Rh1@TiO2
For the grafting of Rh1 onto the TiO2 nanoparticles,
00 mg of sonicated TiO nanoparticles was added to Rh1
1
2
(
10 mg, 0.022 mM) in dry acetonitrile solution (100 mL)
and magnetically stirred in the dark for 60 min to ensure
6
5
it reaches adsorption–desorption equilibrium. The Rh1
adsorbed TiO nanoparticles (Rh1@TiO ꢀ were collected
2
2
by centrifugation at 2500 rpm for 5 min followed by wash-
(
FESEM) images of the TiO nanoparticles were obtained
2
ing the residue with acetonitrile (2×25 mL) and dried in
on FESEM Quanta 250 FEG (FEI) with 10 kV and reso-
IP: 185.251.14.213 On: Wed, 19 Sep 2018 01:37:21
a vacuum desiccator (in the dark) for 4 h to obtain a white
lution 2 nm. The powder X-ray diffra cCt i oo pn y( rXi gR h Dt :) Ap ma t et e rr i nc sa n Scientific Publishers
solid (110 mg, 100% yield) as the final sensing material.
were obtained from powder X-ray diffractome tDe re l (i vM e irne id by Ingenta
Flex II, Rigaku, Japan).
2
.3.4. The General Procedure of Sample Preparation
for Fluorescence Measurement
2
2
.3. Synthesis of Materials
.3.1. Preparation of Rhodamine Hydrazide (Rh1)
1
0 mg of the probe Rh1@TiO was dispersed in 10 mL
2
of deionized water (MilliQ, 18 Mꢃꢀ to get a 1 g/L of
the stock solution as and when required and diluted fur-
ther as per requirement. 1 g/L (10 mM) stock solution
of orthosilicic acid was prepared in deionized water and
diluted further for the preparation of working solutions.
To analyse the effect of different metal ions stock solu-
tions (10 mM, 5 mL) were prepared by dissolving their
respective nitrates/chlorides in deionized water. All analyte
solutions were subjected to 0.22 ꢂm syringe filtration to
avoid any interference by particulate matter in fluorescence
measurement. After analyte addition, each solution was
incubated for 10 min before recording their respective flu-
orescence spectrum at an excitation wavelength of 525 nm
and the emission was recorded from 526 to 650 nm. All
the experiments were executed at room temperature. For
the equivalence study, orthosilicic acid doses were altered
from 0–100 ꢂL of 10 mM (or 0.0–33.3 mg/L) for fluores-
cence measurement.
The chemodosimeter, Rh1 was prepared according to a
5
3ꢁ63
reported method
(Scheme 1). In a 100 ml round bot-
tom flask, rhodamine B (1.0 g, 2.08 mM) was dissolved
in 15 ml absolute alcohol, to which excess of Hydrazine
hydrate (98%, 2.08 g, 4.16 mM) was added under stir-
ring at room temperature. The reaction mixture was then
ꢀ
refluxed for 16 h at 110 C. After completion of the reac-
tion, 10 ml water was added and the residue was fil-
tered, washed with water (3 ×10 mL), and then air-dried.
The crude product was recrystallized from the ethanol-
water mixture to obtain the sufficiently pure product, Rh1
(
650 mg, 67.5% yield).
Cl
N
O
N
N
O
N
NH
2 2 2
NH .H O
N
NH
2
EtOH, 90°C, 16h
COOH
O
2
.3.5. General Procedure for Fluorescence Study
Rh1
The fluorescence study was carried out by addition of
Scheme 1. Synthesis of rhodamine B hydrazide (Rh1).
30 ꢂL of the sensing material, Rh1@TiO (1 g/L stock
2
8144
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Mascarenhas et al.
Water-Dispersible Rhodamine B Hydrazide Loaded TiO Nanoparticles for “Turn On” Fluorimetric Detection
2
solution) in deionized water (3 mL) in a cuvette. To this
solution, increasing amount of orthosilicic acid (1 g/L
stock solution) were added and fluorescence response was
measured after every 10 min, respectively. For equiva-
lent study concentrations of orthosilicic acid were changed
from 0–33.3 mg/L (i.e., 0–105 ꢂL from stock solu-
tion). For each reaction mixture fluorescence response was
recorded. The same procedure was followed for compara-
tive study with other metal ions.
1 g/L of stock solution was added to 2 mL of culture
ꢀ
medium) at 37 C and 5% CO for 30 min. Fluores-
2
cence images were captured by a fluorescence microscope
(Olympus IX51) under a TRITC filter and reported. Each
experimental study was triplicated to ensure enhanced data
reproducibility.
3
. RESULTS AND DISCUSSION
3
.1. Characterizations of the Synthesized Rh1, TiO2
Nanoparticles, and Rh1@TiO2
The synthesized materials were characterized by using sev-
eral instrumental techniques, such as H NMR, C NMR,
ESI-MS, IR, CHN, XRD, TGA, and FESEM. Rh1
2
.3.6. Procedure for pH Dependency Study
For the preparation of acidic pH solutions, sodium acetate
in the acetic acid buffer and for basic pH solutions phos-
phate buffer was used. The pH dependency study was
1
13
1
13
was characterized by H NMR, C NMR, ESI-MS, IR
carried out by addition of 30 ꢂL of Rh1@TiO sensing
2
1
13
and CHN. The H and C NMR spectra of the Rh1
are shown in Figures 1 and 2, respectively and the
obtained data are in well-agreement with the reported val-
material (from 1 g/L stock) in 3 mL of each individual
pH buffer (ranging from 3–10) in a cuvette and the flu-
orescence response was recorded. This was followed by
addition of 30 ꢂL of orthosilicic acid (1 g/L) to each indi-
vidual pH buffer solution containing the Rh1@TiO sens-
ing material and the fluorescence intensity was recorded
after 10 min incubation.
5
0ꢁ61
ues of Rh1.
Mass spectroscopy analysis (m/z = 457
+
[
M + H] ꢀ and CHN analysis (Calculated C, H and N
2
amount for C H N O : C: 73.66; H: 7.06; N: 12.27
28 32
4
2
and data obtained from CHN analysis: C: 73.59; H: 6.99;
N: 12.19) of the product also confirmed the formation of
Rh1 (molecular formula C H N O ꢀ.
