Graphitic carbon nitride for efficient fluorometric quenching bioassay of hydrogen peroxide: Effect of structure on Properties

Article abstract of DOI:10.1016/j.jphotochem.2020.112731

Demand for sensitive and selective sensing of hydrogen peroxide is high because of its importance in various fields. So, metal-free nanostructures of g-C₃N₄ were synthesized and characterized through different techniques. Characterizations showed that g-C₃N₄ physicochemical properties were depended upon the nanostructure. A porous, thin and fluffy structure was got from urea, which possessed a much higher surface area and pore volume as compared to the planar and crystalline structured g-C₃N₄ got from melamine. A fluorescence quenching approach of hydrogen peroxide determination using catalyst nanosheets was used to explore the catalytic activity of these nanostructures. Because of the porosity, structural defects, smaller-crystallite size, and large surface area of the fluffy structured catalyst showed better quenching efficiency as compared to the flat and planar structured g-C₃N_4. An oxygen atom in urea played a role in changing the textural properties to porous and fluffy g-C₃N₄. Based on that, a selective, sensitive, and rapid sensing method was established having a wide linear range of 90–2500 nM, a lower detection limit of 73 nM, and a quantification limit of 220 nM.

Full text of DOI:10.1016/j.jphotochem.2020.112731

Journal Pre-proof
Graphitic Carbon Nitride for Efficient Fluorometric Quenching Bioassay of
Hydrogen Peroxide: Effect of Structure on Properties
Aftab Ahmed (Conceptualization) (Writing - original draft), Akhtar
Hayat (Conceptualization), Mian Hasnain Nawaz (Supervision),
Peter John (Supervision) (Conceptualization), Muhammad Nasir
PII:
S1010-6030(20)30530-X
DOI:
https://doi.org/10.1016/j.jphotochem.2020.112731
JPC 112731
Reference:
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Journal of Photochemistry & Photobiology, A: Chemistry
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Accepted Date:
11 May 2020
21 June 2020
26 June 2020
Please cite this article as: Ahmed A, Hayat A, Nawaz MH, John P, Nasir M, Graphitic Carbon
Nitride for Efficient Fluorometric Quenching Bioassay of Hydrogen Peroxide: Effect of
Structure on Properties, Journal of Photochemistry and amp; Photobiology, A: Chemistry
(2020), doi: https://doi.org/10.1016/j.jphotochem.2020.112731
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Graphitic Carbon Nitride for Efficient Fluorometric Quenching Bioassay of
Hydrogen Peroxide: Effect of Structure on Properties
Aftab Ahmed1, 2, Akhtar Hayat1, Mian Hasnain Nawaz1, Peter John2*, Muhammad Nasir1*
1 Interdisciplinary Research Centre in Biomedical Materials (IRCBM), COMSATS University
Islamabad, Lahore Campus, 1.5 Km Defence Road, off Raiwind Road, Lahore, Punjab, Pakistan,
54000
2 Government College University Lahore, Katchery Road, Anarkali, Lahore, Punjab, Pakistan
54000
Muhammad Nasir: Email; muhammadnasir@cuilahore.edu.pk, Tel; +92 (42) 111-001-007 Ext:
828, 829, Fax; +92 (42) 5321090
Peter John: Email; peterjohn@gcu.edu.pk, Tel; +92 (42) 111-000-010 Ext 262, +92 (42)
99213338
AUTHOR INFORMATION
Corresponding author
*Email: muhammadnasir@cuilahore.edu.pk (M. Nasir)
peterjohn@gcu.edu.pk (P. John)
ORCID iD
Muhammad Nasir: 0000-0002-6742-2721
Graphical Abstract:
1

Schematic illustration of synthesis of Graphitic Carbon Nitride Nanosheets and procedure for
fluorescence quenching based detection of H2O2 in the presence of RhB.
Highlights
 Effect of different precursors on the structure of graphitic carbon nitride was
exploited in term of peroxidase-like-catalytic-activity by using fluorescence
quenching of Rhodamine B to determine hydrogen peroxide
 Nanosheets of porous, folded along with curl edged fluffy g-C3N4 and crystalline
smooth-surfaced planar g-C3N4 were prepared through liquid exfoliation
ultrasonication method
 A possible reaction mechanism for the detection was proposed
2


