Microchemical Journal 181 (2022) 107607

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Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

Amino benzene dicarboxylic acid-derived luminescent nitrogen-doped

Carbon- quantum Dots/anti-TNT antibodies conjugate for detection of
nitroaromatic contaminant in water: A comparative analysis of
chemo-Bio-sensing affinity
Sarita Devi a, Vaishali Garg b, Sachin Tyagi a, c, *
a
CSIR-Central Scientific Instruments Organization, Chandigarh 160030, India
b
University Institute of Engineering and Technology, Panjab University, Chandigarh, India
c
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India

A R T I C L E I N F O A B S T R A C T

Keywords: Water contaminants are posing deadly impacts on living beings’ survival. Nitroaromatic compounds e.g. Trini­
Nitrogen-doped carbon quantum dots trotoluene (TNT), are one among these lethal contaminants and require to be early detected before their con­
Trinitrotoluene sumption. The nitrogen-doped carbon quantum dots (NCQDs) are synthesized from the precursor 2-
Antibody
aminobenzene-1,4-dicarboxylic acid via the hydrothermal route. The as-synthesized NCQDs are conjugated
Fluorescent
Biosensing
with anti-TNT antibodies and characterized using various analytical techniques. The spherical NCQDs are having
In-situ a size of 5.69 nm. At excitation wavelength 347 nm, the emission wavelength of NCQDs is shifted from 445 nm to
443 nm after their conjugation with anti-TNT Ab. The decrease in photoluminescence emission intensity of
NCQDs and NCQDs/Ab with the increased concentration of TNT is regulated by the formation of Jackson-
Meisenheimer and antigen–antibody complex, respectively. The detection limit of NCQDs and NCQDs/Ab is
observed to be 1.39 X 10-5 M and 1.07 X 10-9 M respectively. The stern–volmer quenching constant of NCQDs is
increased from 1.5 X 103 M− 1 to 8.5 X 105 M− 1 after their conjugation with anti-TNT Ab depicting the elevated
affinity of NCQDS/Ab for TNT. The biosensing NCQDs/Ab are found to be more selective towards TNT than the
chemosensing NCQDs probe. In spiked real water samples, the RSD of NCQDs/Ab is found to be ± 1.5%.

1. Introduction for civility destruction has generated significant quantities of explosive

polluted soil and waterways. The discharge of untreated wastewater has
Water contaminants are posing deadly impacts on living beings’ resulted in elevated levels in soil and water. Due to the possible human
survival. A large number of nanomaterials have been utilized for treat­ carcinogenic effects of TNT, highly sensitive and selective TNT detection
ing the water pollutants via different techniques [1] such as Cd/Zr- before its consumption is required [8]. Another major issue with TNT is
bimetallic metal–organic frameworks for organic dye pollutants [2], Pt its possible reduction to aromatic amines, which can be highly
doped Ti/Sb-SnO2 Electrode for phenols [3], Nickel-Nitrogen-Carbon mutagenic.
Catalysts for CO2 reduction [4], nanocellulose-based composite mate­ Among the various nanomaterials, carbon-derived nanomaterials are
rials for water–oil remediation [5], glycine-modified Fe/Zn-layered found superior to the rest of all [9–15]. Carbon-quantum dots (CQDs)
double hydroxides for arsenic removal [6], and CdSe/gold nanoparticles deliver very fascinating characteristics in photostability, high quantum
composites for trinitrotoluene (TNT) [7]. Nitroaromatic compounds e.g. yield (QY), semiconducting properties, broad-spectrum, size-tunable
Trinitrotoluene (TNT), are one among these lethal water contaminants emission properties, and their chemical coupling due to their colloidal
and require to be early detected before their consumption [8]. Over the state, etc. [16]. The large surface area to volume ratio and porosity of
past few decades, owing to the advancement in explosive production at nanoparticles is another unique characteristic for various applications
ammunition plants for military purposes and dam or building explosions [17–19]. It renders them suitable for various applications such as

* Corresponding author at: Analytical Techniques Division, CSIR-Central Scientific Instruments Organization, Chandigarh, India.
E-mail address: [email protected] (S. Tyagi).