2
.3.7. Cytotoxicity Study
28 32
4
2
The crystalline phase and crystallite size of the synthe-
Cytotoxicity of Rh1@TiO was tested using the colouri-
metric MTT assay. HK cells (Human Kidney cells)
were cultured in Dulbecco’s Modified Eagle’s Medium
2
6
4
sized TiO were determined by using XRD. XRD pattern
2
of the synthesised TiO nanoparticles (Fig. 3) showed the
2
IP: 185.251.14.213 On: Wed, 19 Sep 2018 01:37:21
ꢀ
ꢀ
ꢀ
ꢀ
presence of peaks at 2ꢄ = 25.3 , 37.8 , 48.1 , 53.9 , and
Copyright: American Scientific Publishers
55.06 , which are corresponding to the (101), (004), (200),
Delivered by Ingenta
(
DMEM) supplemented with 10% (v/v) fetal bovine serum
ꢀ
ꢀ
4
at 37 C and humidified 5% CO . 5 × 10 cells were
2
(
2
105) and (211) planes of anatase phase [JCPDS Card no.
1-1272], and confirmed the formation of the pure anatase
phase of TiO . The crystallite size of the synthesized TiO
plated per well in a 24-well plate. Rh1@TiO (1 g/L)
2
was dissolved in aqueous PBS (pH 7) to make a stock
solution. Following 24 h incubation, the cells were dosed
with increasing concentrations of the sensing material,
Rh1@TiO (1, 10, 50, 100, and 200 mg/L) formulated by
serial dilution in DMEM. Unexposed controls were main-
2
2
nanoparticles (calculated by using Scherrer equation) was
found to be ∼38 nm.
2
The sensing material, Rh1@TiO was prepared by graft-
2
ing TiO nanoparticles with Rh1. The thermogravimetric
tained. After an additional 24 h incubation, 50 ꢂL of MTT
2
analysis (TGA) was utilized to determine the amount of
(
5 mg/mL in PBS) was added per well and incubated
for 4 h. Following which the media was aspirated from
the wells, 0.5 mL of DMSO was added. Absorbance was
recorded at 570 nm and cell viability was expressed as
a percentage of relative absorbance of the sample versus
unexposed control cells for every probe concentration.
Each set of experiments were triplicated and the average
results are presented.
Rh1 in Rh1@TiO (Fig. 4). TGA thermograms showed
2
that pure Rh1 was fully decomposed within the tem-
ꢀ
perature range of 600 C, whereas TiO
nanoparticles
2
were quite stable in this temperature range. In case
of Rh1@TiO , 8% weight loss was observed when the
2
ꢀ
sample was heated up to 600 C, which indicated that
Rh1@TiO is composed of 8 wt% Rh1 and 92 wt% of
2
TiO . This is in well-agreement with the ratio of the probe,
2
2
.3.8. Fluorescence Imaging of Toxic Silica
Species in Living Cells
Rh1 and TiO used during the synthesis (1:10), and it can
be concluded that almost 90% of the Rh1 successfully
2
For fluorescence imaging of silica (viz. orthosilicic acid)
accumulation in living cells, HK cells were chosen. HK
cells were plated in a 6-well plate in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum for 24 h. The cells were then incubated with
increasing doses of silica (0.1, 1, 10 and 100 mg/L) for
three different time points (1 h, 24 h, and 8 days), fol-
lowed by treatment with the sensing material (100 ꢂL of
adhered onto the surface of TiO
In the IR spectra of Rh1@TiO
of the signature peaks of Rh1 (IR bands at 3340, 3082,
2969, 1774, 1618, 1519, 1272, 1115 cm ꢀ also indicated
the physisorption of Rh1 on TiO2.
2
nanoparticles.
(Fig. 5) the presence
2
−1
SEM micrographs revealed that the diameter of TiO2
nanoparticles was in the range of 25–6 nm, with an average
of ∼35 nm (Fig. 6(A)). SEM images of Rh1@TiO also
2
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8145

Water-Dispersible Rhodamine B Hydrazide Loaded TiO Nanoparticles for “Turn On” Fluorimetric Detection
Mascarenhas et al.
2
IP: 185.251.14.213 On: Wed, 19 Sep 2018 01:37:21
Copyright: American Scientific Publishers
1
1
Delivered by Ingenta
Figure 1. H NMR spectra of Rh1 ( H NMR (400 MHz, CDCl ꢀ: ꢅ (ppm) 1.18 (12 H, t, J = 6.8 Hz), 3.36 (8 H, q, J = 7.2 Hz), 6.32 (2 H, dd,
3
J = 2.8 Hz, J = 8.8 Hz), 6.44–6.49 (4 H, m), 7.11–7.13 (1 H, m), 7.44–7.49 (2 H, m), 7.93–7.98 (1 H, m)).
1
2
indicated that TiO nanoparticles retain their original sur-
as a function of pH in the presence and absence of
2
face morphology after modification with fluorophore unit
orthosilicic acid. As expected, Rh1@TiO started emitting
2
(
Rh1) (Fig. 6(C)).
strong fluorescence signals as the pH of the solution went
below 7. The fluorescence output showed steady rise up to
pH 3 (excited at 525 nm). This was well-expected, con-
sidering the possibility of protonation of the spirolactam
ring of Rh1 which leads to the formation of the fluores-
cent ring-open state in Rh1 under acidic pH. However, the
3
.2. Effect of pH on the Fluorescence Response of the
Sensing Material (Rh1@TiO2)
It was conceived that the fluorophore, Rh1, would respond
to acidic pH by going to its ring-opened form. The
response of Rh1 at different pH was carried out in
CH CN–H O and the fluorescence response was noted.
