Naphthalimide-gold-based nanocomposite for the ratiometric detection of okadaic acid in shellfish

Article abstract of DOI:10.1039/d0tb01195a

Okadaic acid (OA) is one of the known marine biotoxins produced by various dinoflagellates and exists in seafood such as shellfish. The consumption of contaminated shellfish with OA leads to diarrheic shellfish poisoning (DSP), which results in the inhibition of protein phosphatase enzymes in humans. This poisoning can cause immunotoxicity and tumor promotion due to the accumulation of okadaic acid in more than the allowed limit in bivalve molluscs. The reported methods for the detection of okadaic acid include mouse bioassays, immunoassays, chromatography coupled with spectroscopic techniques, electrochemical sensors and immunosensors. We have developed a naphthalimide-gold-based nanocomposite for the detection of okadaic acid. Individually, the organic nanoparticles (ONPs) of synthesized naphthalimide-based receptors and gold-coated ONPs are less sensitive for detection. However, fabrication of the composite of Au?ONPs and ONPs enhance the sensing properties and selectivity. The composite shows a ratiometric response in the UV-Vis absorption spectrum and quenching in the fluorescence profile with a detection limit of 20 nM for OA in aqueous medium. In cyclic voltammetry, a shift was observed in the cathodic peak (-0.532 V to -0.618 V) as well as in the anodic peak (-0.815 V to -0.847 V) with the addition of okadaic acid. To study the quick binding of the composite with OA, a time response experiment was performed. Also, the developed sensor retains its sensing ability in the pH range of 5-9 and in high salt conditions. Our developed composite can be used for the detection of OA in real applications.

Full text of DOI:10.1039/d0tb01195a

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Chaudhary, A. Singh, N. Kaur and N. Singh, J. Mater. Chem. B, 2020, DOI: 10.1039/D0TB01195A.
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Pages 341-528
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ISSN 2050-750X
PAPER
Wei Wei, Guanghui Ma et al.
Macrophage responses to the physical burden of cell-sized
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DOI: 10.1039/D0TB01195A
ARTICLE
Naphthalimide – Gold based Nanocomposite for the Ratiometric
Detection of Okadaic Acid in Shellfish
Meenakshi Verma,a† Monika Chaudhary,b† Amanpreet Singh,c Navneet Kaur,d,* Narinder Singhc,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Okadaic acid (OA) is one of the known marine biotoxin produced by various dinoflagellates and exists in sea food such as
shellfish. The consumption of contaminated shellfish with OA leads to diarrheic shellfish poisoning (DSP) which results in
inhibition of protein phosphatases enzymes in humans. This poisoning can cause immunotoxicity and tumor promotion
due to accumulation of okadaic acid more than allowed limits in bivalve molluscs. The reported methods for the detection
of okadaic acid include mouse bioassays, immunoassays, chromatography coupled with spectroscopic techniques,
electrochemical and immunosensors etc. We have developed naphthalimide-gold based nanocomposite for the detection
of Okadaic acid. Individually, the organic nanoparticles (ONPs) of synthesized naphthalimide based receptor and gold
coated ONPs are less sensitive for detection. However, the fabrication of the composite of Au@ONPs and ONPs enhances
the sensing properties and selectivity. The composite shows ratiometric response in UV-Vis absorption spectrum and
quenching in fluorescence profile with limit of detection 20 nM for OA in aqueous medium. In cyclic voltammetry, a shift
was observed in cathodic peak (-0.532 V to -0.618 V) as well as in anodic peak (-0.815 V to -0.847 V) on addition of Okadaic
Acid. To study quick binding of composite with OA time response experiment was performed. Also, the developed sensor
retains its sensing ability in pH range of 5-9 and high salt effects. Our developed composite can be used for the detection
of OA in real applications.