https://doi.org/10.1016/j.microc.2022.107607
Received 28 March 2022; Received in revised form 15 May 2022; Accepted 16 May 2022
Available online 21 May 2022
0026-265X/© 2022 Elsevier B.V. All rights reserved.
S. Devi et al. Microchemical Journal 181 (2022) 107607

biosensing, optoelectronics, photoelectrodes, etc. Heavy metal-based dicarboxylic acid (ABDC) in ethanol was prepared and vigorously stir­
QDs are unsuitable for bioimaging applications due to their cytotox­ red at 1000 rpm/100 ◦ C for 2 min. This solution was transferred to a
icity. This drawback can be overcome by using metal-free, highly Teflon-lined stainless steel autoclave (TLA) and heated at 180 ◦ C for 12
luminescent organic QDs i.e. carbon derived QDs (CQDs) [20]. This hrs. After overnight colling at room temperature (RT), the resultant
emerging class of CQDs is biocompatible, highly water-soluble, bright product was monitored for the PL emission property. Thereafter, it was
photoluminescence (PL), higher photostability, and easy to function­ centrifuged at 10,000 rpm/10 min. The 6 mL supernatant was trans­
alize due to the presence of edge groups on their surface [21]. These ferred in an activated dialysis membrane and dialyzed for 2.5 hrs against
properties widen their scope of applicability to cellular imaging, drug 300 mL DI water with stirring. The PL emission properties of as-prepared
delivery, catalysis, and metal-ion sensing also [22]. The PL spectrum of NCQDs were also optimized at pH 2, 4, 6, 8, and 10 using 0.1 M HCl and
CQDs can be tuned by their mean size controlled by synthesis parame­ 0.1 M NaOH.
ters such as the precursor to introducing desired functionalities at their
graphitic sheet edges [23]. The PL behavior of CQDs is mediated by their 2.2. EDC/NHS treatment of NCQDs
quantum size, electron-hole recombination, surface defects, and the
triplet ground state of carbene-like free zig-zag sites [24]. Different types The carboxylic groups present on the NCQDs surface were utilized
of synthesis approach e.g. top-down and bottom-up procedures, lead to for their conjugation with anti-TNT antibodies. The NCQDs’ volume was
the product materials possessing diverse properties rendering it difficult fixed while EDC/NHS concentration was varied maintaining the con­
to understand the exact mechanism of their PL [25]. The heteroatom stant volume. The EDC/NHS molarity was kept as1:2. Therefore, a series
doping can be strategized to tune the surface chemistry of CQDs. The of EDC (i.e. 0.5, 0.4, 0.3, 0.2, 0.1, 5x10-2, 1 x10-2, 5 x10-3, 1 x10-3, 5 x10-
functionalization of CQDs with hetero groups such as amine groups re­ 4
, 4 x10-4 and 5 x10-5 M) and NHS (i.e. 1, 0.8, 0.6, 0.4, 02, 0.1, 2x10-2,
duces the surface defects and diminishes the carboxyl groups mediated 1x10-2, 2x10-3, 1x10-3, 2x10-4 and 1x10-4 M) dilutions (5 mL each) was
non-radiative recombination of electron-hole pairs. It ends up in the PL prepared. The NCQDs dilution was mixed with EDC/NHS to achieve
enhancement and blue shift [26]. As of now, the surface passivation of NCQDs/EDC/NHS as 1:1:1 v/v. So, a total of 12 NCQDs/EDC/NHS so­
CQDs is usually performed for doping wherein their edge groups, like lutions were prepared each containing the 300 μL as-prepared NCQDs
carboxyl and hydroxyl groups, are used as reactive sites [27]. Different and 600 μL of respective molar pair of EDC: NHS::1:2 (300 μL each).
passivation reagents have been used such as polyethyleneimine, 1,2-eth­ Then, these 12 solutions were kept at RT for 30 min and ultra-
ylenediamine, branched, or PAMAM-NH2 dendrimer [28–30]. The centrifuged at 4000 rpm/5 min. The precipitate was mixed with 900
requirement of extra reagents can be surpassed by using the sole pre­ μL DI water (precipitate solution: PS). The respective supernatant and PS
cursor compound with both the hetero group and edge groups without were denoted as S1 to S12 and PS1 to PS12 respectively. All the solutions
compromising the desired properties of CQDs. 2-aminobenzene-1,4- were characterized for their UV-absorbance and PL-emission properties.
dicarboxylic acid can be explored to achieve the in-situ amine-func­ The major change was noticed between solutions 10 and 11. Therefore,
tionalized CQDs with higher QY, photostability, and PL properties. intermediate concentration dilutions were also prepared as named 10.1,
Various studies have been reported for the detection of TNT using 10.2, and 10.3 having the EDC/NHS concentrations (in molar) as 4x10-
CQDs via the chemosensing approach due to the formation of the 4
/8x10-4, 3x10-4/6x10-4, and 2x10-4/4x10-4 respectively, and repeated
Jackson Meisenheimer (JM) complex between the electron-rich heter­ the same procedure. The optical properties of all the solutions were
ogoups on CQDs and the electron-deficient aromatic ring of TNT [31]. compared with the reference NCQDs solution (300 μL as-prepared
The presence of three electron-withdrawing nitro groups on the toluene NCQDs + 600 μL DI water).
ring of TNT renders this aromatic ring to be electron-poor enough so an
easy loss of a proton from its methyl group. The hydrogen from the
methyl is exchanged with the hydrogen from the amine groups of 2.3. Preparation of Anti-TNT antibody conjugated NCQDs (NCQDs/Ab)
NCQDs. Finally, the lone pair of electrons from the primary amine
groups present in NCQDs forms the JM complex with an electron-poor Maintaining the constant volume of EDC/NHS treated NCQDs
TNT ring [31]. The antibodies are known for their high specificity for (S10.3), the antibody concentration was varied in each solution. The 25
target analytes hence, they offer highly sensitive detection. The JM μL of anti-TNT Ab dilution was added in 175 μL S10.3. The final volume
complex-based chemosensing and Ag-Ab complex-based biosensing af­ was set to 1 mL by adding 800 μL of DI water. The anti-TNT Ab stock was
finities of single sensing material i.e. CQDs are to be compared in this prepared as 1 mg/mL in 0.01 M PBS. The Ab dilutions (in 0.01 M PBS)
study. The objective of this paper is to synthesize amine-functionalized were prepared in a way that the addition of 25 μL of each one would
CQDs (NCQDs) from the precursor, 2-aminobenzene-1,4-dicarboxylic generate the final anti-TNT Ab concentration as 0.025, 0.05, 0.1, 0.2,
acid for the first time via a bottom-up synthesis approach. These 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8 μg/mL in the final solution. The reference
NCQDs are conjugated with anti-TNT antibodies (NCQDs/Ab) for S10.3 solution was prepared by adding 175 μL S10.3 and 825 μL DI
affinity-based biosensing of TNT. The biosensing efficiency of NCQDs/ water. All the S10.3/Ab-dilutions were compared with reference S10.3
Ab is compared with the chemosensing efficiency of NCQDs. The che­ solution in terms of their UV-absorbance and PL-emission properties.
mosensing is based on a decrease in the PL intensity of NCQDs in pres­ The synthesis and anti-TNT Ab conjugation procedure of NCQDs are
ence of TNT due to the formation of JM complex between the electron- represented in Fig. S1.
rich amine groups of NCQDs and the electron-deficient aromatic ring of
TNT. 2.4. TNT detection using NCQDs and NCQDs/Ab