As the sensing material can be dispersed in water only, the
pH dependency study of the sensing material (Rh1@TiO2ꢀ
was carried out in water and buffer solutions, and pH was
maintained by the addition of acid or base. In the present
study, the chosen pH range was 3.0–10.0. The fluorimetric
response was negligible at the neutral and basic pH, but
surge up in the acidic pH. The highest response was seen
at pH 5 (Fig. 7).
sensing material, Rh1@TiO showed very weakly to neg-
2
ligible fluorescence response at pH ≥ 7, suggesting that
the spirocyclic form of the probe molecule remained intact
at this pH condition. The pH-controlled emission mea-
3
2
surements established that Rh1 and Rh1@TiO could be
2
+
applied for the detection of the incessant release of H
from a hydrated form of silica (viz. orthosilicic acid). Con-
sidering the fact that the detection of silica species bioac-
cumulation in-vitro or in-vivo would require cellular pH,
the media for fluorometric detection of orthosilicic acid
was set at pH 7.0 for further studies.
The response of Rh1@TiO towards orthosilicic acid
2
was also measured at the same pH range. Strongly alka-
line pH was avoided as basic metal silicates precipitate
3.3. Spectrofluorometric and Spectrophotometric
6
5ꢁ66
out at this condition.
The strong acidic condition was
Titrations of Orthosilicic Acid by Rh1@TiO
2
also avoided as cells cannot bear such harsh condition.
Figure 8 shows the fluorescence responses of Rh1@TiO2
To comprehend the toxic silica species (viz. orthosili-
cic acid) sensing abilities of Rh1@TiO , fluorescence
2
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Mascarenhas et al.
Water-Dispersible Rhodamine B Hydrazide Loaded TiO Nanoparticles for “Turn On” Fluorimetric Detection
2
1
3
13
Figure 2.
C NMR spectra of Rh1 ( C NMR (CDCl , 100 MHz): ꢅ (ppm) 12.62, 44.37, 65.91, 97.98, 104.62, 108.03, 122.27, 123.83, 128.08,
3
128.84, 130.05, 130.93, 132.49, 148.88, 151.56, 153.85, 166.13).
IP: 185.251.14.213 On: Wed, 19 Sep 2018 01:37:21
Copyright: American S rc ei se pn ot ni f si ce Po uf bal ni s ha eq ru se ous dispersion of the sensing mate-
Delivered by Ingenta
rial upon steady addition of 0–100 ꢂL of 10 mM
0.0–33.3 mg/L) of orthosilicic acid was measured. The
fluorescence output of fluorophore unit (Rh1) at ꢆmax
85 nm was intensified with the incremental addition
(
=
5
of orthosilicic acid, up to 12 fold, demonstrating quick
recognition efficacy (Fig. 9). This is the outcome of the
transformation of the non-fluorescent spirolactam form of
Rh1 in the sensing material to its ring-opened fluorescent
+
state on reacting with H ions released by orthosilicic
acid. Additionally, a separate time-dependent fluorescence
Figure 3. XRD spectra of the synthesized TiO nanoparticles.
2
Figure 4. TGA curves of TiO2 nanoparticles (black line), probe Rh1
(
red line) and sensing material Rh1@TiO (blue line).
Figure 5. IR spectra of Rh1 and the sensing material, Rh1@TiO2.
2
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Water-Dispersible Rhodamine B Hydrazide Loaded TiO Nanoparticles for “Turn On” Fluorimetric Detection
Mascarenhas et al.
2
Figure 6. FESEM images of (A) anatase TiO2 nanoparticles; (B) free
Rh1 and (C) the sensing material, Rh1@TiO2.
Figure 9. Fluorescence response of Rh1@TiO2 sensing system
10 mg/L) upon addition of orthosilicic acid (0.0–33.3 mg/L) in deionized
(
water at room temperature after 10 min [excitation at ꢆmax = 525 nm].
Inset: Plot of the increment in emission against the concentration of the
analyte.
In a similar study, the chromogenic response of the
Rh1@TiO towards orthosilicic acid was investigated by
2
the absorbance measurements of an aqueous probe solu-
tion on steady addition of 0–100 ꢂL of 10 mM solution
of orthosilicic acid (or 0.0–33.3 mg/L). The absorbance
signal peak, at ꢆ = 558 nm, was found to be inten-
max
IP: 185.251.14.213 On: Wed, 19 Sep 2018 01:37:21
sified with increasing orthosilicic acid doses in solution
Copyright: American Scientific Publishers
(
Fig. 10). Both the investigations strongly portrayed the
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+
efficacy of the probe in H ion detection (from orthosilicic
acid) in aqueous solutions.
Figure 7. The effect of pH on the fluorescence response of Rh1 probe
at different pH in 20% CH CN–H O.
3
2
3
.4. Mechanistic Aspects of Silica Species Sensing by
measurement of an equimolar mixture of Rh1@TiO and
2
Rh1@TiO and Spectral Features
2
orthosilicic acid (both 10 mg/L) revealed that 10 min of
incubation is sufficient in obtaining the highest fluores-
cence response suggesting a quick conversion to the spiro-
The spirolactam form of rhodamine derivatives (viz. Rh1
and sensing material, Rh1@TiO ꢀ is non-fluorescent and
colourless at neutral pH. However, binding with H ions
2
+
+
lactam form approach in the presence of H ions.
(
from orthosilicic acid) induces a strong orange fluores-
cence peak and an intense pink colour due to spontaneous
conversion of the spirolactam form of rhodamine residue
Figure 8. The fluorescence response of Rh1@TiO2 sensing system
(
10 mg/L) after 10 min with and without orthosilicic acid (10 mg/L, 1:1
ratio) in different pH buffers (pH 3.0–10.0) at room temperature (ꢆex
25 nm).
Figure 10. Absorbance response of the Rh1@TiO2 sensing sys-
tem (10 mg/L) after 10 mins of the addition of orthosilicic acid
(0.0–33.3 mg/L) in deionized water at room temperature.
=
5
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2
to show a response after 2 h and surpasses Cu2 after
+
N
O
N
N
O
N
6 h (Figs. 11(B and C)). In 10% CH OH-phosphate buffer
3
2
+
Hg showed a strong response, whereas other metal ions
showed a negligible response (Fig. 12). In both the cases,
H
4
SiO
4
N
NH
2
O
(
or H+)
H SiO did not show any response as the availability of
O
NHNH
2
4 4
+
Rh1 (non-fluorescent)
H was restricted by buffer medium.