This method exhibited a linear range of 90 – 2500 nM and LOD of 73 nM.
ABSTRACT
Demand for sensitive and selective sensing of hydrogen peroxide is high because of its
importance in various fields. So, metal-free nanostructures of g-C3N4 were synthesized and
characterized through different techniques. Characterizations showed that g-C3N4
physicochemical properties were depended upon the nanostructure. A porous, thin and fluffy
structure was got from urea, which possessed a much higher surface area and pore volume as
compared to the planar and crystalline structured g-C3N4 got from melamine. A fluorescence
quenching approach of hydrogen peroxide determination using catalyst nanosheets was used to
explore the catalytic activity of these nanostructures. Because of the porosity, structural defects,
smaller-crystallite size, and large surface area of the fluffy structured catalyst showed better
quenching efficiency as compared to the flat and planar structured g-C3N4. An oxygen atom in
urea played a role in changing the textural properties to porous and fluffy g-C3N4. Based on that,
a selective, sensitive, and rapid sensing method was established having a wide linear range of 90
- 2500 nM, a lower detection limit of 73 nM, and a quantification limit of 220 nM.
KEYWORDS: Fluffy, Porous, Graphitic Carbon Nitride, Hydrogen Peroxide, Rhodamine B,
Fluorescence Quenching.
1.
INTRODUCTION
3

The H2O2 is generated in many physiological enzymatic catalytic processes. It has
become a messenger of several abnormalities in the human body, such as oxidative stress,
diabetes, renal failure, and cancer. [1] It is used in cosmetics and food industry, environmental
protection, pharmaceutical, and clinical fields. This widespread use has made H2O2
determination very necessary. The H2O2 is diagnosed by reacting with natural-enzymes of
horseradish-peroxidase [2] through colorimetric, electrochemical, [3] and luminescence
techniques. [4] Despite high-selectivity of natural-enzymes, the detection process has faced the
limitations of denaturing to environmental conditions, complicated and costly extraction and
purification processes of the natural-enzymes. [5] To overcome these limitations, artificial
enzyme-based sensors were designed and used in the assay of H2O2. Different artificial enzymes,
such as Fe3O4 [6] nanoceria [7] and nitrogen-doped Titania [8] were reported for H2O2 bioassay.
Carbon-based nanomaterials are low-cost, less-toxic, and biocompatible. [9] Graphitic
structured carbon-based nanomaterials like graphitic carbon nitride (g-C3N4) have reported as the
catalyst in many analytical applications. It is used in fields such as photocatalytic production of
hydrogen [10], degradation of organic pollutants [11, 12] pharmaceuticals and personal care
products [13] and in the environmental remediation. The g-C3N4 has unique physicochemical
properties, semiconductor behavior, and the -conjugated network connecting two-dimensional
layers of tris-s-triazine building blocks. It is fabricated and designed in different structures like
nanoflowers, nanorods, and nanotube.
In this study, crystalline and porous g-C3N4 were synthesized from different precursors
through the calcination method with no catalytic components or complex treatment to avoid
toxicity. Characterization showed that porous and fluffy g-C3N4 have small-crystallite domain
and structural-defects such as curling of nanosheets because of highly repeated graphitic-
4

structure as compared to crystalline and planar g-C3N4. Fluffy structure of g-C3N4 improved the
optical properties and enhanced the surface area. Because of selectivity, high sensitivity, and
handling of fluorescence quenching technique, a fluorescence quenching approach was explored
using graphitic carbon nitride to build a detection assay of H2O2. The fluffy structure of g-C3N4
has shown better fluorescence quenching as compared to planar structured g-C3N4. The use of g-
C3N4 in the present fluorescence quenching approach shortened the analysis time, lower limit of
detection, formed broader linear range, and high sensitivity for H2O2 as compared to other
methods of sensor development like colorimetric methods. Furthermore, the fluorescence
quenching method was easier to use as the use of the colorimetric method. This study also helps
in the high throughput synthesis of small-sized g-C3N4 nanosheets of the different nanostructures
and investigating these nanosheets as peroxidase-like-mimics for biotechnology. [14] A
summary of the synthesis process of catalyst and catalytic reaction mechanism between RhB and
H2O2 are shown in Scheme 1.
5

Scheme 1. Schematic illustration of calcination synthesis and reaction mechanism for H2O2
detection
2.
EXPERIMENTATION
Materials and Reagents
2.1
Sodium acetate anhydrous, L (+)-ascorbic acid, and urea were received from Daejung.
Melamine, citric acid, acetic acid, 3, 3ʹ, 5, 5ʹ-tetramethylbenzidine (TMB), and pyrocatechol
were obtained from Sigma Aldrich. Rhodamine B was received from Avonchem. L-cysteine, p-
benzoquinone (BQ), resorcinol, and H2O2 (30% of w/v solution) were obtained from Merck.
Triethanolamine (TEOA), hydrochloric acid, D-glucose, tert-butyl alcohol (TBA), and uric acid
were got from BDH chemicals Ltd. Dimethyl sulfoxide (DMSO) was obtained from Lab-Scan.
Sodium hydroxide was got from Omicron. Phosphate buffer tablets (pH 7.2 ± 2, 10 mM) were
received from VWR. PBS solution was prepared by dissolving PBS tablets in ultra-pure
6

