An organic-inorganic nanohybrid of a calix[4]arene based chromogenic chemosensor for simultaneous estimation of ADP and NADH

Article abstract of DOI:10.1039/c5ra18003a

The versatility in the environmental and biological applications of nanohybrids encouraged us to prepare a novel chemosensor based on an organic-inorganic nanohybrid (H1) employing receptor 1 (R1), which was synthesized via the Schiff's base condensation reaction of a calix[4]arene derivative and an aliphatic amine. Techniques such as DLS and TEM were employed for the characterization of organic nanoparticles (N1) and H1. Further, sensor properties of H1 were explored towards various biologically important molecules in aqueous media using UV-visible spectroscopy. The proposed sensor responded effectively for the selective and simultaneous nanomolar determination of adenosine diphosphate (ADP) and reduced nicotiniamide adenine dinucleotide (NADH). The response was not affected by the presence of each analyte or any other potentially interfering biomolecule or a high concentration of salt. The proposed sensor was also found to show a stable response in an extensive pH range thus widening its practical applicability. H1 was able to detect a minimum concentration (detection limit) of 6.11 × 10^-9 M of ADP and 4.87 × 10^-9 M of NADH. The prepared hybrid was subjected to real sample analysis for the determination of ADP and NADH in samples prepared artificially by adding known concentrations of NADH and ADP in solution and also in a mixture of both.

Full text of DOI:10.1039/c5ra18003a

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An organic-inorganic nanohybrid of calix[4]arene based
chromogenic chemosensor for simultaneous estimation of ADP
and NADH
Received 00th January 20xx,
Accepted 00th January 20xx
Harpreet Kaura†, Jasminder Singhb†, Shweta Chopraa, Pushap Rajb, Narinder Singh*b and Navneet
Kaur*a,c
DOI: 10.1039/x0xx00000x
www.rsc.org/
The versatility in environmental and biological applications of nanohybrids encouraged us for the preparation of a novel
chemosensor based on organic-inorganic nanohybrid (H1) employing receptor 1 (R1), which was synthesized by schiff’s
base condensation reaction of a calix[4]arene derivative and an aliphatic amine. The techniques such as DLS, TEM were
employed for the characterization of organic nanoparticles (N1) and H1. Further, sensor properties of H1 were explored
towards various biologically important molecules in aqueous media using UV-Visible Spectroscopy. The proposed sensor
responded effectively for selective and simultaneous nanomolar determination of adenosine diphosphate (ADP) and
reduced nicotiniamide adenine dinucleotide (NADH) and the response was not affected by the presence of each other or
any other potentially interfering biomolecule or high concentration of salt. The proposed sensor is also found to show a
stable response in an extensive pH range thus widening its practical applicability. H1 was able to detect a minimum
concentration (detection limit) of 6.11 × 10-9 M of ADP and 4.87 × 10-9 M of NADH. The prepared hybrid was subjected to
artificially prepared real sample analysis for determination of ADP and NADH in samples prepared artificially by adding
known concentration of NADH and ADP in solution and also in mixture of both.
homeostasis, the redox couple NAD+/NADH represents a very
INTRODUCTION
important system, being a substrate for a number of
dehydrogenase, hydroxylases and oxidoreductase enzymes in
natural biological systems.6 Aberrant level of various
biomolecules are known to cause many diseases such as
hypoglycemia, hypoxia, ischemia, Parkinson’s disease,7 renal
diabetes, cystic fibrosis, diabetes.8 Change in the level of few
of the biomolecules such as NAD/NADH, adenine, thymine and
guanine is closely related to many diseases like cancer, AIDS,
epilepsy and lupis erythematosus.9-11 Therefore, it is necessary
to develop highly sensitive, prompt, dependable and expedient
method for the detection of biomolecules for various
biochemical studies and clinical diagnosis. Detection of these
substances require complicated separation and purification
steps, as well as elaborated sample preparation, large costly
instruments and skilled labor.12 Number of artificially prepared
receptors employing various organic units such as
benzimidazole, urea, thio-urea, and calixarene moiety have
already been reported in literature.13-14 With-in this context,
the development of receptors for multi-analyte detection in
real samples is still a challenge for researchers. A few
chromogenic and fluorescent sensors for the detection of
more than one analyte simultaneously are reported in
literature. Such receptors are designed and synthesized either
by inserting multi-chromogenic moieties in a single receptor
which necessitate tedious synthesis steps or employing several
detection techniques such as UV-visible, electrochemical and
In recent years, sensors for quantitative recognition of various
biomolecules (amino acids, DNA etc.) increasingly attract much
more interest of researchers due to their imperative
physiological functions and numerous applications in medical
diagnostics, anti-bioterrorism and food safety.1 The standard