Rh2 (fluorescent)
In a separate study, the sensing material, Rh1@TiO was
2
2
+
2+
N
O
N
separately treated with Cu , Hg , and H SiO in water
4
4
-
H+
and the fluorescence response was noted after 10 min,
30 min, and 6 h (Fig. 13). While H SiO started to show
+
H+
N
NH
2
4
4
strong response immediately after 10 min, the fluorimetric
OH
2
+
response of Hg picked up only after 6 h of exposure
2
+
+
Scheme 2. A plausible mechanism of the H mediated ring opening of
Rh1 in the process of detecting orthosilicic acid.
(Fig. 13(C)). Presumably, the approach of Hg ions was
somewhat restricted inside the core of the sensing mate-
rial (Rh1@TiO ꢀ and therefore, the chemodosimeter (Rh1)
2
to its ring-opened form, Rh2 (Scheme 2).23ꢁ59 This phe-
nomenon is justified by the appearance of an anticipated
peak of the ring-opened form of rhodamine B hydrazide
at 586 nm in the emission spectra. The formation of the
ring-opened form (Rh2) was confirmed by carrying out a
reaction of Rh1 in the presence of orthosilicic acid at a
larger scale and taking IR and NMR of the isolated prod-
does not get quick access to these cations to react with.
This resulted in a very low fluorometric response in water
after 10 min.
3.6. Selectivity Study with Interfering Metal Ions
Kidneys, specifically the proximal tubular cells are actively
involved in maintaining a balance of major elements in
5
9
+ + 2+
the body viz. Na , K , Mg and trace elements, like
uct and matching with reported data.
2
+
3+
2+
2+
Fe , Fe , Mn , Zn ions, and thereby inherently con-
tain these cationic species which are essential for various
cellular functions, such as heart and muscle contrac-
tions, nerve signalling, hormone production, anti-oxidative
3
.5. Fluorescence Response at Different
Time Intervals
5
2
The probe Rh1 was previously reported by Czarnik
IP: 185.251.14.213 On: Wed, 19 Sep 2018 01:37:21
for Cu2 and Chang for Hg detection. However, we
+
58
2+
enzyme action, haemoglobin formation, glucose homeosta-
Copyright: American Scientific Publishers
6
9ꢁ70
observed that the sensing material (Rh1@TiO Dꢀ ehl iavr ed rl ye d bys iI sn ,ga en nd t ar enoprotective effects.
These organs addition-
2
2
+
shows any fluorimetric response in the presence of Cu
ally serve as the primary target of metal toxicity owing
to its high reabsorptive and accumulative properties. Some
of the metal species are well-reported to inflict severe
nephrotoxicity when surpassing the permissible levels of
and Hg2 ions under the sensing condition for orthosilicic
+
acid (Figs. 11–14). These observations insisted us to carry
2
+
2+
out a time-dependent study for both Cu and Hg ions
in water. In a separate study, we have repeated the stud-
2
+
2+
2+
2+
2+
3+ 71ꢁ72
Ca , Cd , Co , Hg , Pb , Sr . The specificity
5
2
58
ies of Czarnik and Chang. In the present study, free
probe, i.e., Rh1, was separately treated with Cu , Hg ,
Fe , Zn and H SiO in 20% CH CN-HEPES buffer
of Rh1@TiO sensing material for a toxic hydrated form
2
2
+
2+
of silica (viz. orthosilicic acid) in an aqueous solution was
examined analogously in the presence of various compet-
ing toxic and inherent renal metal ions under comparable
conditions. For this purpose, the fluorescence responses of
3
+
2+
4
4
3
(
at pH 7) and 10% CH OH-phosphate buffer (at pH 7) and
3
the fluorescence response was noted after 30 min, 2 h, and
h. It was observed that Rh1 is a very effective sensor
6
Rh1@TiO after addition of equal mixture (10 mg/L) of
2
2
+
2+
2+
2+
2+
2+
3+
2+
for Cu in 20% CH CN-HEPES buffer with the highest
response after 30 min (Fig. 11(A)). However, Hg started
each metal ion (Ca , Cd , Co , Cu , Fe , Fe , Hg ,
3
2
+
+ + 2+ 2+ 2+ 3+ 2+
Na , K , Mg , Mn , Pb , Sr , Zn ꢀ and orthosilicic
(
A)
(B)
(C)
Figure 11. Time-dependent study of Cu2 , Hg , H SiO and other metal ions against Rh1 after (A) 30 min, (B) 2 h and (C) 6 h in 20% CH CN-
+
2+
4
4
3
HEPES buffer (at pH 7).
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Water-Dispersible Rhodamine B Hydrazide Loaded TiO Nanoparticles for “Turn On” Fluorimetric Detection
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2
(
A)
(B)
(C)
Figure 12. Time-dependent study of Cu2 , Hg , H SiO and other metal ions against Rh1 after (A) 30 min, (B) 2 h and (C) 6 h in 10% CH OH-
+
2+
4
4
3
Phosphate buffer (at pH 7).
(
A)
(B)
(C)
2
+
2+
Figure 13. Time-dependent study of Cu , Hg and H SiO and other metal ions against Rh1@TiO after (A) 10 min, (B) 30 min, (C) 6 h in water.
4
4
2
+
acid (H SiO which acts as a source of H ꢀ in the aqueous
and got similar results as per their reports for Rh1 when
it is free. However, in a separate study, it was observed
4
4
dispersion of sensing material (10 mg/L) were recorded
IP: 185.251.14.213 On: Wed, 19 Sep 2018 01:37:21
2
+
that Rh1@TiO responds to Hg after long exposure in
2
(
Fig. 14).
