A colorimetric nanoprobe based on enzyme-immobilized silver nanoparticles for the efficient detection of cholesterol

Article abstract of DOI:10.1039/c9ra08328f

A large number of cardiovascular diseases have recently become of serious concern throughout the world. Herein, we developed a colorimetric probe based on functionalized silver nanoparticles (AgNPs) for the efficient sensing of cholesterol, an important cardiovascular risk marker. A simple sodium borohydride reduction method was employed to synthesize the AgNPs. The cholesterol oxidase (ChOx)-immobilized AgNPs interact with free cholesterol to produce H₂O₂ in proportion to the concentration of cholesterol, resulting in decreased AgNP absorbance (turn-off) at 400 nm due to electron transfer between the AgNPs and H₂O₂. The response of the sensor can also be observed visually. The absorption intensity of the AgNPs is recovered (turn-on) upon the addition of sodium dodecyl sulfate due to the inhibition of ChOx. This on-off mechanism was effectively applied to detect cholesterol within the concentration range 10-250 nM with a low detection limit of approximately 0.014 nM. Moreover, the selectivity of the sensor toward cholesterol was analyzed in the presence of a range of interfering organic substances such as glucose, urea, and sucrose. Finally, the potential of the proposed sensor was evaluated using real samples.

Full text of DOI:10.1039/c9ra08328f

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A colorimetric nanoprobe based on enzyme-
immobilized silver nanoparticles for the efficient
Cite this: RSC Adv., 2019, 9, 42085
detection of cholesterol
a
a
a
b
Lakshita Dewangan, Jyoti Korram, Indrapal Karbhal, Rekha Nagwanshi,
c
a
Vinod K. Jena and Manmohan L. Satnami
*
A large number of cardiovascular diseases have recently become of serious concern throughout the world.
Herein, we developed a colorimetric probe based on functionalized silver nanoparticles (AgNPs) for the
efficient sensing of cholesterol, an important cardiovascular risk marker. A simple sodium borohydride
reduction method was employed to synthesize the AgNPs. The cholesterol oxidase (ChOx)-immobilized
AgNPs interact with free cholesterol to produce H
resulting in decreased AgNP absorbance (turn-off) at 400 nm due to electron transfer between the
AgNPs and H . The response of the sensor can also be observed visually. The absorption intensity of
2 2
O in proportion to the concentration of cholesterol,
2 2
O
the AgNPs is recovered (turn-on) upon the addition of sodium dodecyl sulfate due to the inhibition of
ChOx. This on–off mechanism was effectively applied to detect cholesterol within the concentration
range 10–250 nM with a low detection limit of approximately 0.014 nM. Moreover, the selectivity of the
sensor toward cholesterol was analyzed in the presence of a range of interfering organic substances
such as glucose, urea, and sucrose. Finally, the potential of the proposed sensor was evaluated using real
samples.
Received 12th October 2019
Accepted 5th December 2019
DOI: 10.1039/c9ra08328f
rsc.li/rsc-advances
ꢀ
1
below 160 mg dL
are classied as hypocholesterolemia.
1
. Introduction
Therefore, it is essential to detect both high and low levels of
15
Cholesterol is an important component of cell membranes that cholesterol.
16–22
is transported via blood plasma in mammals. Abnormal levels
Various methods such as electrochemical sensing
uo-
2
3–25
26–28
of cholesterol can lead to atherosclerosis, hypertension, stroke, rometric analysis,
chromatographic analysis,
molecular
29
30
and other issues. Furthermore, high levels of cholesterol in the imprinting, surface plasmon resonance, eld-effect tran-
31
32
blood and body tissues have perilous effects such as coronary sistor sensors,
heart and peripheral arterial diseases, diabetes, hypertension, methods
chemiluminescence,
have been reported to detect cholesterol and H O .
2 2
and colorimetric
33,34
1–4
cardiac arrest, and anemia. Therefore, in medical diagnostics Among these methods, colorimetric detection is of great
and therapeutics, the accurate monitoring of cholesterol levels interest due to its simplicity, low cost, high sensitivity, and good
1,5–7
14,35–38
in blood plasma is important.
Recently, hypercholesterol- selectivity.