sea food such as shellfish.6 Food contamination may diversely
effect agriculture and food industry and also it may affect
economic, production and recreational areas (e.g. agricultural
and shellfish industry). These pollutants can enter the chain
Introduction
from agricultural activities (pesticides/herbicides), side
products from industries, pharmaceutical compounds
(drugs/antibiotic), terrorists and military activities (explosives
molecules)7 or toxins produced by aquatic animals or
phytoplankton blooms.8-9
Toxins are natural secondary metabolites usually produced by
microorganisms, fungi, animals or plants, are endangering
food, water resources and human health.1 Owing to their
carcinogenic and mutagenic nature these mycotoxins can
adversely affect the functioning of kidney, liver,
gastrointestinal tract, nervous system and blood-vascular
system. Mycotoxins also causes damages to the reproductive
function and leads to hormonal imbalance.2 These mycotoxins
consist of diverse substances having molecular weight ranging
from 0.14 kDa to 150 kDa.3 However, the toxicity rely on many
factors e.g. quantity, application, and the particular mode of
action within the organism. Existence of such toxic materials
makes them strong candidates that can be used as biological
warfare agents and exhibit a risk for human beings.4-5 Food and
water containing traces of mycotoxins can seriously endanger
the public health. Food such as cereals, fruits, wines etc,
generally contains mycotoxins. The marine toxins exist in the
Marine toxins form an interesting group of complex organic
compounds having long-lasting impact on human beings. The
increased emergence of toxins is a possible result of raised
ocean temperature that promotes algal blooms. On the basis
of their chemical structure, point of origin and characteristics
these can be classified into six groups. These groups include (i)
diarrheic shellfish poisoning (DSP, mainly due to okadaic acids
and dinophysistoxins), (ii) paralytic shellfish poisoning (PSP,
due to saxitoxins), (iii) amnesic shellfish poisoning (ASP, due to
domoic acids), (iv) neurotoxic shellfish poisoning (NSP, due to
brevetoxins) (v) azaspiracid poisoning (AZP, due to
azaspiracids), and (vi) ciguatera fish poisoning (CFP, due to
ciguatoxins).10 However, their increasing level in warming
marine environment necessitates considerable monitoring of
toxins in sea products.11
a. Department of UCRD, Chandigarh University, Ghraun, Mohali, - 140413, India
b. Centre for Biomedical Engineering, Indian Institute of Technology Ropar,
Roopnagar, Punjab-140001, India
c. Department of Chemistry, Indian Institute of Technology Ropar, Roopnagar,
Punjab-140001, India
d. Department of Chemistry, Panjab University, Chandigarh – 160014, India.
†Equal Contribution
Diarrheic shellfish poisoning (DSP) is caused by consumption of
okadaic acid (OA) contaminated shellfish by human beings.
The main symptoms of DSP in humans are vomiting,
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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abdominal pain, nausea and diarrhea.12-13 Okadaic acid (OA) is visible absorption spectroscopy, fluorescence spectroscopy
View Article Online
a well-known marine toxin which is produced by algae of the and cyclic voltametery techniques were used for the detection
DOI: 10.1039/D0TB01195A
genera Dinophysis and Prorocentrum.8 It is a lipophilic as well of OA.
as heat-stable phycotoxin which can accumulate in different
types of shellfish, generally in scallops, mussels, oysters and
clams.14 Inhibition of protein phosphatases, PP1 and PP2A
Experimental
enzymes is supposed to be the reason of toxicity of OA.15,16
According to the European Union, 160 g of OA per kg is the
highest allowed limit in the bivalve molluscs.17 Higher than this
allowed limit of OA may lead to immunotoxicity and tumor
promotion.18 Hence, OA contaminated shellfish is not only
harmful for human health but also for shellfish industry,
worldwide. Thus, development of detection methods for OA is
need of the time. Till 2011, the traditional method for the
detection of OA, the mouse bioassay was utilized as suggested
by European Commission (EC). However, mouse bioassay had
some drawbacks associated with it such as low selectivity,
sensitivity, inconvenience of usage and time consumption.19,20
Therefore, EC recommended other methods such as protein
General Information: 1,8-naphthalic anhydride, bromine, 4-
picolylamine, 2-fluorophenyl isothiocyanate, chloroauric acid,
ascorbic acid and ethylenediamine were purchased from
Sigma-Aldrich / local suppliers and were used without further
purification. The toxins Okadaic Acid, Domoic acid, Brevetoxin
2, Brevetoxin 3 and Azaspiracids 1 were purchased from
Sigma-Aldrich. Milli-Q water (resistivity = 18.2 MΩ.cm, at 25°C)
was used to prepare all aqueous solutions. Purified solvents of
commercial grade were purchased from Spectrochem. Jeol
1
Instrument was utilized to carry out the H NMR and 13C NMR
1
chracterizations, working at 400 MHz for H NMR and at 100
MHz for 13C NMR. All chemical shifts were recorded in ppm
relative to trimethylsilane as internal reference. The UV –
visible absorption spectra were recorded on Shimadzu 2400
spectrophotometer using quartz cuvettes of 1 cm path length.
The fluorescence experiments were conducted at room
temperature on a Perkin Elmer LS-55 Spectrophotometer with
a fixed scanning speed and emission slit width (10 nm). Particle
size was determined with dynamic light scattering using the
external probe feature of Metrohm Microtrac Ultra Nanotrac
particle size analyzer. TEM images were recorded on FEI
(Tecnai G2 F-20) instrument that worked at 200 kV and has the
resolution of 0.27 nm (point to point). Electrochemical studies
were performed using Epsilon BASi instrument at a scan rate
of 50.0mV/s. EDAX images were recorded with Jeol JSM-
6610LV scanning electron microscope operating at 15 keV.
phosphatase
chromatography
inhibition
assays
or
immunoassays,21
fluorescence/mass
coupled
to
spectrometry,22 electrochemistry, particularly enzyme-linked
immunosorbent assay (ELISA) etc.23 Though, still there is a vital
need to develop novel detection methods/techniques which
can not only perform rapid but also should be simple to use,
sensitive and cost-effective for detection of OA in shellfish.