As represented in Figure 14, the sensing material,
Copyright: American Scientific Publishers
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water (Fig. 13). Presumably, the approach of larger metal
ions is somewhat restricted inside the core of the sensing
Rh1@TiO exhibited negligible to no response for most
2
of the potential rival metal ions and generated a strong
fluorescence response upon interaction with orthosili-
cic acid at the working pH. Notably, low response
material (Rh1@TiO ꢀ and therefore, the chemodosimeter
2
(Rh1) does not get quick access to these cations to react
with. This results in very low fluorometric response at pH
7 after 10 min. The fluorescence output in the presence
of these cations is still very nominal under the sensing
condition making the probe practically unperturbed by the
presence of the interfering metal ions. This fact is further
established through competition experiments conducted in
the presence of large excess (5 times of orthosilicic acid)
2
+
2+
2+
aroused out of interaction with Hg , Zn , Fe
or
Co under the sensing condition. This is in sheer con-
2
+
5
2
2+
trast with the reported results by Czarnik for Cu
5
8
2+
and Chang for Hg with Rh1. We repeated their studies
Figure 15.
A plot of relative fluorescence intensity versus con-
Figure 14. Maximum fluorescence responses of Rh1@TiO (10 mg/L),
centration of analyte obtained from a solution of sensing material
2
which were recorded after 10 min of the addition of equal amount of
various analytes (metal ions), in deionized water at room temperature.
Rh1@TiO (10 mg/L) and lower concentration range of orthosilicic acid
(0.1–1 mg/L) after 10 min of interaction.
2
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2
from this, the detection limit was calculated to be 8.4 ppb
−8
or 8ꢇ4 ×10 M of orthosilicic acid.
3
.8. Determination of Cytotoxicity of Rh1@TiO and
2
Fluorescence Imaging of Toxic Silica Species in
Live Cells
If a sensing system is intended to apply to live biologi-
cal species, it is essential to check its biocompatibility and
cytotoxicity. As a model human kidney cell system viz.
HK cells (Human Kidney cells) was chosen for cytotox-
icity study and detection of the toxic silica sp. (specifi-
cally orthosilicic acid) accumulation via cellular imaging.
Accordingly, the cytotoxicity of different concentrations of
Figure 16. Cytotoxicity study of the sensing material, Rh1@TiO2 on
HK cells at varying doses for 24 h.
Rh1@TiO to HK cells was assessed using MTT assay to
determine cell sustainability at an ideal probe concentra-
tion. For this, 70% confluent HK cells were exposed to
2
of all metal ions and orthosilicic acid (Fig. 14). As antic-
ipated, the orthosilicic acid induced fluorescence response
of the sensing material, Rh1@TiO was unaltered by the
2
enhancing Rh1@TiO sensing system concentrations, seri-
2
existence of the competing metal ions, suggesting that the
ally diluted in DMEM (i.e., 1, 10, 50, 100, and 200 mg/L)
in a 24 well plate, and incubated for 24 h. Unexposed con-
trol was simultaneously maintained. The percentages of
viable cells relative to untreated controls were determined
and plotted (Fig. 16). The cell viability was ascertained
to be greater than 90% on 24 h exposure to the highest
+
presence of H ions amidst several other cations is uni-
vocally identified by the sensing material with no loss in
fluorescence output. The response of the sensing mate-
rial towards orthosilicic acid amongst diverse nephrotoxic
and inherent renal metallic species in an aqueous solution
extends its application for selective in-vitro detection of
toxic silica species accumulation in affected kidney cells
or elsewhere.
Rh1@TiO concentration (200 mg/L). This suggests that
2
even high Rh1@TiO concentrations are non-toxic to the
2
living human cells and can be employed for detection of
toxic silica species viz. orthosilicic acid bioaccumulation
IP: 185.251.14.213 On: Wed, 19 Sep 2018 01:37:21
3
.7. Determination of the Detection Limit
in living organisms.
The limit of detection (LOD) is an essential prop De r et yl i vo ef r ae d by I nS gi ne cn et ano cytotoxic effect of Rh1@TiO was detected,
Copyright: American Scientific Publishers
2
sensing system, assessed to gauge its utility for real sam-
a median concentration of Rh1@TiO (i.e., 50 mg/L) was
2
ple analysis. To elucidate the LOD, the fluorescence output
employed for fluorescence monitoring of orthosilicic acid
deposition in HK cells, which were exposed to increasing
doses of orthosilicic acid. The orthosilicic acid deposition
over the 1st time point was monitored by pre-treating four
sets of HK cells with increasing doses of orthosilicic acid
(0.1 mg/L (Fig. 17), 1 mg/L, 10 mg/L and 100 mg/L)
of Rh1@TiO at lower concentrations of orthosilicic acid
2
in deionised water was plotted (Fig. 15). Under prevail-
ing conditions, the sensing material, Rh1@TiO responds
2
linearly to the varying concentration of orthosilicic acid
2
(
0.1–1 mg/L or 0.3–3.0 ꢂL in 3 mL) with R = 0.9973 and
Figure 17. Time-dependent fluorescence detection of toxic silica species (specifically orthosilicic acid) deposition in HK cells treated with low
orthosilicic acid (0.1 mg/L) utilizing sensing material, Rh1@TiO . HK cells incubated with 0.1 mg/L orthosilicic acid for three different exposure
2
periods viz. 30 min (A), 24 h (B) and 8 days (C) followed by treatment with 50 mg/L of Rh1@TiO2 sensing material (for 30 min) after each dosing
period. Images recorded after sensing material exposure in the absence (a, c) and presence (b, d) of orthosilicic acid at each temporal point. Scale bars
are 200 ꢂm.