amount of biomolecules is essential for organisms, while the
amount too high or too low will lead to certain diseases. For
example, adenosine diphosphate (ADP) in the form of ADP-
ribose is known to play important role in different cellular
processes such as extracellular cell signaling, DNA repair,2 gene
regulation and apoptosis,3 ADP also plays an imperative
function in normal hemostasis and thrombosis, ADP has a
crucial role to play in enzymes functioning as bacterial toxins
and metabolic regulators,4 nicotinamide adenine dinucleotide
(NAD) play a part in regulation of energy metabolism, DNA
repair and transcription,5 and in maintaining cellular redox
fluorescence detection method.15-16 Lately,
a shift from
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designing and fabricating a selective receptor to differential Synthesis of the
Journal Name
R
1
was carried out by reacting 3-
receptor has facilitated the recognition of more than one (dimethylamino)-1-propylamine with calix[4]arene-based
analyte concurrently using one chromophore. Various dipodal aldehyde in acetonitrile at room temperature using
colorimetric detection techniques employing nanomaterials previously reported literature method22
Scheme 1[SI]), which
1
(
(nanoparticles, nanorods, quantum dots) have been used in in turn was synthesized by reacting p-tert-butyl calix[4]arene
order to get quick, precise, and cost-effective response in and 2-(2-bromoethoxy)benzaldehyde in acetonitrile as
samples of biological and environmental origin these days.17 depicted in scheme 1. The R
1 (Figure 1) thus formed was white
Nanomaterials, particularly hybrid nanostructures are in color, having a yield of 70% and was characterized using
massively employed these days to meet high-end elemental analysis.
requirements such as inexpensive, efficient system having high
sensitivity and selectivity towards particular ion or molecule.
Metallic nanoparticles, particularly, gold nanoparticles (AuNPs)
are one of the most explored nanostructures lately owing to its
high biocompatibility, conductivity, fluorescent quenching and
excellent plasmonic coupling. These exhibit distinctive features
such as localized surface Plasmon resonance (LSPR)18 and a
very high molar extinction coefficient. As per Beer-Lambert
Law, these show the possibility of reaching a much lower limit
of detection (LOD). AuNPs are employed these days for the
detection of various biomolecules owing to their comparable
sizes. Quantum dots is another class of nanomaterial
possessing quantum confined charge carriers, emitting
fluorescence with a high quantum yield and possessing
remarkable electro-chemiluminescent properties.19 These are
known to conjugate with biomolecules (proteins and DNA)
with high sensitivity. Organic nanoparticles (ONPs) are a
recently explored class of soft matter which finds application
in sensitive and selective chemo-sensing. So, herein, we
Figure 1: Design of Receptor 1
Fabrication of Organic nanoparticles (N1) of receptor 1
Organic nanoparticles (ONPs) are fine suspensions of solid
particles composed of organic compound in aqueous medium
ranging in diameter from 10 nm to 1 µm. Because of their
optical properties23 and competency to generate intense
electromagnetic fields, these particles have imperious
relevance in various fields such as conducting materials,
sensors,24 medicine,25 biotechnology and innumerable other
applications like catalysis, diagnostics26 and photo-thermal
therapeutics. Of all the procedures known in literature for the
fabrication of ONPs, single step re-precipitation technique is
usually followed because it is economical and can be easily
develop
a simple, prompt and responsive colorimetric
chemosensor for the estimation of ADP and NADH
simultaneously by employing a calix[4]arene derivative and
using the interparticle plasmon coupling induced by the
aggregation of AuNPs due to binding with a particular
analyte.20-21 Receptor
1 based on calix[4]arene molecule
substituted at 1,3-alternate positions on the lower rim is
designed in such a manner so as to devise a molecule having
tunable cavity size. Such a molecule is bound to have two
different cavities competent of accommodating two analytes
simultaneously. Because of the presence of the podand arms
at the lower rim, the cavity of the calix[4]arene molecule
becomes more rigid and is therefore able to selectively and
reproduced. Keeping that in mind, organic nanoparticles (N1
of R were obtained by employing single-step re-precipitation
method. For this, 1 mL (0.6 M) of the compound dissolved in
)
1
ꢀ
promptly bind
a particular analyte of biological and
pure tetrahydrofuran (THF) was injected manually into
deionised water (100 mL) taken in 250 mL beaker, using a
micro syringe at a constant rate under sonication for atleast
environmental importance. Also, the substituents at the lower
rim bear various binding sites which proficiently bind specific
analyte. We report development of organic-inorganic hybrid
half an hour. The change in photophysical profile of R1 was
(H1) based chemosensor using receptor 1 (R1) for the
studied using UV-Visible and Fluorescence Spectroscopy
(Figure 2a and 2b).