2 2
H O is generated during the catalytic oxida-
emia has been identied as the main reason for human death. tion of substrates by ChOx, glucose oxidase, and xanthine
Thus, controlling blood cholesterol level has become a global oxidase, among others. Therefore, the accurate determination
challenge. Most cholesterol biosensors involve the enzymatic of H
2
O
2
is useful for indirectly measuring the levels of target
1,5
oxidation of cholesterol by cholesterol oxidase (ChOx).
Consequently, the production of hydrogen peroxide (H
) is Zhang et al. reported a MXene-Ti
Low levels of cholesterol the colorimetric determination of cholesterol with a linear
may be associated with medical conditions such as cancer, range of 10–100 mM and a limit of detection (LOD) of 1.9 mM. Li
molecules such as glucose, cholesterol, and xanthine. Recently,
39
O
2
C /CuS nanocomposite for
3 2
2
8–14
quantied by various procedures.
40
depression, anxiety, hopelessness, nervousness, confusion, et al. developed a F ¨o rster resonance energy transfer-based
agitation, and hypocholesterolemia. Total cholesterol levels sensor for the determination of cholesterol. They obtained
ꢀ
1
ꢀ1
a linear range of 10–210 mM L with a LOD of 343.48 nM L
.
Cholesterol was also detected using an electrochemical tech-
nique with a LOD of 0.03 mM and a linear range of 0.06–15 mM.
The sensor developed in this study improves upon the above
detection methods in terms of its cost-effectiveness, low limit of
detection (ꢁ0.014 nM), and linearity in the cholesterol
concentration range of 10–250 nM.
a
41
School of Studies in Chemistry, Pt. Ravishankar Shukla University, Raipur, C.G.,
India-492010. E-mail: manmohanchem@gmail.com
b
Department of Chemistry, Govt. Madhav Science P. G. College, Ujjain, M.P., India-
4
c
56010
Department of Chemistry, Govt. Nagarjuna P. G. College of Science, Raipur, C.G.,
India-492010
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In this study, a biosensing probe was developed for the 2.2 Instrumental details
determination of cholesterol based on the immobilization of
ChOx on modied silver nanoparticles (AgNPs), which
enhance the peroxidase-like catalytic activity. ChOx speci-
All optical experiments were performed using a Thermo-
scientic Evolution 300 spectrophotometer. The Fourier-
transform infrared (FTIR) spectroscopy was conducted using
a Nicolet iS10 FTIR spectrometer (Thermosher) with KBr
pellets to identify the functional groups of the AgNPs and ChOx-
conjugated AgNPs. The AgNP morphology was investigated by
transmission electron microscopy (TEM; JEOL, JEM-2100F) with
an accelerating voltage of 200 kV.
cally catalyzes the oxidation of cholesterol to produce H
2
O
2
in
0
the presence of oxygen. Hence, the AgNPs (Ag ) are oxidized to
+
Ag by H
2 2
O . As a result, the absorbance of the AgNPs at
4
00 nm decreases, resulting in a distinct color change from
yellow to colorless. In the presence of different concentrations
of cholesterol, the etching of AgNPs by H O and corre-
2
2
sponding decrease in absorption intensity were exploited for
the determination of cholesterol. The developed method has
some advantages over existing methods because no additional
chromogenic agent is necessary as a dye. The developed
method also shows high sensitivity based on the high molar
extinction coefficient of AgNPs, is easy to implement, allows
rapid detection with the naked eye, and can be applied to
2
.3 Preparation of cholesterol solution
The cholesterol catalytic reaction was carried out by adding
different concentrations of cholesterol (10–250 nM) dissolved in
0.1 mL Triton X followed by mild heating to dissolve the
cholesterol. Aer the complete dissolution of cholesterol,
ꢀ
1
0.1 mol L phosphate buffer solution (pH 7.5) was added. The
+
prepared cholesterol solutions were further used for enzymatic
assay.
complex samples. The amount of released Ag is low because
the concentration of AgNPs used is low (0.2 nM). In the human
+
body, Ag is commonly present in the bloodstream (<2.3 b. mg
ꢀ
1
2.4 Synthesis of AgNPs
L
) and key tissues such as the liver and kidney without any
association with disease or disability. Therefore, the oxidative
Ag concentration in the proposed method is negligible. For
45
AgNPs were synthesized by Martin's method. Briey, triso-
+
dium citrate (0.25 mM) was added to 100 mL of an aqueous
colorimetric sensors based on metallic nanoparticles, the
optical properties are mainly determined by surface plasmon
resonance. The optical responses of the nanoparticles depend
solution of AgNO
trations of NaBH
3
(0.25 mM). Aer 3 min, different concen-
were added; the NaBH concentration of
4
4
9.5 mM was determined to be optimal for AgNP formation. The
4
2
on the nanoparticle size, shape, geometry, and environment.
colorless solution became yellow when the AgNPs were formed.