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2
for 1 h, followed by PBS washing to remove the non-
sensing-system) and fluorescence images were captured
(Fig. 18). These steps were replicated for the separate
sets of cells pre-incubated with orthosilicic acid for 24 h
and 8 days, respectively. At each time point, set of HK
accumulated species. This was followed by incubation
ꢀ
with 50 mg/L of the Rh1@TiO for 30 min at 37 C and
2
5
% CO succeeded by a triple PBS wash (to remove excess
2
IP: 185.251.14.213 On: Wed, 19 Sep 2018 01:37:21
Copyright: American Scientific Publishers
Delivered by Ingenta
Figure 18. Dose and time-dependent monitoring of toxic silica species (viz. orthosilicic acid) accumulation in live HK cells with the sensing material,
Rh1@TiO . Phase contrast (b–d) and fluorescence images (f–h) of HK cells on treatment with 1 mg/L (b, f), 10 mg/L (c, g) and 100 mg/L (d, h) of
2
orthosilicic acid for three different exposure time points viz. 1 h (A), 24 h (B) and 8 days (C) followed by 30 min incubation with Rh1@TiO (50 mg/L)
2
at each temporal point. Images of HK cells (a, e) were captured at each time point in the absence of orthosilicic acid and presence of Rh1@TiO2 to
serve as controls. Gradual escalation in fluorescence intensity noted with increasing orthosilicic acid doses and exposure period signifying progressive
intracellular accumulation of toxic silica species inflicting chronic nephrotoxicity. Scale bars are 200 ꢂm.
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2
cells were separately incubated with solely 50 mg/L of
cross the cellular surface of nephrotoxic HK cells interact-
ing with bioaccumulated orthosilicic acid produced under
silica stress conditions. The synthesized sensing material
Rh1@TiO in the absence of orthosilicic acid for 30 min to
2
serve as probe controls negating the possibility of intrinsic
fluorescence of the Rh1@TiO sensing material, justified
Rh1@TiO offers several advantages like good water dis-
2
2
by the absence of fluorescence as recorded by fluorescence
microscope (Fig. 18).
As depicted in Figure 18, significant orange fluo-
rescence was observed from the intracellular region of
nephrotoxic HK cells contaminated with orthosilicic acid
persibility, the absence of organic solvents during fluo-
rimetric studies, quick turn-on type signal transduction,
low-level imaging. This sensing material has the potential
to be used in the medical tool for early identification of
silica-induced nephrotoxicity with effective detection and
imaging of toxic silica species deposition in the exposed
kidney cells intending to adopt necessary prophylactic
measures to reduce the burden and risk of chronic kidney
disease development.
and consequently treated with the probe Rh1@TiO . It can
2
be considered that the nanosize of the probe helps to
smoothly cross the cell membrane and subsequent inter-
act with the accumulated silica (in the form of orthosilicic
acid) to get converted to its ring-opened fluorescent form
of Rh1. The fluorescence intensity was apparently a func-
tion of the dose and time of orthosilicic acid exposure
manifested by an enhanced response on exposure to the
highest orthosilicic acid dose (100 mg/L) for the longest
period (8 days) due to more and more interaction with
Abbreviations
CKD—Chronic Kidney Disease; DMEM—Dulbecco’s
Modified Eagle’s Medium; HK—human kidney cells;
H SiO —orthosilicic acid; MTT—3-(4,5-Dimethylthiazol-
4
4
2
-yl)-2,5-Diphenyltetrazolium Bromide; PBS—Phosphate
+
intercellular H ions leading to fluorescent ring-opened
buffered saline; P-XRF—portable X-ray fluorescence
state of the probe. However, the cell density diminishes
on exposure to higher doses of silica species (orthosili-
cic acid) over a longer period of time highlighting dose
and time-dependent toxicity. Notably, the sensitivity of this
analytical tool was found to be very high as the accumu-
lation of low concentration orthosilicic acid (0.1 mg/L) in
HK cells is good enough to generate sufficiently strong flu-
spectrometer;
Rhodamine
RA—Rheumatoid
hydrazide; Rh1@TiO —Rhodamine
arthiritis;
Rh1—
B
2
hydrazide adsorbed onto TiO nanoparticles; SiO —Silica;
2
2
SLE—systemic lupus erythematosus; TiO —titanium
2
dioxide.
Declaration of Interest
IP: 185.251.14.213 On: Wed, 19 Sep 2018 01:37:21
orescence signals from the intra-cellular region (Fig. 17).
Copyright: American S Tc ihe en at iuf it ch oPr su bd lei sc lha er er sn o personal, financial or organisational
This study provides substantial evidence on the ability of
Delivered by Ingenta
conflict of interests.
this sensing material to detect silica species accumulation
in human kidney cells on chronic exposure to increasing
dosages that inflicts severe nephrotoxicity ultimately man-
ifesting as Chronic Kidney Disease. A low-level detection
and imaging of silica in affected HK cells may lead to the
Acknowledgments: Amrita Chatterjee thanks UGC-
DAE (CSR-KN/CRS-91/2016-17/1132) and DST-SERB
(project No. EMR/2016/001416) for financial support.
Starlaine C. Mascarenhas and Ram U. Gawas are grateful
to BITS, Pilani for research fellowships. The authors are
thankful to the central NMR Facility of BITS Pilani, Pilani
campus for NMR spectral data and Central Sophisticated
Instrumentation Facility (CSIF) of BITS Pilani KK Birla
Goa Campus for LC-MS data and FESEM images.
conclusion that the sensing material, Rh1@TiO can even-
2
tually be employed for in-vitro monitoring of toxic silica
species bioaccumulation in any biological samples.
4
. CONCLUSIONS
In summary, the present work demonstrates an effec-
tive strategy to detect and image the toxic silica species
(
orthosilicic acid) accumulation in the live cells. The
References and Notes
sensing material Rh1@TiO2 was prepared by simple
physisorption of a rhodamine derivative, which acts as a
chemodosimeter based pH sensor, onto the biocompati-
1. J. Marin-Carbonne, F. Robert, and M. Chaussidon, Precambrian Res.
247, 223 (2014).
2
3
. J. T. Cornelis and B. Delvaux, Funct. Ecol. 30, 1298 (2016).
. M. M. Mendez-Vargas, F. B. Baez-Revueltas, P. Lopez-Rojas,
J. HoracioTovalin-Ahumada, J. O. Zamudio-Lara, I. A. Marin-
Cotoñieto, and F. Villedaf, Rev. Med. Inst. Mex. Seguro. Soc. 51, 384
ble, non-toxic TiO nanoparticles. This newly developed
2
water-dispersible fluorescent nanoprobe has demonstrated
its capability to detect orthosilicic acid in aqueous media,
and in biological systems via interaction with the inces-
santly released H ions. The H ions generate the fluores-
cent ring-opened form of the fluorophore and result in a
strong orange fluorescence output.