Immunosensors are another tool used for detection of OA,23
however, these, require the use of costly antibodies, enzyme
and reporter reagents having limited stability and special
storage conditions.
Nanotechnology has been widely being utilized in various
fields of science such as catalysis, biosensing, optronics and
medicinal chemistry. Nanomaterials based biosensors have
found extensive use in different areas that not only limit to
pathogen detection but play a crucial role in the diagnosis.24,25
Hence, nanomaterials based biosensors may offer
considerable advantages in terms of sensitivity over the
currently used techniques. Out of various components
employed in biosensors, gold nanoparticles are attracting
utmost attention in the area of biomedical research due to
their good biocompatibility, significant optical and
photothermal properties.26,27 In addition to this, the surface
functionalization of gold nanoparticles is quite simple due to
various shapes of gold nanoparticles.28 The surface
functionalization allows us to tune the properties of
nanoparticles and widens the region towards various
applications.29
Synthesis and characterisation of 5: The bromination of 1,8-
naphthalic anhydride (8) was done as per method reported in
our previous work.30 In brief, a solution of 1,8-naphthalic
anhydride (1.98 g, 10.0 mmol) in KOH (2.8 g in 12 ml water)
was prepared and to this solution, 2 ml of bromine was added
drop-wise followed by stirring for 30 min at room temperature
(Scheme 1). The reaction mixture was then heated to 60 oC for
6 h. Light brown coloured precipitates separated out on
completion of the reaction. The reaction mixture was acidified
using hydrochloric acid (HCl) and then filtered the solid residue
which was further heated with 5% NaOH solution. The resulted
solution was filtered and filtrate was neutralized with diluted
solution of HCl. Filtered to collect the solid precipitates and
washed it with cold water which resulted into brown powder
of 4-bromo-1,8-naphthalic anhydride (7). To synthesize 5;
compound 7 (2.77 g, 10 mmol) was dissolved in ethanol and
then 4-picolylamine (1.08 g, 10 mmol) was added to it. This
solution was refluxed for 8 h and cooled to room temperature
on completion of reaction. Precipitates of compound 5 was
separated out, filtered and dried as brown coloured powder.
Yield = 89%. mp = 167-169 °C. 1H NMR (400 MHz, CDCl3) δ 8.63
(d, J = 4 Hz, 2H, ArH), 8.53 (d, J = 4 Hz, 2H, ArH), 8.26 (d, J = 8
Hz, 2H, ArH), 7.79 (t, J = 8 Hz, 2H, ArH), 7.37 (d, J = 4 Hz, 1H,
ArH), 5.37 (s, 2H, picolylamine-CH2). 13C NMR (100 MHz, CDCl3)
δ 150.10, 134.58, 133.35, 131.84, 130.42, 129.56, 127.19,
123.33, 42.73. MS (EI) : m/z 367.05 (M++1).
In this work, we have synthesized a composite to detect OA
(Figure 1A) using a naphthalimide based receptor (Figure 1B)
and gold nanoparticles. The organic receptor was processed to
its nanoparticles (ONPs) (Figure 1C) via reprecipitation
method. These ONPS were then coated on gold nanoparticles
(Au@ONPs) (Figure 1D) via reported methods (Figure 1E). All
the materials prepared were characterised using high
resolution transmission electron microscopy (HRTEM),
dynamic light scattering (DLS) to check the size and surface
morphology. Then composite of both Au@ONPs and ONPs has
been prepared which was used to detect okadaic acid. UV –
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Synthesis and characterisation of 3: Compound 5 (3.67 g, 10 Preparation of Au@ONPs
mmol) was refluxed for 8 h with ethylenediamine (2) (10
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DOI: 10.1039/D0TB01195A
mmol) using ethanol as reaction solvent. On completion of
Gold based organic – inorganic nanohybrid particles have
unique synthetic properties. The gold nanoparticles have an
absorption maximum near 530 nm, and TEM and DLS
experiments were performed for the size and shape of the
nanohybrids. Common method used for the fabrication of the
nanohybrid35 uses heating of the solution of ligand and HAuCl4
under rapid stirring, followed by addition of reducing agent
like trisodium citrate, sodiumborohydride and ascorbic acid. In
present work, we have prepared Au@ONPs via wet chemical
method to prepare using ascorbic acid as reducing agents.