Citrate was used as a stabilizing agent to prevent the aggrega-
tion of Ag atoms.
AgNPs show good optical performance, as expected given their
extremely high dielectric constant or polarizability. AgNPs
have high optical radiation efficiency. As the particle size
increases, the re-radiation of energy from the AgNPs to the
2.5 Functionalization of AgNPs and colorimetric sensing of
4
3
surrounding medium is expected to become dominant. The
spherical AgNPs with diameters of approximately 13–15 nm
showed maximum absorbance at 400 nm, indicating good
agreement between the optical properties (surface plasmon
resonance), AgNP geometry, and the polarization of incident
light. We also studied the inhibition of ChOx using a logic
gate, validating the developed method. The anionic surfactant
sodium dodecyl sulfate (SDS) was used as an inhibitor with
a maximum inhibition of approximately 90%. The developed
method was successfully applied in the detection of free
cholesterol in bovine serum albumin (BSA) and milk.
Cholesterol could be efficiently detected by the naked eye
without the need for any specialized instruments.
cholesterol
AgNPs were reacted with a mixture of freshly prepared sulfo-
NHS (0.1 mM) and EDC solution (0.4 mM) for 30 min to
induce the coupling reaction. Sulfo-NHS-terminated AgNPs
were isolated by centrifugation and re-dispersed in phosphate-
buffered saline (PBS; 0.1 M, pH 7.5). Subsequently, a working
solution (1.67 mL EDC) and 6.67 mL sulfo-NHS were added into
an aqueous solution of AgNPs (25 mL). The solution was kept at
room temperature for 24 h. Aer synthesis, the AgNPs were rst
functionalized with EDC/NHS and then immobilized with
ChOx. Upon the addition of cholesterol, cholestenone and H
are produced, which react with the AgNPs, as shown schemat-
ically in Fig. 1. As a result, the color of the solution changes
4
4
46
2 2
O
0
from yellow to colorless due to the oxidation of Ag in the AgNPs
+
to Ag by accepting electrons from H
2
O
2
. The changes in
2. Experimental
ꢂ
absorbance at 400 nm were measured at 37 C.
2
.1 Materials and reagents
Cholesterol, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC), N-hydroxysuccinimide (NHS), ChOx, Milk and BSA were dissolved separately in 5 mL of KOH/ethanol
silver nitrate (AgNO ), trisodium citrate, sodium solution and saponied in a water bath for 1 h. Next, 5 mL water
hydroxide, sodium borohydride (NaBH
), and sodium phos- and 10 mL n-hexane were added into the sample solution, and
2.6 Analysis of cholesterol in real samples
3
2 2
H O ,
4
phate (dibasic) were purchased from Sigma-Aldrich (Bangalore, the mixture was centrifuged at 5000 rpm for 5 min. Finally, the
India). All experiments were performed using Milli-Q ultrapure n-hexane was separated, and the solvent was evaporated under
water, and the reagents used were of analytical grade. All a stream of nitrogen. The residue was re-dissolved with the
glassware was rinsed in aqua regia prior to use.
previously mentioned mixture of isopropanol and Triton X-100.
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Fig. 1 Mechanism of the production of H
2
O
2
by AgNPs immobilized with ChOx in the presence of cholesterol and the optical response of the
nanoprobe.
The diluted samples and ChOx (3.0 mU) in phosphate buffer absorbance of the AgNPs occurred at 400 nm, and the density
ꢂ
ꢀ13
ꢀ3
were then incubated at 37 C for 1.5 h. The mixture of AgNPs, was 2.21 ꢃ 10
mol cm (Fig. 2A). No major changes in
cholesterol (250 nM), and phosphate buffer was then added to absorbance were observed when EDC/NHS and ChOx were
the above reaction solution, and the ultraviolet (UV)-visible added to the AgNPs solution (Fig. 2A). Aer the addition of EDC/
spectra were recorded.