simultaneous detection of ADP and NADH in an aqueous
medium having no interference from any other potentially
Organic ligand dissolved in pure THF shows low absorption
peaks in UV-visible spectra. As the amount of water was
increased, ONP formation occurred and the solution became
slightly cloudy, indicating uniform distribution of nanoparticles
in aqueous medium. p-tert-butyl calix[4]arene molecule in
CHCl3 is known to show appreciable absorption bands at 230
and 280 nm due to n-π* transitions and these absorption
bands are expected to shift towards longer wavelengths as
polarity of the solvent is increased.27 The UV-Visible profile of
interferent biomolecules. R
1 was obtained by Schiff’s base
condensation reaction of 3-(dimethylamino)-1-propylamine
and 1, 3-disubstituted p-tert butyl calix[4]arene.
RESULTS AND DISCUSSION
Synthesis and characterization of sensor
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N1 shows peaks at 286 nm and 317 nm attributed to n-π* It is quite evident that with the increasing concentration of
transitions, while emission profile showed peaks at 303 nm water, the intensities of the peaks increased considerably and
and 372 nm. Both the spectral profiles showed marked best peaks are obtained with composition of 1:99, THF: water.
enhancement in the peak intensity which can now be used to Therefore providing us with best suited media for formation of
estimate the changes in photophysical profile of the R1
.
optimum sized ONPs. The optimum sized ONP were confirmed
using TEM analysis and DLS (Dynamic Light Scattering) and had
a size of 12 nm (Figure 5a and 5b).
Figure 2: (a) UV-Visible, and (b) Fluorescence Spectra of Ligand in THF and ONPs in
water showing noticeable enhancement in peak intensity as ONPs are fabricated.
Figure 3: (a) UV-Visible Spectra, (b) Fluorescence Spectra of N1 with increasing
concentrations of R1.
Analysis of N1 particle size was confirmed by TEM
(Transmission Electron Microscopy) which showed the
development of nanoparticles, spherical in shape having a size
of 12 nm (Figure 5a). The concentration of R1 for the
fabrication of ONPs was required to be optimized by varying
the injected amount of R1 in 100 ml of distilled water in order
to achieve more intense peaks both in UV-visible and
Fluorescence spectra. It is quite evidently perceived from the
results (Figure 3a & 3b) using both UV-visible spectroscopy and
fluorescence spectroscopy that as the amount of R1 was
Fabrication of organic-inorganic hybrid (H1)
Many different techniques have been developed to generate
organic-inorganic hybrids (H1) using reduction of gold as gold
nanoparticles (AuNPs) on surface of organic nanoparticles.26,28-
30
For this, stock solutions of HAuCl4 (1.0 × 10−3 M), and
ascorbic acid (1.0 × 10−3 M) were made using de-ionized water,
and subsequent dilutions (100 times) were made from the
stock solutions and were then tried in different ratios along
with previously prepared N1 (0.1 µM) to obtain H1. It was
deduced that HAuCl4, N1 and ascorbic acid when mixed in the
ratio of 9:1:9 leads to the formation of H1. Formation of H1
from N1 happened with dramatic change in color from
colourless to pink (Figure S2). Both UV-Visible and
fluorescence spectra (Figure 6a and 6b) confirmed the
formation of H1, as a clear surface plasma resonance (SPR)
band appeared in visible region at around 528 nm due to
reduction of Au (III) to Au (0) over organic ligand, while the
increased from 0.2 ꢀM to 0.6 ꢀM the intensity of the peaks
increased. On further increasing the amount of R1 the
intensity of the peaks decreased considerably which is ascribed
to increase in particle size leading to agglomeration. The same
has been confirmed using DLS studies (Figure S1). In order to
explore the effect of amount of water on the photophysical
profile of R1, various compositions of THF:water were
examined by UV-Visible and fluorimetric profiles of the same
(Figure 4a and 4b).