TEM analysis was further used to study the size and shape of
the Au@ONPs. Ascorbic acid solution (0.1 mM) was prepared
by dissolving ascorbic acid in type 1 water. Gold solution (0.1
mM) was prepared by dissolving HAuCl4 in water. The required
Au@ONPs were prepared by mixing the gold solution, ONPs
and ascorbic acid in a ratio of 9:1:9, respectively. Appearance
of pink colour confirmed the formation of Au@ONPs. The
prepared Au@ONPs were characterized using DLS and TEM
analysis.
reaction, reaction mixture was allowed to cool to room
temperature naturally. Yellow coloured compound
3
precipitated out and was filtered and dried. Yield = 82%. mp =
159-161 °C; 1H NMR (400 MHz, CDCl3) δ 8.63 (m, 2H, ArH), 8.53
(dd, J = 1.6 Hz, 2H, ArH), 8.26 (dd, J = 1.6 Hz, 2H, ArH), 7.80 (t, J
= 8 Hz, 2H, ArH), 7.34 (dd, J = 8 Hz, 1H, ArH), 5.37 (s, 2H,
picolylamine-CH2), 3.90 (t, J = 4 Hz, 2H, ethylenediamine-CH2),
3.44 (t, J = 4 Hz, 2H, ethylenediamine-CH2). 13C NMR (100 MHz,
DMSO-d6) δ 164.43, 163.43, 151.71, 150.15, 147.26, 135.04,
131.69, 129.53, 127.90, 125.05, 122.60, 122.16, 108.03,
104.72, 42.74, 42.38, 38.36. MS (EI) : m/z 347.20 (M++1).
Synthesis and characterisation of 1:
Compound 3 (0.346 g, 1 mmol) was dissolved in dry DCM and
to this solution, 2-fluorophenyl isothiocyanate (2) (0.153 g, 1
mmol) was added and reaction mixture was stirred at room
temperature for 8 h. On completion of reaction, solvent was
evaporated under vacuum. The crude product was then
purified using column chromatography to get yellow coloured Chemosensing response of materials:
solid as product 1. Yield = 78%. mp = 187-189 °C. 1H NMR (400
MHz, DMSO-d6) δ 9.45 (s, 1H, -NH), 8.69 (d, J = 8 Hz, 1H, ArH),
1mM stock solutions of ONPs and Au@ONPs were prepared in
8.43 (dd, J = 1.6 Hz, 1H, ArH), 8.24 (d, J = 8 Hz, 1H, ArH), 8.01
(s, 1H, ArH), 7.95 (s, 1H, ArH), 7.70 (t, J = 8 Hz, 1H, ArH), 7.52 (t,
J = 8 Hz, 1H,ArH), 7.23 (dd, J = 8 Hz, 3H, ArH), 7.13 (m, 1H,
ArH), 6.95 (d, 1H, ArH), 5.19 (s, 2H, picolylamine-CH2), 3.82 (t, J
= 4 Hz, 2H, ethylenediamine-CH2), 3.59 (t, J = 4 Hz, 2H,
ethylenediamine-CH2). 13C NMR (100 MHz, DMSO-d6) δ 182.10,
164.37, 163.38, 151.60, 150.15, 147.31, 135.07, 131.61,
130.18, 129.51, 128.93, 127.89, 124.97, 122.58, 122.15,
120.76, 116.45, 108.02, 104.68, 55.45, 42.92, 42.72, 42.36. MS
(EI) : m/z 500.19 (M++1).
water. A composite solution of ONPs and Au@ONPs was
prepared with varied ratios of both materials and the most
suitable are achieved with the solution where ONPs:Au@ONPs
has ratio of 4:6 by v/v was chosen for further studies. All
solutions were shaken properly for a sufficient time and kept
undisturbed for 30 min to confirm the homogeneity of
solutions, before recording the spectra. The spectroscopic
studies were executed at 25 ± 1 ◦C. ONPs, Au@ONPs and their
composite were studies for their interaction with various
toxins in aqueous medium. Composite of ONPs and Au@ONPs
exhibited tremendous change in the UV-Vis absorption spectra
on interaction with Okadaic Acid (OA) as comparative to ONPs
and Au@ONPs. Titrations of OA with the composite were done
by the addition of successive amounts of OA solution to the
composite solution in a 10 mL flask and recording of UV-Vis
absorption spectra incremental addition. Similarly, titration
experiments of OA with composite were performed in using
fluorescence spectroscopy and cyclic voltametery.
Preparation of organic nanoparticles (ONPs)
There are various methods to develop organic nanoparticles;
researchers use the method according to their usage.
Depending on the fabrication method the properties of the
nanoparticles such as size and morphology may vary. The
choice of the solvents, temperature and pH govern the
fabrication process of the nanomaterials. In present work we
have fabricated the ONPs using bottom – up strategy. 1mM
solution of the synthesized receptor was prepared in DMSO.
The solution was then slowly injected into the water using
microsyringe under ultrasonication. The solution was further
sonicated for 30 minutes to assure the formation of stable
ONPs. This technique is the single step process called re-
precipitation. The formation of ONPs was monitored using
external probe of a particle size analyzer. Different solvents
and concentrations were used to optimize the desirable result.
Size distribution of the ONPs was continuously monitored
using DLS analysis. TEM analysis was further used to study the
size and morphology of the ONPs.