NHS, the stability or dispersion of the AgNPs was enhanced. In
the presence of ChOx, the surface plasmon resonance absor-
2 2
bance at 400 nm decreased due to the formation of H O , which
induced the oxidation of AgNPs from Ag to Ag (surface
etching), and the color changed from yellow to colorless
2
.7 Selectivity of the nanoprobe
0
+
The selectivity of the sensor toward cholesterol was investigated
in the presence of interfering bioanalytes including ascorbic
acid, urea, glucose, galactose, and L-cysteine. The UV-visible
spectra of the AgNPs immobilized with ChOx (3.0 mU) were
recorded in presence of these biomolecules under similar
experimental conditions. The conditions and interfering ana-
lyte concentrations were the same as those of cholesterol
(Fig. 2A). The photographs of the corresponding solutions are
shown in the inset of Fig. 2A.
The FTIR spectra were measured to analyze the surface
structure and surface modication of the AgNPs (Fig. 2B and C).
The FTIR spectrum of trisodium citrate on AgNPs exhibited nas
ꢀ
ꢀ
ꢀ1
(
s
COO ) and n (COO ) stretching bands at 1585 and 1382 cm ,
(
3
concentration ¼ 250 nM). The solutions were incubated at
respectively. The typical signals of EDC/NHS were also observed
ꢂ
7 C for 1 h. If an analyte possessed an interfering effect, it
ꢀ1
at around 3420 and 1560 cm ; these peaks are assigned to –OH
would enhance the absorption intensity. Thus, recording the
absorption intensities of the probe in the presence of biomol-
ecules and cholesterol reveals the selectivity of the sensing
platform.
and –COOH, which are required for the immobilization of
enzymes. The surface modication of AgNPs with ChOx was
conrmed by the characteristic FTIR bands of –C]O (amide I)
ꢀ1
and –N–H (amide II) at 1650 and 1550 cm , respectively
(Fig. 2C).
The size of the nanomaterial plays an important role in
3
. Results and discussion
sensing. Spherically shaped AgNPs with sizes of 10–20 nm show
higher sensitivity than the bulk material due to their large
surface-to-volume ratios. In general, the sensing behavior of
3.1 Structural and morphological analyses of functionalized
AgNPs
AgNPs were obtained via a simple reduction method and then AgNPs changes with the particle size, and the surface plasmon
characterized by the UV-visible spectrophotometry, TEM, and resonance and scattering of light depend upon the nanoparticle
FTIR spectroscopy. Fig. 2A shows the UV-visible absorption size. Due to the size of the AgNPs being smaller than the
spectra of AgNPs with surface modication. The maximum
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Fig. 2 (A) UV-visible spectra of (a) AgNPs, (b) AgNPs conjugated with EDC/NHS and ChOx, and (c) AgNPs and ChOx in the presence of
cholesterol. (B) FTIR spectra of trisodium citrate and citrate-capped AgNPs. (C) FTIR spectra of AgNPs (a) conjugated with EDC/NHS, (b) in the
presence of H O , and (c) in the presence of cholesterol. TEM images of (D) AgNPs, (E) AgNPs conjugated with EDC/NHS and immobilized with
2 2
ChOx, and (F) AgNPs conjugated with EDC/NHS and immobilized with ChOx in the presence of cholesterol.
wavelength of the incident light and because of their large of agglomerated AgNPs with larger sizes of approximately
surface area, electrons from the conduction band of the AgNPs 150 nm; this is attributed to the production of H O by the
2 2
are easily ejected to generate secondary electrons. The mono- catalytic reaction of cholesterol with immobilized enzyme on
dispersed and spherically shaped functionalized AgNPs with an the AgNP surfaces.
average diameter of 13–15 nm were characterized by TEM
Fig. 2D). No obvious changes in AgNP size were observed aer tively charged molecules. The –OH groups on the surfaces of the
immobilization with ChOx (Fig. 2E). Fig. 2F shows a TEM image AgNPs can be covalently conjugated with any molecules
The synthesized AgNPs were generally surrounded by nega-
(
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4
7
containing –COOH groups via EDC/NHS coupling reaction.
decreased (Fig. 4A) with good linearity over the concentration
ꢀ
The EDC molecule activates the –COO functional group to range (inset of Fig. 4A). This can be attributed to the formation
form ester linkages with –OH groups. EDC can also form amide of dissolved Ag in proportion to the amount of added H O .