deionized water of conductivity (<18 Ω cm-1), made in Elga Pure Ultra deionizer. All other
reagents and chemicals were analytical-grade and used as got without further purification.
2.2
Apparatus
Scanning electron microscope images were got at 10 kV by using Vega 3 LMU Tescan
SEM, attached with an energy dispersive spectrometer for EDX analysis. For SEM images and
EDX analysis, samples were coated with a layer of gold, and coated samples were mounted on
Aluminium support by using double adhesive tape. The adsorption isotherm data were used to
measure the BET surface area (SBET) by using a Tri-Star II (3020 2.00, USA) nitrogen gas
adsorption apparatus. The pore size distribution was calculated via the Barret-Joyner-Halenda
(BJH) method. XRD patterns were got with Rigaku D / max 2500 / PC XRD (Rigaku Corp
Japan), equipped with a graphitic monochromator (40 kV, 40 mA). A nickel filtered copper-Kα
radiation source (λ = 1.5418Å) was used during the sample analysis. Fourier transform infrared
spectra were got from 660 – 4000 cm-1 on a Thermo Fisher Scientific FTIR spectrophotometer
(Nicolet 6700). Ultraviolet-visible diffuse reflectance spectra were obtained on a Perkin Elmer
Lambda 35 UV-Vis DRS spectrophotometer. UV-Vis absorption spectra were got on a Perkin
Elmer Lambda 25 UV-Vis absorption spectrophotometer (UV - 25, Perkin Singapore). A
bandwidth of 1 nm with a scan-rate of 960 nm min-1 was used for scanning absorption-spectra in
the range from 400 - 800 nm. Photoluminescence spectra were scanned using a Reni Shaw PL-
InVia-laser-scanning-confocal-microscope at a 457 nm excitation wavelength of the laser.
Fluorescence measurements were performed on an Agilent Cary Eclipse fluorescence
spectrophotometer.
2.3
Synthesis of g-C3N4
7

Differently, structured g-C3N4 was synthesized by a simple calcination method. For fluffy
g-C3N4, 80 gram (g) of urea was grounded to a fine powder in a pestle mortar. Fine grounded
urea powder was transferred to 100 mL ceramic crucible and covered the crucible with semi-
closed ceramic lid. The urea was calcinated in the muffle-furnace with a temperature ramp of
5oC min-1 up-to 550oC and maintained this temperature for 4h. As-prepared product was washed
with distilled water several times to remove any impurities and unreacted nitrogenous materials
from the sample. The purified product was dried in an oven at 60oC for 10h to get amorphous
powder. The final dried product got from urea was labeled as PCN. Crystalline g-C3N4 got from
melamine (10 g) as a precursor under the same synthesis protocol as PCN and labeled as CCN.
Milky dispersion of exfoliated nanosheets containing 2.5 milligrams (mg) solid catalyst in one
mL of the PBS was prepared via ultra-sonication at 25oC for 15 min. These nanosheets-
suspensions were stored at 4oC in the refrigerator for further analysis.
2.4
Oxidation Reaction over PCN and CCN
Chromogenic substrates like terephthalaldehyde,
[15] 2, 2′-azino-bis(3-
ethylbenzothiazoline-6-sulfonic acid) diammonium salt, [16] and plasmonic-hot-electron transfer
induced multicolor-chromogenic system [17] are reported for sensor development. TMB can act
as peroxidase-substrate for the reaction with H2O2 in the presence of a catalyst. Oxidized-TMB
showed the peroxidase-like activity of the catalyst in the reaction. So, an oxidation reaction
between TMB and H2O2 was studied for determining our catalyst peroxidase-like activity.
Typically, 40 L of H2O2 solution (final concentration of 400 μM) was added to 1870 mL of
sodium acetate buffer (pH = 5.0). Then, 60 L of catalyst (PCN, CCN with a final concentration
of 28 g mL-1) was added in the reaction mixture. The resultant reaction mixture was sonicated
for 5 minutes. Finally, 30 L of TMB solution (final concentrations of 50, 100, 200, 300, and
8