The studies were carried out on human kidney cells
at biological pH (pH 7). The cell imaging studies con-
firmed that this probe is biocompatible and can easily
(2013).
4. K. M. Bang, J. M. Mazurek, J. M. Wood, G. E. White, and S. A.
Hendricks, Morb. Mortal. Wkly. Rep. 64, 117 (2015).
+
+
5
6
7
. K. Tsuchiya, M. Toyoshima, Y. Kamiya, Y. Nakamura, S. Baba, and
T. Suda, Internal Med. 56, 1701 (2017).
. V. Malmstrom, A. I. Catrina, and L. Klareskog, Nat. Rev. Immunol.
17, 60 (2017).
. S. Lee, H. Matsuzaki, N. Kumagai-Takei, K. Yoshitome, M. Maeda,
Y. Chen, and Y. Nishimura, Environ. Health. Prev. 19, 322 (2014).
J. Nanosci. Nanotechnol. 18, 8142–8154, 2018
8153

Water-Dispersible Rhodamine B Hydrazide Loaded TiO Nanoparticles for “Turn On” Fluorimetric Detection
Mascarenhas et al.
2
8
. I. Marie, J. F. Gehanno, M. Bubenheim, A. B. Duval-Modeste,
P. Joly, S. Dominique, P. Bravard, D. Noel, A. F. Cailleux, J. Weber,
and P. Lagoutte, Autoimmun. Rev. 13, 151 (2014).
43. S. Singh and H. S. Nalwa, J. Nanosci. Nanotechnol. 7, 3048 (2007)
44. V. Kumar, M. Banerjee, and A. Chatterjee, Talanta 99, 610
(2012).
9
. K. Steenland and E. Ward, Cancer. J. Clin. 64, 63 (2014).
45. D. G. Khandare, V. Kumar, A. Chattopadhyay, M. Banerjee, and
A. Chatterjee, RSC Adv. 3, 16981 (2013).
1
0. S. Sen, R. Mitra, S. Mukherjee, P. K. Das, and S. Moitra, Curr.
Respir. Med. Rev. 12, 56 (2016).
46. D. G. Khandare, H. Joshi, M. Banerjee, M. S. Majik, and
A. Chatterjee, RSC Adv. 4, 47076 (2014).
47. S. Hazra, S. Balaji, M. Banerjee, A. Ganguly, N. N. Ghosh, and
A. Chatterjee, Anal. Methods 6, 3784 (2014).
1
1
1. N. Ghahramani, Int. J. Occup. Environ. Med. 1, 108 (2010).
2. M. L. Millerick-May, S. Schrauben, M. J. Reilly, and K. D.
Rosenman, Am. J. Ind. Med. 58, 730 (2015).
1
1
3. M. Ricco, E. Thai, and S. Cella, Ind. Health 54, 74 (2016).
4. T. Nishizono, S. I. Eta, H. Enokida, K. Nishiyama, M. Kawahara,
and M. Nakagawa, Int. J. Urol. 11, 119 (2004).
48. D. G. Khandare, H. Joshi, M. Banerjee, M. S. Majik, and
A. Chatterjee, Anal. Chem. 87, 10871 (2015).
49. A. Chatterjee, D. G. Khandare, P. Saini, A. Chattopadhyay, M. S.
Majik, and M. Banerjee, RSC Adv. 5, 31479 (2015).
50. D. G. Khandare, M. Banerjee, R. Gupta, N. Kumar, A. Ganguly,
D. Singh, and A. Chatterjee, RSC Adv. 6, 52790 (2016).
51. A. Chatterjee, M. Banerjee, D. G. Khandare, R. Gawas, S. C.
Mascarenhas, A. Ganguly, R. Gupta, and H. Joshi, Anal. Chem.
89, 12698 (2017).
1
1
1
5. S. Mascarenhas, S. Mutnuri, and A. Ganguly, Chemosphere 177, 239
(2017).
6. S. Kumar, Y. Milstein, Y. Brami, M. Elbaum, and R. Elbaum, New
Phytol. 213, 791 (2017).
7. E. McLarnon, S. McQueen-Mason, I. Lenk, and S. E. Hartley, Front.
Plant. Sci. 8, 1199 (2017).
1
1
2
2
8. B. Tesson and M. Hildebrand, Plos One 5, e14300 (2010).
9. T. Ueno and T. Nagano, Nat. Methods 8, 642 (2011).
0. T. Terai and T. Nagano, Pflug. Arch. Eur. J. Phy. 465, 347 (2013).
1. M. Beija, C. A. Afonso, and J. M. Martinho, Chem. Soc. Rev.
52. V. Dujols, F. Ford, and A. W. Czarnik, J. Am. Chem. Soc. 119, 7386
(1997).
53. X.-F. Yang, X.-Q. Guo, and Y.-B. Zhao, Talanta 57, 883 (2002).
54. X.-F. Yang, X.-Q. Guo, and Y.-B. Zhao, Anal. Chim. Acta 456, 121
(2002).
38, 2410 (2009).
2
2
2. J. Chan, S. C. Dodani, and C. J. Chang, Nat. Chem. 4, 973 (2012).
3. H. N. Kim, M. H. Lee, H. J. Kim, J. S. Kim, and J. Yoon, Chem.
Soc. Rev. 37, 1465 (2008).
55. Y. Xiang, L. Mei, N. Li, and A. Tong, Anal. Chim. Acta 581, 132
(2007).
56. X. Chen, and J. Zou, Microchim. Acta 157, 133 (2007).
57. Z. Zhang, Y. Zheng, W. Hang, X. Yan, and Y. Zhao, Talanta 85, 779
(2011).
58. K. N. Kim, M. G. Choi, J. H. Noh, S. Ahn, and S.-K. Chang, Bull.
Korean Chem. Soc. 29, 571 (2008).
59. M. Pandurangappa and S. K. Kumar, Anal. Methods 3, 715
(2011).