Results and discussion
Synthesis and Characterization of materials:
Receptor 1 has been designed with excellent fluorescent
properties to target the desired analyte. The synthesis of
desired receptor was achieved through the bromination of 1,8-
naphthalic anhydride using a method reported in literature.30
Compound 5 was synthesized by reacting 7 with 1 equivalent
of picolylamine in ethanol. After 8 h of refluxing, the reaction
mixture was cooled to room temperature, which yielded the
brown precipitates of compound 5 (Figure S1-3).
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DOI: 10.1039/D0TB01195A
N
N
N
6
2
F
4
N
O
O
O
O
N
O
O
N
O
O
O
O
O
N
O
S
H2N
C
NH2
EtOH, reflux
KOH, Br2
60o
NH2
N
C
DCM, RT
EtOH, reflux
8
5
7
1
3
HN
Br
Br
HN
NH
NH2
S
N
H
F
Scheme 1. Synthesis scheme of receptor 1
Compound 3 was synthesized by refluxing 5 with 1 equivalent required anti-solvent under continue sonication. The
of ethylenediamine for 8 h, in presence of ethanol. Yellow formation of ONPs is thought to be due to variation in
colored precipitates of compound 3 were separated out on solubility of organic compound in two solvents. Introduction of
completion of reaction (Figure S4-6). Synthesis of receptor 1 the organic compound to water surrounding may alter their
was achieved by reacting compound 3 with 2-fluorophenyl microenvironment that lead the formation of supramolecular
isothiocyanate at room temperature using DCM as reaction aggregates and formation of nanoparticles by non-covalent
solvent. After 8 h, the solvent was evaporated under vacuum molecular interactions (e.g. Vander Waal and ᴫ-ᴫ
to get the crude product which was purified using column interactions).33,34
chromatography to collect the pure product as yellow solid.
Spectroscopic data confirmed formation of receptor 1 (Figure
S7-9).
Preparation of organic nanoparticles (ONPs)
Organic nanoparticles and inorganic nanoparticles differ in
terms the fabrication principles. Where inorganic
nanoparticles are usually prepared by the precipitation of
inorganic salt, organic nanoparticles uses number of organic
molecules, through self-organization or chemical binding.
Formulation of organic nanoparticles (ONPs) requires organic
compounds having ability to arrange themselves in a three
dimensional system.31 There are basically two common
approaches to synthesize ONPs: “top-down” and “bottom-up”
approach. “Top-down” approaches for the synthesis of ONPs,
commonly include lithography and mechanical milling. These
techniques are quite complicated for use on regular basis as
they are not only time consuming but also are energy intensive
and have disadvantages like inappropriate control of particle
size and introduction of impurities. Due to the tendency of
aggregation, “top-down” approach is inappropriate to prepare
nanoscale products. Contrary, the bottom-up approach deals
with the organized assembly of tiny building blocks, e.g. atomic
and molecular aggregates into larger structures (such as
organic lattices, clusters, synthesized macromolecules, and
supramolecular structures). The “bottom-up” techniques are
further classified into two categories viz. re-precipitation and
condensation methods. Among these diverse methods, the re-
precipitation method has been extensively used for
Figure 1 (A) Structure of Okadaic acid (B) Structure of synthesized receptor (C)
preparation of organic nanoparticles as the factors like shape,
size and dispersity can be controlled by altering the condition
of reprecipitation.32 In this method, first the organic
compound is completely dissolved in a suitable organic solvent
and then this concentrated solution is slowly injected to
Representative diagram of ONPs (D) Representative diagram of Au@ONPs (E)
Schematic diagram showing the preparation of Au@ONPs and (F) The
mechanism for the detection of okadaic acid.
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Figure 2 (A) TEM images of ONPs. (B) UV-Vis absorbance spectra of ONPs and Au@ONPs (C) XRD patterns of ONPs (D) TEM images of Au@ONPs (E) XRD patterns of
Au@ONPs (F) 13C spectra of ONPs and Au@ONPs (G) Change in UV-Vis absorbance spectra of ONPs on addition of OA (10 µM) (H) Change in UV-Vis absorbance
spectra of Au@ONPs on addition of OA (10 µM) and (I) Changes in cyclic voltammetry of ONPs on addition of OA (10 µM).