+
2
2
couplings (CONH). Hence, EDC induces the binding of the Similarly, Fig. 4B depicts the decrease in the absorbance of
2 2
enzyme (ChOx) on the surfaces of the modied AgNPs. The AgNPs with reaction time in the presence of 150 mM H O .
4
8–50
ꢀ
–
COOH groups on the AgNPs are activated by EDC/NHS. This is According to He et al.,
AgNPs accept electrons from O c to
2
a well-known technique to conjugate carboxyl groups with form highly reactive “charged” nanoparticles that induce the
+
amine groups in enzymes. A possible mechanism for the reduction of both oxygen and Ag , resulting in the reformation
conjugation of EDC/NHS and immobilization of ChOx is shown of AgNPs. The in situ-formed AgNPs following the electron
+
in Fig. 3.
transfer from charged AgNPs to Ag may either result in the
formation of larger particles or more particles (increased
density). An increase in particle size would be expected to
induce a redshi in the AgNP peak (Fig. 4), while an increase
in particle number would be expected to result in an increase
in absorbance.
3.2 Effect of H
2 2
O concentration
The dissolution of citrate-coated AgNPs by H
efficient, even at low H
between AgNPs with H O is given as follows:
2 2
O appears to be
2
O
2
concentrations. The general reaction
2 2
0
+
ꢀ
2
Ag + H O / Ag + O + 2H O
(1) 3.3 Cholesterol sensing assay
2
2
2
0
The detection capability of the proposed sensor was evaluated
2 2
Ag is expected to be dissolved by H O due to the catalytic
for H
Fig. 4D). The absorbance of the AgNPs gradually decreased
with increasing H concentration from 10 to 150 mM. The
absorbance vs. the H concentration is plotted in the inset of
2 2
O and cholesterol under the optimum conditions
reaction involving the production of radicals and oxygen, as
oxygen was excluded under the experimental conditions. The
intermediates may include hydroxyl radicals (OH ) with very
short lifetimes. The decomposition of H O upon contact with
AgNPs was followed over time. The results showed that H O
in the concentration range of 10–150 mM was consumed within
approximately 40 min in the presence of 0.02 nM AgNPs. The
pH dependence of this reaction was observed by the rapid
decomposition under alkaline pH and slower reaction rate
2
under acidic pH. The produced superoxide anion O can
further react with AgNPs and Ag to again form AgNPs or
(
ꢀ
2 2
O
O
2 2
2
2
Fig. 4A, showing a good linear relationship (R ¼ 0.9867) in the
concentration range of 10–150 mM. Similarly, as shown in
Fig. 4B, absorbance decreased gradually with time when the
H O concentration was 150 mM. ChOx is commonly used in
2
2
2
2
cholesterol detection. ChOx can catalyze the oxidation of
cholesterol in the presence of oxygen to produce H
enzyme-catalyzed reaction is described by eqn (2):
2
O
2
. The
+
ChOx
ƒ!
recombine to form H O . Therefore, aer an increase in H O
concentration, the absorption intensity of the AgNPs
2
2
2
2
Cholesterol þ O þ H O
cholestenone þ H O
(2)
2
2
2
2
Fig. 3 Activation of functional groups on the surfaces of AgNPs using EDC/NHS and conjugation with ChOx.
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Fig. 4 UV-visible spectra of AgNPs (A) in the presence of different concentrations of H
O
2 2
(10–150 mM) and (B) at different time intervals (0–40
min) in the presence of 150 mM H
absorbance vs. time in the presence 150 mM H
presence of various concentrations of cholesterol (1–50 nM) and (E) AgNPs conjugated with EDC/NHS immobilized with ChOx in presence of
50 nM cholesterol. The inset of (D) shows the change in absorbance as a function of the cholesterol concentration. (F) Plot of absorbance vs.
O
2 2
. The inset of (A) shows the change in absorbance as a function of H
2 2
O concentration. (C) Plot of
2 2
O . UV-visible spectra of (D) AgNPs conjugated with EDC/NHS and immobilized with ChOx in
2
time for AgNPs immobilized with ChOx in the presence of 250 nM cholesterol. The insets of (C) and (F) show zoomed in images of linear areas of
the plots.