2
2
2
4. Q. A. Best, R. Xu, M. E. McCarroll, L. Wang, and D. J. Dyer, Org.
Lett. 12, 3219 (2010).
5. M. Tian, X. Peng, J. Fan, J. Wang, and S. Sun, Dyes Pigments
95, 112 (2012).
6. L. Liu, P. Guo, L. Chai, Q. Shi, B. Xu, J. Yuan, and W. Zhang,
Sensor. Actuat. B-Chem. 194, 498 (2014).
2
2
2
7. M. A. Brzezinski and D. J. Conley,IPJ.:P1hy8c5ol..2 350 1, .4 15 4( 1. 29 91 4 3) . On: Wed6,0 1. 9M S. Ae spa n 2o ,0 M1 .8 D 0o 1i ,: K3 .7 B: 2a b1a , M. Taniguchi, M. Shibano, S. Tanaka,
8. M. Kucki and T. Fuhrmann-Lieker, J. R. SocC. Ionpteyrfraicgeh9t,:7 A2 7m ( 2e0 r1 i 2c ) a. n Scien Mt i .f i Sc a kPa gu ubc lh i is, hM e.r Ts akaoka, M. Hirata, R. Yanagihara, R. Nakahara,
9. S. Su, W. Wu, J. Gao, J. Lu, and C. Fan, J. Mater. Chem. 22, 18101
Y. Hayashi, T. Yamaguchi, H. Matsumura, and Y. Fujita, J. Biosci.
Delivered by Ingenta
(2012).
Bioeng. 114, 98 (2014).
3
3
3
0. C. E. Rowland, C. W. Brown, J. B. Delehanty, and I. L. Medintz,
61. M. Kang, D. Peng, Y. Zhang, Y. Yang, L. He, F. Yan, S. Sun, S. Fang,
P. Wang, and Z. Zhang, New J. Chem. 39, 3137 (2015).
62. P. Praikaew, T. Duangdeetip, N. Chimpalee, C. Wainiphithapong,
P. Swanglap, and N. Wanichacheva, Mater. Express 5, 300 (2015).
63. L. Wang, B. Zheng, Y. Zhao, J. Du, and D. Xiao, Anal. Methods
4, 2369 (2012).
Mater. Today 19, 464 (2016).
1. P. Kumar, K. H. Kim, V. Bansal, S. Kumar, N. Dilbaghi, and Y. H.
Kim, Trend. Anal. Chem. 86, 235 (2017).
2. L. Walekar, T. Dutta, P. Kumar, Y. S. Ok, S. Pawar, A. Deep, and
K. H. Kim, Trend. Anal. Chem. 97, 458 (2017).
3
3
3
3. J. Yao, M. Yang, and Y. Duan, Chem. Rev. 114, 6130 (2014).
4. Q. Liu, Q. Zhou, and G. Jiang, Trend. Anal. Chem. 58, 10 (2014).
5. J. Schneider, M. Matsuoka, M. Takeuchi, J. Zhang, Y. Horiuchi,
M. Anpo, and D. W. Bahnemann, Chem. Rev. 114, 9919 (2014).
6. H. Chen, C. E. Nanayakkara, and V. H. Grassian, Chem. Rev.
64. B. Naik, C. H. Manoratne, A. Chandrashekhar, A. Iyer, V. S. Prasad,
and N. N. Ghosh, J. Exp. Nanosci. 8, 462 (2013).
65. E. R. Spada, E. A. Pereira, M. A. Montanhera, L. H. Morais, R. G.
Freitas, R. G. Costa, and F. R. de Paula, J. Mater. Sci-Mater. El.
28, 16932 (2017).
66. A. Adan, Y. Kiraz, and Y. Baran, Curr. Pharm. Biotechnol. 17, 1213
(2016).
67. D. Hermosilla, R. Ordonez, L. Blanco, E. de la Fuente, and
A. Blanco, Chem. Eng. Technol. 35, 1632 (2012).
68. N. A. Milne, T. O’Reilly, P. Sanciolo, E. Ostarcevic, M. Beighton,
K. Taylor, M. Mullett, A. J. Tarquin, and S. R. Gray, Water Res.
65, 107 (2014).
69. U. C. Gupta and S. C. Gupta, Pedosphere 24, 13 (2014).
70. A. Agarwal, P. Khanna, D. K. Baidya, and M. K. Arora, J. Clin.
Endocrinol. Metab. 1, 57 (2011).
71. A. Wilk, E. Kalisi n´ ska, D. I. Kosik-Bogacka, M. Romanowski,
J. Rozanski, K. Ciechanowski, and N. Lanocha-Arendarczyk, Envi-
ron. Geochem. Hlth. 39, 889 (2017).
3
3
3
3
4
4
4
112, 5919 (2012).
7. T. Rajh, N. M. Dimitrijevic, M. Bissonnette, T. Koritarov, and
V. Konda, Chem. Rev. 114, 10177 (2014).
8. A. A. Keller, H. Wang, D. Zhou, H. S. Lenihan, G. Cherr, B. J.
Cardinale, and Z. Ji, Environ. Sci. Technol. 44, 1962 (2010).
9. Z. Ji, X. Jin, S. George, T. Xia, H. Meng, X. Wang, and A. E. Nel,
Environ. Sci. Technol. 44, 7309 (2010).
0. C. Zhao, F. U. Rehman, Y. Yang, X. Li, D. Zhang, H. Jiang, and
C. Liu, Sci. Rep. 5, 11518 (2015).
1. J. S. Taurozzi, V. A. Hackley, and M. R. Wiesner, Nanotoxicology
7, 389 (2013).
2. Y. Zhao and H. S. Nalwa (eds.), Nanotoxicology: Interactions of
Nanomaterials with Biological Systems, American Scientific Pub-
lishers, Los Angeles (2007).
72. S. E. Orr and C. C. Bridges, Int. J. Mol. Sci. 18, 1039 (2017).
Received: 10 June 2018. Accepted: 2 July 2018.
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J. Nanosci. Nanotechnol. 18, 8142–8154, 2018