600 M) was added to the reaction mixture at room temperature. The reaction color was changed
to blue after a few seconds. The absorbance of the blue-color reaction mixture was monitored at
654 nm in 3 mL aliquots. Kinetic parameters were determined by using the Line-weaver-Burk
plots of the double‐reciprocal (1 / velocity against 1 / [substrate]) of Michaelis-Menten equation
as follow
[ ]
푉푚푎푥푖푚푎푙 × 푆
푉 =
[ ]
퐾푚 + 푆
Where Vmaximal was maximum reaction rate, V was initial velocity, Km was Michaelis-
Menten constant, and [S] was the concentration of the reacting-substrate.
2.5
Estimation of H2O2 by Fluorescence Methods
Chromogenic RhB can act as a fluorescence indicator for a reaction. In the present study,
RhB was used as a fluorescence changing agent for varying catalytic activity of nanosheets in
sensor development. Effect of reaction temperature, reaction time, pH, nanosheets concentration,
and RhB dye concentration on sensor performance was also tested.
Fluorescence analysis was performed by incubating nanosheets and dye-containing
reaction mixture with H2O2. For H2O2 detection, 28 µg mL-1 of nanosheets and 60 ng mL-1 of
dye were mixed with different concentrations of H2O2 ([H2O2]) in 3 mL of the final volume. The
[H2O2] was varied in the range from 30 nM to 80000 nM. After incubation for one min at 24oC,
reaction mixtures were added in 3 mL cuvette, and fluorescence emissions at 577 nm were
recorded under an excitation wavelength of 554 nm. For fluorescence quenching of dye in
nanosheets mixture by H2O2, change in fluorescence intensity at 577 nm was measured by using
equation (∆F577 = F0 − F577). The F0 and F577 nm showed the fluorescence emission
9

intensities of reaction mixtures containing dye and nanosheets in the absence and presence of
H2O2 at 577 nm.
Other interferential experiments were performed under the same reaction conditions as
above. For interferential-compound analysis, calcium chloride, potassium chloride, sodium
chloride, glucose, resorcinol, uric acid, L-cysteine, ascorbic acid, dopamine, citric acid, and
pyrocatechol were used in place of H2O2.
The mechanism of RhB oxidation was investigated by the addition of reactive-species
scavengers in the reaction mixture. The main oxidative-species was detected by taking TEOA (5
mM) as hole-radical removal, TBA (10 mM) as an •OH radical scavenger, and BQ (5 mM) as a
super-oxide scavenger in the reaction mixture.
3.
RESULTS AND DISCUSSIONS
Characterizations
3.1
The nature of crystallization was found through XRD patterns. [18] XRD patterns of the
prepared samples are displayed in Fig. 1. Both PCN and CCN have shown two diffraction peaks.
PCN peak at 27.4o gave a d-space of 0.324 nm, but the CCN peak gave a d-space of 0.34 nm.
The PCN and CCN peaks at 27.4o were indexed to (002) plane of inter-planar graphitic layer
system (JCPDS 87-1526). This stacking-difference in the d-space of (002) peak of PCN and
CCN represented a stranger binding between layers and more electron localization in PCN. PCN
small-peak at 13.1o with a d-spacing of 0.663 nm was indexed to (100) diffraction planes of the
g-C3N4, which showed the stacking of the aromatic conjugated system (JCPDS 87-1526).
Because of tilted angularity and structural defects, the d-spacing of the second (100) peak in
PCN was smaller than that of the d-spacing of the tris-s-triazine unit of CCN as 0.713 nm. PCN
10

peaks were narrow and less-intense than CCN peaks, because of higher-polymerization, higher-
porosity, smaller-crystalline domains, structural defects such as curling of nanosheets, and
irregular repetition between graphitic layers. These factors could cause the higher catalytic
activity of PCN. So, XRD spectra have confirmed a typical of g-C3N4 in PCN and CCN samples
as reported by Wang et al. [19]
PCN
CCN
(002)
750
600
450
300
150
0
(100)
15
30
45
60
75
2 (Deg.)
Fig. 1. XRD patterns of PCN and CCN
FTIR spectroscopy was used to detect the functional groups in the samples. [20] FTIR
spectrum of PCN and CCN are displayed in Fig. 2. CCN spectrum showed peaks at 3099, 1620,
11

1536, 1397, 1313, 1229, 874 and 800 cm-1. In contrast, the PCN spectrum showed a shift to
higher wavenumber numbers for the peaks at 1627, 1558, 1410, 1320, and 1236 cm-1 as
compared to corresponding CCN peaks. The higher shift in these PCN peaks positions may be
attributed to the curled with porous structured nanosheets in PCN but planar with the crystalline
structure in CCN. Broadband at 3099 cm-1 in the CCN spectrum and 3180 cm-1 in the PCN
spectrum were assigned to stretching-vibrational modes of incompletely condensed NH2 and NH
bonds. This peak broadness may be attributed to water coordination with free N-H bonds.
However, this peak shift to higher-wavenumber was assaigned to more uncondensed N-H bonds
in PCN. Several bands in 1200 – 1700 cm-1 showed the characteristics of aromatic stretching
modes of CN in aromatic heterocycle. The CCN peak at 1620 cm-1 and the PCN peak at 1627
cm-1 was assigned to NH bonds of the primary amine and C=N heterocycle. The higher shift of
22 cm-2 in the PCN peak (1558 cm-1) as compared to CCN (1536 cm-1) of stretching vibration of
repeated units of triazine may be assigned to curling structure of nanosheets in PCN. The small
band at 881 cm-1 for PCN and 874 cm-1 for CCN in the fingerprint region showed the out-of-
plane C-H bonds in the aromatic domain. Peak around 800 cm-1 in CCN and 804 cm-1 in PCN
spectra were corresponded to breathing vibrational modes of the s-triazine ring. All these
findings have confirmed graphitic like structure in both PCN and CCN samples.
12