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emission spectra demonstrated quenched emission bands of
N1 at 303 nm and 372 nm attributed to deactivation of the
surface of N1 by the immobilization of AuNPs over its surface.
This was further confirmed using TEM (Transmission Electron
Microscopy) which showed an appearance of black spots on
the surface of N1 having a size of 18-22 nm and also
confirmed by DLS studies (Figure S3).
Figure 5: (a) TEM images of ONPs in water (N1), and (b) DLS histogram of N1
RECOGNITION STUDIES
Binding of biomolecules with organic-inorganic hybrids (H1)
In order to study the binding ability of the developed R1 in the
form of H1 towards various biomolecules in an aqueous
medium, solution of H1 was taken in a volumetric flask (5 mL)
and was subjected to 100 µL (5µM) solution of different
biomolecules (NAD, NADH, NADP, AMP, ADP, ATP, Uracil,
Adenine, Cytosine and Guanine) and their UV-visible spectra
were noted. It was perceived that upon addition of
biomolecules to the fixed concentration of H1 (6µM),
negligible change in UV-visible profile with all biomolecules
except ADP and NADH (Figure 7).
Figure 6: Comparison of (a) UV-Visible, and (b) Fluorescence Spectra of inorganic
organic hybrid nanoparticles (H1) and organic nanoparticles (N1)
Interaction with NADH with H1 showed a shoulder peak at 648
nm, whereas addition of ADP showed a new peak at 712 nm,
along with decrease in absorbance peak at 528 nm. Binding of
the receptor with anion leads to stabilisation of the excited
state in comparison with the ground state. This leads to
bathochromic shift in the absorption spectrum.31 These
spectroscopic changes may be assigned to the formation of a
charge transfer complex. The observed bathochromic shift
both in case of ADP and NADH may be attributed to
Figure 4: Effect of various composition of solvent (Water:THF) on (a) UV-Visible
absorption (b) fluorescence spectra of N1.
It is quite evident from Figure 6 that the absorption band has
clearly shifted from UV region to visible region, which clearly
justified our decision to prepare inorganic-organic nanohybrids
from organic nanoparticles for sensor applications.
intermolecular hydrogen bonding between receptor
1 in the
form of H1and biomolecules. To authenticate the binding
behaviour of H1 with both ATP and NADH, titrations were
performed by adding small aliquots of both separately in 10
mL solution of H1 (Figure 8a & 8b).
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Figure 8: UV-visible Spectra of H1 in the presence of increasing concentrations (a) (0-
100 nM) of ADP, and (b) (0-80 nM) of NADH (Inset A: Calibration plot for ADP, Inset B:
calibration plot for NADH)
Figure 7: UV-Visible profile of H1 in the presence of 5µM of various biomolecules
It is quite evident from the calibration curves that H1 responds
linearly for ADP and NADH with a linear dynamic range of 0-
100 nM for ADP and 0-80 nM for NADH, having a regression
coefficient of 0.9896 and 0.9783, respectively. H1 was able to
detect a minimum concentration (detection limit) of 6.11 × 10-
Further, in order to project it as a sensor for simultaneous
determination of ADP and NADH, simultaneous binding assay
was performed (Figure S4
& S5). The experiment was
performed by adding one component in excess as interferent
and doing the titration with the second component as analyte
of interest and calibration curves were plotted for both NADH
and ADP, in presence of each other and compared with the
calibration curve of NADH and ADP in absence of interferent. It
is quite evident from the calibration plots (Figure S4 & S5) that
calibration curve remains the same i.e. neither slope nor
intensity changes on addition of one component as
interferent. The differential behaviour of H1 with NADH and
ADP respectively may be ascribed to distinct binding sites in
the molecule for both NADH and ADP. For a sensor to have
practical application in real sample analysis beside selectivity,
it should be able to work in complex environment like in the
presence of various potential interferent, varying pH and salt
concentration. In order to achieve this, interference studies
were carried out and UV-vis spectra were noted. In 10 mL
volumetric flasks, H1 containing 0.1 µM of ADP and NADH
respectively, were subjected to various other biomolecules
(NADP, NAD, AMP, ATP, Uracil, Adenine, Guanine, and
Cytosine) and their UV-vis profiles were investigated (Figure 9).