Subsequently, the produced H O can act as a mediator to method, the developed cholesterol sensing system was
2
2
construct an oxidase-based sensor. As shown in Fig. 4D, the compared with some previous methods (Table 1). The detection
absorbance of the AgNPs decreased gradually with increasing limit of our method was better than those of previously reported
cholesterol concentration. The detection limit for cholesterol methods. Some studies found that the sensitivity could be
was as low as 0.0149 nM. The absorption of the AgNPs gradually increased by using smaller nanoparticles with maximum
decreased with time from 0–23 min when the cholesterol surface area. The AgNPs used in this study (sizes of 15–20 nm)
concentration was 250 nM. The plot of absorbance vs. time showed good reactivity due to their large surface areas; however,
showed a good linear correlation (R ¼ 0.9925; insets of Fig. 4E the presence of cholesterol produced H O , and the AgNP
2
2
and F). To better understand the performance of the proposed particle size increased due to particle aggregation.
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Table 1 Comparison of the developed method with previously reported methods for the determination of cholesterol
S. No.
Method
Cholesterol sensor
MXene-Ti /CuS nanocomposites
Linear range
LOD
Reference
1
2
3
4
Colorimetric
Colorimetric
Colorimetric
Fluorescence
3
C
2
10–100 mM
4–100 mM
0.1–0.8 mM
0.06–15 mM
1.9 mM
3.01 mM
78 mM
40
14
52
41
ChOx-PB/MWCNT
CuO graphene nanospheres
Ag nanocluster-decorated MoS
nanosheets
2
0.03 mM
5
6
7
Fluorescence
Electrochemical
Colorimetric
Au@CQD
CuO nanoparticles
AgNPs
1–6.25 mM
1–15 mM
1–6.5 nM
2.5 mM
0.43 mM
0.0149 nM
9
51
Present method
3
.4 Inhibition of enzyme
disturbing the noncovalent forces. These forces include
hydrogen bonding, hydrophobic interactions, and ionic inter-
actions, which are responsible for the three-dimensional
structures of the native proteins.
The activity of ChOx from Streptomyces was studied in the
presence of SDS using AgNPs as an assay. The optical absorp-
tion spectra (Fig. 4 and 5) show the formation of AgNPs with
a single narrow surface plasmon located at 400 nm, which is
characteristics of spherically shaped nanoparticles with diam- 3.5 Implementation of a logic system
eters of 15–20 nm. The addition of H O reduced the peak
2
2
Aqueous solutions of ChOx immobilized on AgNPs in the
presence of cholesterol and AgNPs immobilized with ChOx and
cholesterol in presence of SDS can be used to construct an
absorbance-based logic system. For logic gate implementation,
the combination of AgNPs and cholesterol is considered as the
system, while ChOx and SDS are the two inputs. The output ‘1’ is
obtained for the AgNPs when the input is (0,0), corresponding
to the absence of ChOx and SDS. The addition ChOx but not SDS
is the input (1,0), which gives output ‘0’. When SDS is present
but ChOx is absent, the input is (0,1), and the output ‘1’ is ob-
tained. When both ChOx and SDS are present, the input is (1,1),
and the output ‘1’ is obtained. Fig. 6A shows the truth table
followed by the logic system. Fig. 6B depicts the absorbance
intensity for different input signals, and the correspond logic
symbol is shown in Fig. 6C.
intensity, which is attributed to the reduction in the free elec-
tron density of Ag due to the possible oxidation of AgNPs. The
addition of SDS denatured the enzyme; hence, the production of
was arrested, and the oxidation of AgNPs to Ag was
interrupted. The intensity of the absorption peak of the AgNPs
increased with increasing SDS concentration.
The enzyme was fully inactivated in the presence of SDS. The
effect of SDS was analyzed by incubating ChOx with various
concentrations of SDS (10–250 mM) for 60 min at 37 C. Func-
tionalized AgNPs immobilized with inhibited ChOx (3.0 mU)
2 2
were added into a cholesterol solution. H O was not produced
to cause the aggregation of AgNPs. Incubated enzyme solution
was used to determine the residual enzyme activity in compar-
ison to the enzyme activity without incubation with SDS. When
the SDS concentration increased, the absorption intensity of the
AgNPs also increased (Fig. 5A) with a good linear range (Fig. 5B).