0.48
0.36
0.24
0.12
0.00
s-triazine ring
1200-1700
PCN
CCN
800
C-H
CN in aromatic heterocycle
N-H
700
1400
2100
2800
3500
Wavenumber (cm-1)
Fig. 2. FTIR spectrum of PCN and CCN
The effect of nanostructuring on optical properties like absorbance-edge and bandgap
was determined by scanning UV-Vis DRS spectra of the samples, and resultant spectra are
displayed in Fig. 3. All prepared samples have shown intrinsic semiconductors like absorption in
the blue region of the visible light spectrum. Absorption edges for PCN and CCN were
determined as 450 and 461 nm with corresponding band gaps of 2.75 and 2.69 eV using equation
Eg = 1240/, [21] where Eg the bandgap and  the absorption cut off of the semiconductor
nanomaterials. Less-connected planar structure, the disorder in smaller crystalline domains, and
13

quantum confinement effect were considered as the main reasons for the higher bandgap of PCN
as compared to CCN.
Fig. 3. UV-Vis DRS spectra of PCN and CCN
The texture and morphology of PCN and CCN were determined through the SEM. [22]
The SEM images in Fig. 4 show the morphological and structural differences between PCN and
CCN. PCN exhibited a fluffy-like structure with a small and irregular dense-texture as compared
to CCN texture. These fluffy nanosheets were connected in such a way that they have left a small
14

hollow space between them. In comparison, CCN exhibited a uniform surface texture, and g-
C3N4 crystalline sheets were looked like large-sized aggregates. A rocky and flowery surface of
CCN was comprised of irregular flakes with an edge size of several nanometers. SEM images
showed a structure in the prepared samples, which may be exfoliated to layers and nanosheets by
ultra-sonication.
Fig. 4. SEM images of PCN and CCN
EDX analysis was used to find the purity and elemental composition of the samples. [23]
EDX patterns of the PCN and CCN are displayed in Fig. 5. Carbon (C) and nitrogen (N) peaks in
the EDX spectrum existed at their corresponding energy (KeV), which confirmed the g-C3N4 in
both PCN and CCN. Except for C and N, no other elemental peak was observed in the spectrum
of PCN and CCN, which showed that no impurities were present in synthesized samples.
15

Fig. 5. EDX spectra of PCN and CCN
Table 1 shows the elemental composition in weight percentage (%) and the atomic
percentage for PCN and CCN samples. C/N stoichiometric atomic ratio was calculated as 0.85
and 0.71 for PCN and CCN. The high deviation of the C/N ratio of PCN from the theoretical
value of 0.75 has confirmed a non-perfect structure of the framework in PCN. Low C/N ratio of
CCN from the theoretical C/N ratio of 0.75 can be related to the defects hindered charge
separation and low charge transport property. [24]
16

Table 1. Weight (%) and atomic (%) of different elements present in the prepared samples
Sample
PCN
Elements
Weight (%)
Atomic (%)
C/N ratio (atomic)
C
N
C
N
41
59
36
64
45
53
40
56
0.85
0.71
CCN
The textural-properties, specific surface area, and pore size distribution of PCN and CCN
were investigated by the nitrogen adsorption isotherm method. [25] Fig. 6 displays the nitrogen
adsorption isotherm in the relative pressure (P/Po) range of 0.01 - 1 for the prepared samples.
Higher nitrogen adsorption of PCN as compared to that of the CCN showed the mesoporous
morphology in PCN. Inset of Fig. 6 shows narrow pore-size-distribution curves of the as-
prepared PCN and CCN samples in the mesoporous range from 20 to 200 Å. Large pores size of
about 30 Å and 120 Å in the BJH pore-size-distribution curve of PCN has confirmed nanopores
and mesopores in PCN.
17

PCN
CCN
8
6
4
2
0
40
80
120
160
200
Pore Width (Å)
0.0
0.2
0.4
0.6
0.8
1.0
Relative Pressure (P/Po)
Fig. 6. Nitrogen adsorption isotherms (77K) and BHJ pore size distribution of PCN and CCN
A summary of the comparison of the specific surface area (SBET), total pore volume, and
pore width of PCN and CCN are listed in Table 2. PCN exhibited greater SBET (89.9 m2/g) than
that of CCN (11.8 m2/g). This enlarged surface area can be attributed to porous, thin, and curled
nanosheets in fluffy PCN as compared to crystallized, planar, and large layered nanosheets of
CCN. Urea was decomposed into more bubbles as compared to melamine during the thermal
polymerization process, which can form the porous and fluffy structure in PCN. A larger surface
area of the PCN showed that catalysts might offer more reactive sites for the reactions between
18