One can target solid suspensions ranging from 10 nm to 100 The designing of gold coated nanomaterials is interesting part
nm, using this method. In this work, the compound showed of supramolecular chemistry as it improves the molecular
solubility in DMSO over various solvents (DMF, acetonitrile, recognition and sensing. For the present work, solutions of
THF etc.). The synthesized receptor 1 was dissolved in DMSO ONPs and HAuCl4 were mixed and ascorbic acid was added
and with continuous sonication, it was slowly injected into slowly with continue sonication.35 The appearance of pink
water. The size of the ONPs was confirmed using TEM analysis colour validated the formation of gold nanoparticles. TEM
and it came out to be around 30 nm (Figure 2A). ONPs analysis showed the size of Au@ONPs to be 40-50 nm (Figure
exhibited absorbance peak at 450 nm (Figure 2B) and λmax at 2D). Further, the formation of Au@ONPs was confirmed with
535 nm (Figure S10). Also, the EDAX analysis data confirmed UV-Vis absorption studies which showed the signature peak at
the presence of carbon, nitrogen, oxygen, fluorine and sulphur 530 nm (Figure 2B) and λmax at 635 nm (Figure S12). To confirm
(Figure S11). XRD pattern of ONPs are shown in figure 2C.
the stability of Au@ONPs with time, samples were kept
undisturbed for few days and their UV-Vis absorption studies
were carried out. No change in the absorption profile of ONPs
confirmed the stability of Au@ONPs. Again, the EDAX analysis
Preparation of Au@ONPs
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data confirmed the presence of carbon, nitrogen, oxygen and
gold (Figure S13). XRD pattern of ONPs are shown in figure 2E.
13C spectra of ONPs and Au@ONPs were also recorded in D2O
(Figure 2F) which show clear shifts in Au@ONPs from ONPs.
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DOI: 10.1039/D0TB01195A
A
B
Composite
Composite+10 µM OA
Addition of
0-10 µM OA
Chemosensing response of materials
Composite
Composite+10 µM OA
C
D
ONPs of receptor 1 and Au@ONPs were prepared to examine
their recognition behaviour towards mycotoxins. The sensing
response of ONPs and Au@ONPs over various metal ions (Li+,
Na+, K+, Cs+, Mg2+, Ca2+, Sr2+, Ba2+, Al3+, Cr3+, Mn2+, Fe2+, Co3+,
Cu2+, Zn2+, Ag+, Cd2+, Hg2+ and Pb2+), different tetrabutyl
Addition of
0-10 µM OA
-
ammonium anionic salts (Cl-, Br-, F-, ClO4-, CN-, NO3 , CH3COO-
-
and PO4 ), biomolecules (ATP, ADP, AMP, NADP and NADH) as
well as marine toxins (Okadaic Acid, Domoic acid, Brevetoxin 2,
Brevetoxin 3 and Azaspiracids 1) was evaluated. All the studies
were carried out at 25 ± 1 ◦C.
Composite +10 µM OA
Composite
E
F
Addition of
0-10 µM OA
Chemosensing response of composite:
In present scenario, various studies have been focused on the
synthesis and application of nanoscale materials having unique
features owing to their size quantization effects. Gold
nanoparticles are attracting the attention, as their colloidal
solutions show particular colours as their surface electrons
exhibit collective oscillations due to visible light that results in
colorimetric change for a variations in the interparticle
distance. Particularly, gold nanoparticles when combined with
some biomolecules, are supposed to develop new recognition
systems for certain reactions between biomolecules and these
composites have various applications in biotechnology.36
Figure 4 (A) Changes in UV-Vis absorption spectra of composite on addition of
OA (10 µM) (B) Changes in UV-Vis absorption spectra of composite on addition
successive amounts of OA (C) Change in fluorescent spectra of composite on
addition of OA (10 µM) (λex = 450 nm) (D) Changes in fluorescent spectra of
composite on addition successive addition of OA (λex = 450 nm) (E) Changes in
cyclic voltammetry of composite on addition of OA (10 µM) (F) Changes in cyclic
voltammetry of composite on successive addition of OA.
Reports are present in literature where immunoassays based
on the aggregation of gold nanoparticles modified with
antibody have been demonstrated by specified binding with an
antigen. In another example Mirkin et al. demonstrated the
polynucleotides detection using specific sequences by enabling
the association of gold nanoparticles bearing corresponding
nucleotide chains via hybridization with the target
polynucleotides.37 Many other reports are present for
detection of heavy metals, antibody and lectin which utilize
aggregation of nanoparticles in solutions. Naoki et al. have
reported a composite of gold nanoparticles and molecularly
imprinted polymer as a sensing material having a biomolecule-
like activity.38 Pachfule et al. also reported organic – inorganic
composite based on gold-nanoparticle for the reduction of
nitrophenol.39 Considering the above discussion, we have
thought to prepare a composite material using ONPs and
Au@ONPs. To prepare the composite material of ONPs and
Au@ONPs, various ratios of both the materials have been
prepared and then their UV-Vis absorption and emission
profile were tested for the best match. The best results are
achieved with the solution where ONPs:Au@ONPs has ratio of
4:6 by volume respectively.
Figure 3 (A) TEM images of composite (B) XRD patterns of composite (C) IR
spectra of ONPs, Au@ONPs and composite (D) Linear regression graph between
concentrations of OA added to composite.
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disappeared. UV-Vis absorption titrations were performed by
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gradual addition of Okadaic Acid to the composite solution.