+
H
2
O
2
53
ꢂ
The enzyme activity was highest in Triton X-100 (>80%); 3.6 Kinetic properties of immobilized ChOx
however the presence of only 0.1% SDS fully inactivated the
The Michaelis–Menten and Lineweaver–Burk plots were con-
enzyme. SDS is used in cosmetics and toothpastes; therefore,
there are many sources of SDS to enter the human body. SDS is
an anionic detergent that denatures native proteins by
structed (Fig. 7) to study the kinetics of immobilized ChOx at pH
ꢂ
7.5, a temperature of 37 C, and various substrate concentrations.
m
K is parametric statistic that describes the affinity between the
Fig. 5 (A) UV-visible spectra of AgNP-EDC/NHS + ChOx + cholesterol with different concentrations of SDS. (B) The linear calibration curve
between absorbance and SDS concentration. (C) Inhibition percentages of ChOx in the presence of different concentrations of SDS.
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Fig. 6 Logic gate implementation: (A) truth table for the applied logic gate; (B) absorbance for different input signals; and (C) a representation of
the logic gate relating to the applied truth table [NOT, OR].
The rate of H
cholesterol concentration. Since H
2
O
2
production increased with increasing
is the basic quenching
2 2
O
unit in this assay, the extent of absorption quenching essen-
tially represents the variation in the enzymatic activity of
ChOx. The enzyme/substrate kinetics can be assessed by the
Michaelis–Menten kinetic parameters Km and Vmax. The value
of K
m
reects the affinity of the enzyme for the substrate and
does not depend on the enzyme concentration, whereas Vmax
denes the maximum rate of the enzymatic reaction. The
effect of the substrate concentration on the reaction rate
catalyzed by immobilized ChOx was studied using different
initial concentrations of cholesterol. The enzyme/substrate
kinetic parameters were calculated from the Lineweaver–
Burk plot as follows:
Fig. 7 The Lineweaver–Burk plot for the determination of the enzy-
matic activity of functionalized AgNPs immobilized with ChOx.
1
K
m
1
1
¼ V
þ V
:
(3)
v
0
max ½Sꢄ
max
enzyme and substrate in an enzymatic reaction. A lower Km value
corresponds to a higher affinity between the enzyme with
3.7 Optimization
substrate, indicating biocompatibility and facilitating the enzy- The experimental concentrations of NaBH and ChOx along
4
matic reaction. Vmax is the maximum rate of enzymatic reaction with the pH and temperature prior to analysis were opti-
+
when the enzyme is saturated with substrate. The value of K
m
is mized. The reduction of Ag was evaluated using different
calculated from the intercept and slope of the Lineweaver–Burk concentrations of NaBH4 ranging from 2.5 to 9.5 mM
plot. The changes in absorbance with time were plotted as (Fig. 8A). The maximum absorption of AgNPs was achieved
a function of cholesterol concentration. A decrease in K indicates using a NaBH concentration of 9.5 mM. The effect of pH on
m
4
a faster reaction rate, whereas an increase in Km suggests that the enzymatic activity of ChOx was optimized in the pH range
a larger substrate concentration is required to achieve the same of 5–9 (PBS solution). The optimal reaction solution pH of 7.5
reaction rate observed for the free enzyme. The Michaelis–Menten was adopted in further experiments (Fig. 8D). Aer adding
constant K
indicating the intensive enzyme activity of ChOx-immobilized solution, the maximum activity was observed at 3.0 mU,
AgNPs. The catalytic efficiency (Vmax/K of the nano- resulting in the maximum production of H O and etching of
m
for cholesterol was determined to be 196.07 nM, different concentrations of ChOx (0.1–3.0 mU) to the reaction
m
)
2
2
bioconjugate was higher than that of the free enzyme, largely due AgNPs (Fig. 8B). The effects of temperature were also inves-
ꢂ
to the lower K
m
of the immobilized enzyme. Generally, the kinetic tigated (Fig. 8C), and good response was obtained at 37 C.
parameters (Km and Vmax) of an enzyme change aer immobili- Thus, the temperature for subsequent experiments was
ꢂ
zation, indicating a change in affinity for the substrate.
37 C.
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Fig. 8 UV-visible spectra of (A) AgNO
3
with various concentrations of NaBH
4
and (B) AgNPs with various concentrations of ChOx in the presence
of cholesterol. Relative enzymatic activity of ChOx as a function of (C) temperature and (D) pH.