H2O2 and RhB, which can be expected to improve the catalytic activity of PCN. BJH adsorption
volume of pores for PCN and CCN were calculated as 0.30 cm3/g and 0.038 cm3/g. The higher
BJH adsorption cumulative volume of pores has confirmed porosity in PCN.
Table 2. The SBET and pore properties of PCN and CCN
Samples
PCN
SBET (m2/g)
89.9
Pore Volume (cm3/g)
Pore Width (Å)
0.3
146
178
11.8
0.038
CCN
Semiconductors have a high rate of electron-hole pair’s recombination. Recombination
rate and the effect of defects on the bandgap were determined through PL spectra. [26] PL
spectra of the PCN and CCN samples are displayed in Fig. 7. PL emission peaks at the
wavelengths (of 604 and 570 nm for PCN and CCN can be attributed to electrons-holes pair’s
recombination. The difference in PL emission peak position represented variable sub-gaps
defects distribution in both the samples. PCN peak intensity was smaller than CCN peak
intensity because of the low recombination rate, and high separation of photo-induced electrons-
holes pairs. These factors have confirmed the improved electron transferability of PCN, which
was helpful in the sensing process.
19

Fig. 7. PL spectra under an excitation wavelength of 457 nm
3.2
Catalytic Activity of the Nanosheets through Fluorescence Quenching Method
The activities of the as-prepared samples were investigated using catalytic-oxidation of
RhB. The graphitic structure shows a useful electron transfer process through the graphitic-
carbon network on the micro-level, [27] and such catalyst could reduce H2O2 by the donation and
transfer of electrons. The activity of nanosheets was measured by the oxidation of RhB dye in
H2O2 oxidant using the fluorescence quenching approach. Fig. 8A shows the pre-scan excitation
and pre-scan emission spectra of RhB dye (60 ng mL-1) in PBS (pH 7.2, 10 mM) solution at
20

room temperature. RhB solution exhibited an absorbance peak at 554 nm and an emission peak
at 577 nm under an excitation of 340 nm. Fig. 8B shows that RhB under an excitation
wavelength of 554 nm exhibited a fluorescence emission peak at 577 nm (curve a). This peak
intensity was enhanced upon the H2O2 addition (curve b) in the reaction mixture because of the
H2O2 chromophoric effect. Nanosheets and RhB incubation resulted in a dynamic equilibrium.
RhB was adsorbed and aggregated onto the surface of the nanosheets, which decreased the
fluorescence emission intensity (curves c and e) of the reaction mixture. H2O2 in reaction
mixtures of RhB with nanosheets of PCN and CCN has reduced more emission intensities
(curves d and f). This decreased intensity was because of a redox reaction between RhB and
H2O2 under the catalytic influence. H2O2 was decomposed to radical and ions. Hydroxyl radical
(•OH) was the main species [28] that oxidized chromogenic dye RhB to decrease its fluorescence
intensity. [29] The decrease in fluorescence intensity of the reaction mixture containing PCN
nanosheets (curve d) was higher than that of the reaction mixture containing CCN nanosheets
(curve f). This emission difference can be attributed to the structure and morphology of PCN and
CCN. Thin, porous, and fluffy with curled edged nanosheets of PCN have large surface areas,
charge transportability, and more catalytic sites. Because of that, PCN nanosheets have shown
better catalytic activity as compared to crystallized, flat, and planar CCN nanosheets. So, the
sensor was designed using PCN nanosheets.
3.2
Mechanism of RhB Oxidation
Trapping experiments were performed by using different trapping-agents to determine the
active-species during the oxidation process of RhB. TBA, TEOA, and BQ were added in the
reaction mixtures to explore the oxidative species. Fig. 8C shows a comparison of the catalytic-
activity of blank reaction-mixture with the catalytic-activities of different reaction-mixtures
21