9
M of ADP and 4.87 × 10-9 M of NADH. The lower detection
limit was calculated by using standard IUPAC 3σ
method.32
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NADH, respectively. A3 and A4 were made by dissolving both
NADH and ADP in same solution for knowing the workability of
H1 in simultaneous estimation of NADH and ADP. The results
thus obtained are given in Table 1
:
Table1: Analysis of artificially made samples of NADH and ADP using H1
Added
Recovered
Percentage
Recovery
S.No
1
Sample Name
A1(ADP)
Conc.
(nM)
4.5
Conc. (nM)
4.46 ± 0.03
3.64 ± 0.04
99.1%
2
3
A2(NADH)
3.7
98.38%
A3(ADP + NADH)
12.9
6.8
12.73 ±0.06,
6.63 ± 0.04
98.68%
97.5%
4
A4 (ADP + NADH)
7.6
7.49 ± 0.03
98.55%
97.84%
14.8
14.48 ± 0.07
It is quite evident from Table 1 that H1 can be used for real
sample analysis of various spiked samples of analytical
importance for NADH and ADP with accuracy of more than
95%. The hybrid can also be successfully employed for
simultaneous estimation NADH and ADP, without any lose in
its selectivity or sensitivity.
CONCLUSIONS
Receptor R1 was synthesized by Schiff’s base condensation
reaction of a calix[4]arene-based dipodal aldehyde and 3-
(dimethylamino)-1-propylamine. The receptor was then
subjected to organic nanoparticles (N1) and further to organic-
inorganic nanohybrids (H1). Sensor activities of H1 were
investigated by subjecting it to various biomolecules in
aqueous media using UV-visible spectroscopy. It was perceived
that the receptor worked well for the simultaneous
determination of ADP and NADH even in presence of each
other as interferent molecules. Proposed sensor is stable in a
wide pH range, hence could be used for simultaneous
determination of above mentioned biomolecules in aqueous
medium for various environmental and biologically important
samples.
Figure 9: UV-vis absorption spectra showing negligible interference due to different
biomolecules in the recognition behaviour of H1 towards (a) ADP, and (b) NADH
respectively.
As can be clearly seen from the UV-vis spectra, none of the
biomolecules pose any interference to the recognition
behaviour of the receptor in the form of H1. The effect of
varying pH on the recognition behaviour of H1 and UV-vis
spectrum is recorded (Figure S6). It is quite apparent that UV-
visible profile of H1 remains unaltered in wide pH range of 2 to
12. Hence, the proposed sensor is stable in this pH range and
can be applied for use in samples of biological and
environmental utility without losing its sensitivity or
selectivity. In order to investigate the salt effect on recognition
behaviour of H1, tetrabutylammonium perchlorate salt is
added in increasing amount (1-100 equiv.) to H1 taken in 5 mL
volumetric flask and UV-Visible spectra were recorded (Figure
S7). It was perceived that even the presence of 100
equivalents of salt does not alter the response of H1. Hence, it
can be said that prepared nanohybrid complex (H1) can be
used for real sample analysis of NADH and ADP in samples of
biological and environmental importance.
EXPERIMENTAL
Materials and Methods
All reagents viz
. p-hydroxybenzaldehyde, 1, 2-dibromoethane,
p
-tert butyl phenol, formaldehyde (37%), N, N-
dimethylpropane-1, 3-diamine, K2CO3, NaOH (analytical grade)
were purchased from Loba Chemie and used without further
purification. Solvents such as diphenyl ether, ethyl acetate,
acetic acid were procured from SD Fine and were used as such,
whereas acetonitrile (HPLC Grade) was obtained from Fisher
Scientific which was further dried by distillation method before
use. All biomolecules viz.ADP, NADP, NADH, NAD, ATP, AMP,
cytosine, Adenine, Guanine and uracil were obtained from
Sigma Aldrich. De-ionized bi-distilled water was used for
Spiked sample analysis
The prepared hybrid was scrutinized using spiked samples (A1-
A4) of NADH. A1 and A2 were made by dissolving ADP and
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preparation of organic-inorganic nanohybrids and solutions of is prompt and simple yielding reproducible results and mono-
all biomolecules.
dispersed nanoparticles.