3.8 Analysis of real samples
3.9 Interference effect
To verify the reliability of the developed method for cholesterol The selectivity of the developed probe (AgNPs/ChOx) for
determination in clinical diagnostics, the AgNP-based sensor cholesterol detection was evaluated in the presence of potential
was applied to determine cholesterol in BSA and milk. Known interfering substances (ascorbic acid, urea, galactose, glucose,
amounts of cholesterol (1, 5, and 10 mM) were added to real BSA citric acid, oxalic acid, NaCl, KCl, glycine, sucrose, and choles-
and milk samples. The original and combined cholesterol terol). As shown in Fig. 9, the absorbance intensity at 400 nm
concentrations in the real samples were determined from the decreased dramatically in the presence of cholesterol. In the
absorption spectra of the AgNPs aer treatment with the real presence of these interferential species, no notable changes
samples. The results are listed in Table 2. The recovery ranged
from 99.2% to 102.2% with good relative standard deviations
(<2.60%). The results demonstrate that the developed sensor is
applicable to the detection of cholesterol in real samples with
good precision and accuracy.
Table 2 Determination of spiked cholesterol in BSA and milk samples
Measured cholesterol
Sample Spiked (mM) (mM)
RSD (%)
Recovery (%) (n ¼ 5)
BSA
1
5
0
1
5
0
1.352
5.890
11.981
2.510
6.121
11.825
100.8%
99.2%
102.2%
101.6%
96.2%
0.89
0.98
1.88
0.45
2.59
1.34
1
1
Milk
101.3%
Fig. 9 AgNP absorbance in the presence of various potentially inter-
fering biomolecules during cholesterol sensing.
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observed in the absorbance of the AgNPs. Thus, the developed 11 T. Lin, L. Zhong, H. Chen, Z. Li, Z. Song, L. Guo and F. Fu,
assay exhibited high selectivity for the determination of
cholesterol.
Microchim. Acta, 2017, 184, 1233–1237.
12 Y. Zhang, Y. N. Wang, X. T. Sun, L. Chen and Z. R. Xu, Sens.
Actuators, B, 2017, 246, 118–126.
1
3 V. Sharma and S. M. Mobin, Sens. Actuators, B, 2017, 240,
38–348.
4
. Conclusions
3
1
4 Y. He, X. Niu, L. Shi, H. Zhao, X. Li, W. Zhang, J. Pan,
X. Zhang, Y. Yan and M. Lan, Microchim. Acta, 2017, 184,
We have demonstrated the practicability of a new colorimetric
enzymatic assay based on AgNPs as a nanoprobe. H results
in the aggregation and enlargement of AgNPs, affecting the UV-
visible absorbance of the AgNPs. This response can be exploited
2 2
to detect H O and cholesterol sensitively. The probe showed
good performance for cholesterol detection with high sensi-
tivity, selectivity, and stability along with a relatively low LOD.
The new method was successfully applied to the determination
of cholesterol in milk and BSA samples. To the best of our
knowledge, this is the rst work reporting AgNPs as a colori-
metric assay for the detection of cholesterol. The developed
probe provides a cost-effective and time-saving strategy for
colorimetric assays. Furthermore, the sensing platform can be
2 2
O
1
–9.
15 M. H. Criqui, Circulation, 1994, 90, 2591.
16 P. Gomathi, D. Ragupathy, J. H. Choi, J. H. Yeum, S. C. Lee,
J. C. Kim, S. H. Lee and H. D. Ghim, Sens. Actuators, B, 2011,
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A. P. Turner and M. Y. Vagin, Anal. Chem., 2014, 86, 9540–
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1
8 X. H. Liu, Z. H. Nan, Y. Qiu, L. C. Zheng and X. Q. Lu,
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1
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utilized for various biological molecules with different H O -
54
55
20 F. Xie, X. Cao, F. Qu, A. M. Asiri and X. Sun, Sens. Actuators, B,
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producing oxidases. The detection of glucose, oxalic acid,
sucrose, and uric acid is possible using our approach.
2
2
2
2
1 Z. Wang, F. Xie, Z. Liu, G. Du, A. M. Asiri and X. Sun, Chem.–
Eur. J., 2017, 23, 16179–16183.
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Conflicts of interest
There are no conicts to declare.
Acknowledgements
24 A. Mondal and N. R. Jana, Chem. Commun., 2012, 48, 7316–
318.
7
Lakshita Dewangan is thankful for a PhD scholarship from Pt.
Ravishankar Shukla University, Raipur. All authors are thankful
to the National Chemical Laboratory, Pune for TEM analysis
and NCNR Pt. Ravishankar Shukla University, Raipur for FTIR
analysis.
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