containing TEOA, TBA, and BQ. Upon the addition of scavengers in the reaction-mixture of
RhB, H2O2, and PCN, the catalytic-activity of the reaction-mixtures were suppressed. Trapped
reactive-species can cause a decrease in catalytic-activities by suppressing RhB-oxidation. Most-
lowering in F577 nm was obtained for TBA containing reaction mixture, because of impairing of
•OH. Relative-catalytic-activities of the TEOA reaction mixture were slightly decreased but not
affected by BQ. These results showed that •OH radicals and holes were the active species for the
reaction between RhB and H2O2 in the PCN catalyst.
Structural properties and high surface area of the PCN catalyst could improve the
adsorption of H2O2, which could increase the dissociation of H2O2 to radicals. [30] Furthermore,
due to energy transfer between the excited RhB and PCN catalyst, electron-hole pairs were
created in the catalyst. These electron-holes pairs of PCN might activate the H2O2. The excited
electron of the PCN may also reduce H2O2 to produce hydroxyl radicals. [30, 31] The proposed
reaction mechanism of dye oxidation involved the generation of radicals. It can be expressed in
equation form as follows:
RhB + Visible light → RhB∗
RhB∗ + PCN → RhB + PCN (e− + h+)
−
•
−
( )
PCN e + H2O2 → OH + OH + PCN
PCN(h+) + H2O → OH• + H+ + PCN
2H+ + 2OH− → 2H2O
OH• + RhB → CO2 + H2
+
−
−
+
(
)
h + e → e + h Negligible Recombination Reaction
22

Fig. 8. Pre-scan of RhB under excitation at 340 nm (A), comparison of catalytic activity of PCN
and CCN nanosheets (B), Effect of scavengers on the reactive species in the reaction (C).
Reaction conditions were [RhB] = 60 ng mL-1, [PCN] = 28 µg mL-1 and [H2O2] = 2500 nM in
PBS (10 mM, pH 7.2) solution at room temperature.
3.3
Catalytic Activity of the Nanosheets through Colorimetric Method
The intrinsic-peroxidase-like activity of PCN was investigated by performing reactions at
slightly acidic conditions of sodium acetate buffer (pH 5.0). Fig. 9A shows the high intrinsic
peroxidase-like activity of PCN toward TMB oxidation in constant 400 µM of H2O2. Different
control experiments without H2O2 or PCN showed no absorption peak and no activity toward
23

TMB oxidation (300 µM). The absorption intensity of the reaction mixture of PCN was higher
than the CCN containing reaction mixture. The absorption intensity of PCN can be attributed to
the structural, optical, and electronic properties of the catalyst. Inset of Fig. 9A shows the digital
image of the blue color of the different reaction mixtures towards TMB oxidation after 1h of
reaction. The reaction mixture of PCN has shown more-blue color as compared to the rest of the
reaction mixture. This naked-eye observation also confirmed the higher peroxidase-like activity
of PCN.
The optimum concentration of PCN nanosheets is determined from Fig. 9B. The optimal
PCN amount was 28 μg mL-1. Fig. 9C shows the relationship between the peroxidase-like
activity of PCN and TMB substrate. PCN peroxidase-like activity was increased with TMB
concentration. The steady-state kinetics was used to investigate the mechanism of PCN
peroxidase-like-catalytic action with varied TMB concentrations at constant experimental
conditions. Steady-state kinetics analysis showed that the PCN catalyst followed the typical
Michaelis−Menten equation towards TMB oxidation. Different kinetics constants are determined
towards TMB oxidation at pH of 5.0 and compared with Horseradish Peroxide (HRP) enzyme
kinetics parameters in Table 3.
Table 3. Comparison of kinetics parameters of HRP with PCN towards TMB substrate
Catalyst
PCN
Km (mM)
Vmax
Ref.
0.159
1.3 (10-8 Ms-1)
Present work
0.434
10 (10-8 Ms-1)
[32]
HRP Enzyme
24

Michaelis-Menten constant (Km) of HRP was 0.434 mM toward TMB substrate, which
was higher than our PCN system parameters (0.159 mM). This Km value of PCN catalyst may
represent a strong affinity for TMB as compared to natural enzymes for TMB. Fig. 9D displays
the reaction mechanism of TMB oxidation.
Fig. 9. Absorption spectra of reaction mixtures containing H2O2, TMB, and catalytic-
nanomaterials in acetate-buffer solution (pH 5.0) after 1h of reaction time (A). Effect of PCN
concentration (B) and TMB concentration on peroxidase-like activity (C). A mechanism for
the reaction between TMB and H2O2 under the peroxidase-like PCN catalyst (D). Reaction
conditions were [TMB] = 300 µM, [PCN] = 28 µg mL-1 and [H2O2] = 400 µM in sodium
acetate buffer (10 mM, pH 5.0) solution at room temperature.
25