UV-Visible absorption spectra were recorded on Spectroscan
30 spectrophotometer from Biotech Engineering Management Recognition studies
Co. Ltd. (UK). The fluorescence measurements were performed
on RF-5301 PC spectrofluorophotometer from Shimadzu. The The UV-Visible Spectroscopy technique was used for the
particle sizes and size distribution of the ONPs and AuNPs were recognition studies of prepared H1. A solution of H1 taken in 5
determined on Metrohm Microtac Ultra Nanotrac Particle Size mL volumetric flask and100
ꢀL various biomolecules (5ꢀM)
Analyzer (dynamic light scattering). Transmission electron (NADH, NADP, NAD, AMP, ATP, ADP, Uracil, Cytosine, Adenine
micrographs (TEM) were noted on Hitachi (H-7500) instrument and Guanine) were added into it, volumetric flasks were
working at 120 kV having a resolution of 0.36 nm (point to shaken well and equilibrated for half an hour at temperature
point) with 40-120 kV operating voltage. For sample 25 ± 1 °C before recording the spectrum. To confirm the
preparation, a carbon-coated copper grid (400-mesh) was binding, titrations were carried out with NADH and ADP for
used. Elemental analysis was carried out on Fisons instrument which small aliquots of both the biomolecules were added into
(Model EA 1108 CHNO).
10 mL H1 taken separate volumetric flasks. In order to
evaluate interference due to the presence of other
biomolecules, interference studies were carried out. For this,
UV-vis spectra were recorded for solutions of H1 containing
Synthesis of receptor 1
Calix[4]arene-based dipodal receptor (I) was synthesized as NADH and ADP, respectively without and with different
depicted in Scheme 1. The calix[4]arene-based dipodal biomolecules. In order to propose the receptor for
aldehyde 1 was prepared by the reported method22 from
p
-
simultaneous determination of ADP and NADH without having
tert butyl calix[4]arene and 2-(2-bromoethoxy) benzaldehyde any interference due to each other when present in excess
(Scheme 1). Receptor was having a yield of 70%, prepared by amount, the experiment was performed in which one analyte
the schiff’s base condensation reaction of 3-(dimethylamino)- was present in excess in a given volume of H1 and titration
1-propylamine and dipodal aldehyde 1. The receptor thus was performed with the another analyte. The results showed
1
1
obtained was white in color having a yield of 70% and was no discernible interference of one analyte in the detection of
then characterized by elemental analysis. Expected other. In addition, effect of high concentration of salt was
percentage: C = 77.66, H = 8.69, N = 5.03, O = 8.62; Obtained studied by adding different concentrations of tetrabutyl
percentage: C = 77.89, H = 8.46, N = 5.16, O = 8.54.
ammonium perchlorate salts (0-100 equiv.) Experiments
showing the response time were also carried out by recording
the spectra of H1 at various concentrations with respect to
time. The pH titration was also performed, so as to investigate
Synthesis of organic nanoparticles (N1) of receptor 1
Organic nanoparticles of receptor
reprecipitation method.1 mL of stock solution of
tetrahydrofuran) was injected with a micro syringe at a steady
1
(
N1) were primed using the consequence that varying pH may bring to the
1
(in performance of H1
.
rate into 100 mL de-ionized water under vigorous stirring. The Spiked sample analysis
solution thus obtained was sonicated for half an hour at
constant temperature to ensure the formation of N1. Size To extend the use of H1 as sensor and for real sample analysis,
distribution of N1 formed were scrutinized using particle size the solutions of known concentrations (A1-A4) of NADH and
analyzer Dynamic Light Scattering (DLS) technique.
ATP were made and scrutinized using the prepared H1.
Synthesis of organic-inorganic hybrids (H1)
ACKNOWLEDGEMENT
Organic-inorganic nanohybrids (H1) were prepared by mixing
N1 in a particular ratio with reduction product of HAuCl4 using
ascorbic acid. For this, 250 mL aqueous solutions of HAuCl4 (1
mM) and ascorbic acid (1 mM) were prepared using de-ionized
water from stock solutions of HAuCl4 (10.0 × 10−3 M) and
ascorbic acid (10.0 × 10−3 M). The solutions were equilibrated
at ambient temperature for 2 hours and were then mixed
along with N1 to obtain H1. Appearance of pink color in the
solution confirmed the formation of H1. There are various
factors which control the size distribution of the hybrid
nanoparticles in the solution such as ratio of the three
components i.e. gold, ascorbic acid and the nanoparticles, the
order in which the reagents are added and physical conditions
such as temperature. The methodology we have followed here
This work was supported with research grant from CSIR, New
Delhi through research project sanctioned to NK (Project No.
02(0216)/14/EMR-II). HK will like to acknowledge DST-
INSPIRE for fellowship.
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Table of contents
Nanohybrids of calix[4]arene based receptor has been employed for selective and
simultaneous estimation of ADP and NADH.