DOI: 10.1039/D0TB01195A
Results are plotted in figure 4B. Similarly, addition of 10 µM of
Okadaic Acid showed quenching in the fluorescence profile
(Figure 4C). To check the binding efficacy of the composite,
successive addition of small aliquots of OA to composite
solution was done (Figure 4D). This depicted that toxin is
binding linearly with composite having a linear regression of
0.967 (Figure 3D). Titrations showed good linearity of about
96.7% in the range (1-8.5 µM). The limit of detection (LOD) of
composite can be defined as 3xSD/m, where SD is the standard
deviation and m is the slope of the regression line. The
detection limit of composite for OA is obtained to be 20 nM.
The binding ratio of the nanocomposite with the marine toxin
okadaic acid was calculated from Job’s Plot. For doing so,
intensity ratio of the absorbance (450 nm) was plotted against
the [composite]/ [composite
+
Okadaic Acid]. The
stoichiometry of the composite with okadaic acid was found to
be 1:1 as shown in figure S18.
Figure 5 (A) Competitive binding studies of composite containing OA over other
selected toxins (B) Effect of pH on fluorescence intensity of composite (C) Plot of
the intensity ratio of composite and OA at different concentrations as a function
of time in seconds (D) Salt perturbation studies of the composite with the
respective fluorescence spectrum recorded upon the addition of 100 equiv of
Cyclic voltammetric studies (CV) were also carried out for the
composite material at a scan rate of 50.0 m where Ag/AgCl
was used as reference electrode, Pt disc as working electrode,
Pt wire depicted as counter electrode and NaClO4 working as a
supporting electrolyte. In case of CV, a shift was observed in
cathodic peak, as it changed its position from -0.532 V to -
0.618 V with enhancement in current on addition of Okadaic
Acid. Similarly, anodic peak showed a change from -0.815 V to
-0.847 V with a modulation in the peak (Figure 4E and 4F).
tetrabutyl
ammonium
perchlorate.
Characterisation of the composite
Once, the ratio of ONPs : Au@ONPs was decided, the
composite material was fully characterised using various
techniques. Particle size and shape of the composite material
was determined using TEM analysis. As is clear from figure 3A,
TEM analysis showed different particle sizes of the composite
material is 10-40 nm. XRD patterns of composite are shown in
figure 3B. A comparison of IR spectra of ONPs, Au@ONPs and
composite has been shown in figure 3C.
The prime concern about a composite was that it should be
selective towards a particular toxin, therefore to check the
selectivity towards Okadaic Acid; a competitive binding assay
was performed by adding various analytes to the composite
solution. To check the selectivity of the composite material,
competitive binding test was performed. To perform this study
composite solution was taken in different volumetric flasks
and then added the 0.5 µM of Okadaic Acid solution to each
flask. Then the addition of remaining metal solutions,
tetrabutyl ammonium anionic salts, biomolecules and
remaining marine toxins to the volumetric flasks was done
followed by recording emission spectra for each solution after
shaking the solutions. From comparison of emission spectra of
composite + OA alone and of composite + OA in the presence
of remaining marine toxins (Figure 5A), biomolecules and
tetrabutyl ammonium anionic salts (Figure 6) and metal
solutions (Figure S17) it was observed that addition of various
analytes did not pose any interference to the spectrum and
hence the composite can be used for selective determination
of Okadaic Acid. The performance of the composite for target
analyte may be affected by the pH value of the environment
around it due to various reasons. The effect of pH on the
emission response of composite was therefore investigated.
The experiments were carried out at a pH range from 5.0 to
9.0 (Figure 5B). Both acidic and basic titrations were conducted
by changing pH of composite solution using sodium hydroxide
and hydrochloric acid and then emission spectra were
recorded at various pH values to study the effect of pH on the
emission response of host solution. For composite solution, in
both acidic (pH<7) and basic conditions (pH>7), the pH has
Chromogenic, fluorescent and electrochemical recognition of
okadaic acid:
The recognition behaviour of composite material towards
various metal ions (Li+, Na+, K+, Cs+, Mg2+, Ca2+, Sr2+, Ba2+, Al3+,
Cr3+, Mn2+, Fe2+, Co3+, Cu2+, Zn2+, Ag+, Cd2+, Hg2+ and Pb2+),
-
-
tetrabutyl ammonium anionic salts (Cl-, Br-, F-, ClO4 , CN-, NO3 ,
CH3COO- and PO4 ), biomolecules (ATP, ADP, AMP, NADP and
-
NADH) and marine toxins (Okadaic Acid, Domoic acid,
Brevetoxin 2, Brevetoxin 3 and Azaspiracids 1) was evaluated
by the addition of the analytes to a fixed amount of composite.