3.4
Optimization of Reaction Conditions
Fluorescence quenching of dye depends upon the reaction time, reaction temperature, pH,
nanosheets concentrations, and dye concentrations. These reaction conditions are optimized for
the development of an excellent sensor. [33] The ΔF577 nm represented the catalytic activity of
PCN nanosheets. The influence of reaction conditions on ΔF577 nm was determined by taking
[H2O2] as 2500 nM in the reaction mixture.
Temperature and time effect on the catalytic-activity of PCN nanosheets between 5 –
70oC and 0 – 30 min are displayed in Fig. 10A-10B. The greatest fluorescence quenching was
observed if the reaction performed at 20oC for one min. Thus, one min and 20oC of reaction time
and reaction temperature were adopted in this H2O2 assay. Hydrochloric acid and sodium
hydroxide solutions were used to adjust the pH of the PBS solution. The pH was optimized
between 3.0 – 10.0 and the effect of change in pH on ∆F577 nm are displayed in Fig. 10C. It is
cleared from Fig. 10C that the fluorescence quenching of reaction mixture occurred in neutral
and basic conditions. The ∆F577 nm was the greatest at pH of 8.0 because of the most and
constant fluorescence quenching of the reaction system. So, 8.0 was the optimal value of pH, but
later experiments were performed at neutral pH.
The efficiency of the assay is dependent upon the catalyst concentration. So, the optimal
nanosheets concentration was determined to get better assay performance. Fluorescence
quenching of reaction mixture was determined by increasing [PCN] nanosheets between 0 – 240
µg mL-1 and resultant graph are displayed in Fig. 10D. Higher [PCN] nanosheets resulted in the
lowering of fluorescence quenching signals. A significant increase in fluorescence quenching
was observed when [PCN] varied from 0 to 28 µg mL-1. Beyond 28 µg mL-1 of [PCN], there was
no significant change in the ∆F577 nm. These results showed that PCN could catalyze the reaction
26

between RhB and H2O2 systems. For the maximum quenching efficiency, 28 µg mL-1 of PCN
was enough. This low concentration of catalyst could reduce the assay cost and could enhance its
sensitivity. Thus, 28 µg mL-1 of PCN nanosheets was selected for the high fluorescence
quenching efficiency of the RhB and H2O2 systems.
The amount of dye also affects the assay performance. Effect of [RhB] on fluorescence
quenching of reaction-mixture was studied from 0 to 70 nM, and resultants graphs are displayed
in Fig. 10E. A constant fluorescence quenching of reaction mixture was found at 60 ng mL-1 of
[RhB]. At high [RhB], the catalytic activity of the catalyst was inhibited, which could result in a
decrease in the ∆F577 nm. So, 60 ng mL-1 of [RhB] was recommended for getting a high
sensitivity in this H2O2 assay.
27

50
40
30
20
10
45
40
35
30
25
20
A
C
B
D
13
26
39
52
65
0
6
12
18
24
30
Temperature (oC)
Time (min)
48
40
32
24
16
8
40
30
20
10
0
0
45
90 135 180 225
3.0
4.5
6.0
7.5
9.0 10.5
[PCN] (g/mL)
pH of PBS
E
40
30
20
10
0
0
15
30
45
60
[RhB] (ng/mL)
Fig. 10. Effect of temperature (A), time (B), pH (C), [PCN] (D), and [RhB] (E) on fluorescence
quenching response under an excitation of 554 nm. Reaction conditions were the same as
mentioned in Fig. 8.
28

3.5
H2O2 Detection using PCN Nanosheets
The influence of various [H2O2] on fluorescence quenching is required for developing the
H2O2 assay. [34] The effect of various [H2O2] on fluorescence intensity of RhB and PCN
nanosheets system in PBS solution under an excitation wavelength of 554 nm is displayed in Fig.
11A. Fluorescence intensity at 577 nm was decreased with an increase in [H2O2] from 0 to 2500
nM, reached to least intensity value, and then it was increased. The first decrease in fluorescence
emission-intensity can be related to the oxidation of RhB molecules by first [H2O2]. However,
the final increase in emission-intensity can be related to the hindrance in the catalytic-activity of
PCN nanosheets at high [H2O2]. Quenching efficiency depends on transferring electrons and
energy from PCN nanosheets to the quencher. A dose-response curve was required to find the
relationship between ΔF577 nm and [H2O2]. The dose-response curve of the developing sensor in
Fig. 11B shows that ∆F577 nm was varied with the change in [H2O2]. The ∆F577 nm was increased
with increasing in [H2O2], and the maximum ∆F577 nm was observed at 2500 nM of [H2O2].
Thus, 2500 nM of [H2O2] was chosen as the best value from this experiment. A calibration curve
was constructed at various [H2O2] by performing a series of reactions under the optimal reaction
conditions, and resultants graphs are depicted in Fig. 11C. The graphical curve was linear from
90 to 2500 nM for H2O2 with a linear-fitting-regression equation ∆F577 nm = 0.0167[H2O2] +
2.35512 and a correlation coefficient (R2) of 0.9923. The limit of detection (LOD = 3σ/K, where
sigma ( was standard-error along Y-axis and K was the slope of linear-regression fitting curve)
was calculated as 73 nM. Quantitation limit (LOQ = 10σ/K) was calculated as 220 nM.
29