The composite exhibited absorption at 450 nm and 530 nm
(Figure S15) whereas the fluorescence emission peak displayed
at 535 nm. To the fixed volume (5 ml in volumetric flask) of
composite solution, 10 µM of each analyte was added. The
absorption spectrum depicted weak or insignificant variation
in absorption peak when different analytes were added to
composite expect Okadaic Acid. A significant change in UV-Vis
absorption profile of the composite occurred on addition of
Okadaic Acid (Figure 4A). The absorbance peak corresponding
to 450 nm showed hyperchromic shift whereas peak
corresponding to 530 nm showed quenching and merely
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DOI: 10.1039/D0TB01195A
ARTICLE
Figure 6 Selectivity test of composite for OA: detection over several analytes. The concentrations of all the compounds are the same, and measurements are
performed under identical conditions.
little or no effect on the emission spectra of composite. The participates in electron transfer process to the HOMO of the
fluorescence emission spectrum results revealed that the nanocomposite and thus quenches the fluorescence intensity.
composite can be used over a pH range from 5 to 9. Besides Then mechanism has been shown in the figure 1F.
high selectivity and sensitivity, another necessity for a
The detection of okadaic acid was performed in completely
chemosensor is a short response time to screen the analyte of
aqueous medium. As the ONPs and gold based nano-
interest. To examine the response time, fluorescence emission
composite were prepared in water so we have performed the
spectra of composite was studied by variation in the
effect of polarity in the presence of DMSO and DMF solvents.
concentration of OA in the composite solution. To perform this
As shown in figure S19, we can see that low percentage of
experiment, composite solution was taken in three volumetric
DMSO or DMF does not affect the fluorescence profile of the
flasks and then different concentration of OA was added to
composite’s binding with okadaic acid. When the DMSO is
each flask. After fixed intervals of time, the fluorescence
more than 12% the fluorescence intensity starts decreasing.
spectra of all samples were recorded. The response time of the
Similarly for the DMF, more than 10% of DMF affect the
composite towards OA is concentration-independent, as it was
fluorescence profile hence the detection of the okadaic acid.
observed that the time required to reach equilibrium does not
The figure 6 shows the selectivity of our sensor (composite)
affect with OA concentrations. In all cases, however, the stable
over other toxins, anions and biomolecules. These selectivity
reading could be achieved within 60 seconds after that
tests were performed at 0, 1, 5 and 10 µM concentrations of
negligible change in fluorescence intensity was observed
all the analytes. To the solution of okadaic acid, the
(Figure 5C). Thus, the composite can be used for real time
abovementioned concentrations of the analytes were added
monitoring of OA. Similarly to evaluate the effect of salt
and their fluorescence profile was recorded. The results of the
concentration on fluorescence spectroscopy, increasing
figure 6 show that our sensor selectively binds with the sensor
concentration of electrolyte i.e. tetra butyl ammonium
and shows quenching of the fluorescence intensity of the
perchlorate (TBAP), were added to composite solution. It was
composite. Whereas with the rest of the biomolecules such
perceived that even on addition of 100 equivalents of TBAP,
ATP, ADP, AMP, NADP and NADH, composite do not show any
fluorescence emission spectra of composite remain almost
changes in the fluorescence intensity hence do not bind with
undisturbed (Figure 5D).
the composite. Similarly, the anions such as phosphate,
The composite is capable of successful selective and sensitive
hydrogen sulphate, chloride, bromide, iodide and nitrate ions
detection of the marine toxin okadaic acid. The fluorescence
do not bind with the composite and no change in the
quenching is observed due to the photo-induced electron
fluorescence profile was observed. These results show the
transfer (PET)40-42 from the okadaic acid to the composite. In
selective binding of the composite with okadaic acid; hence we
the absence of the okadaic acid the electron transfer in
can successfully say that our fabricated sensor can be used for
inhibited and PET is OFF and high fluorescence is observed.
selective and selective detection of okadaic acid in aqueous
Okadaic acid being electron rich molecule acts as donor and
medium.
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The biological toxicity of our fabricated nano composite was ability was retained in pH range of 5-9 and also under high salt
View Article Online
studied by cell viability test with HeLa cells. The HeLa cells effects suggesting the workability of the sensor for the
DOI: 10.1039/D0TB01195A
were cultured in MEM media with 10 % fetal bovine serum in detection of OA in real applications.
the presence of 5% carbon dioxide. The incubation of the HeLa
cells was carried out at 37oC for 24 hours. The nano composite
with and without binding with okadaic acid was incubated with
Conflicts of interest
HeLa cells for 24 hours at 37oC and the cell viability was
There are no conflicts to declare.
observed. We observed that 90-95 % of HeLa cells recovered
from the composite in the absence of okadaic acid. In the
presence of composite + okadaic acid the cell viability was
found to be 85-90% as shown in the figure S20.
Acknowledgements
Authors acknowledge Indian Institute of Technology Ropar for
providing the infrastructure to carry out the experiments.
Real Sample Analysis
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composite for the sensing of okadaic acid. The table 1 of the
real sample analysis is shown below and same has been added
to the supplementary information of the manuscript.
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