2. Experimental The as-synthesized NCQDs and NCQDs/Ab (with 12.8 μg/mL Ab

concentration) were utilized for TNT detection. A stock 1000 ppm TNT
2.1. Synthesis of NCQDs was prepared. The TNT dilutions were prepared from this stock in such a
way that the addition of 10 μL in 1 mL of NCQDs solution will result in
The optimization and selection procedure of the final solvent and final TNT concentration as 4.4X 10-6, 4.4 X 10-5, 8.8 X 10-5, 13.2 X 10-5,
precursor reagent for CQDs is elaborated in supplementary data (Fig. S2, 17.6 X 10-5, 22 X 10-5, 26.4 X 10-5, 30.8 X 10-5, 35.2 X 10-5, 39.6 X 10-5,
Fig. S3, and Fig. S4). For this, the precursor concentration and other 4.4 X 10-4, 8.8 X 10-4, 13.2 X 10-4, 17.6 X 10-4 and 22 X 10-4 M. While the
synthesis parameters were kept constant to monitor the effect of solvent final TNT concentration in NCQDs/Ab was 4.4 X 10-8, 2.2 X 10-7, 4.4 X
only. 10-7, 6.6 X 10-7, 8.8 X 10-7, 1.1 X 10-6 and 1.32 X 10-6 M. The PL emission
For the synthesis of NCQDs, 10 mg/mL 2-aminobenzene-1,4- properties of these samples was studied and evaluated.

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S. Devi et al. Microchemical Journal 181 (2022) 107607

2.5. Selectivity of NCQDs and NCQDs/Ab towards TNT requirement of any kind of external template for their stabilization. It
renders this process unique, more economical, and beneficial for prac­
The selectivity performance of these sensing probes was evaluated tical applications. The morphological and size data as acquired from the
against TNT analogs, DNT, DNBA, NB, NP, RDX, and PETN. For this, the TEM measurements at RT is shown in Fig. 1. As shown in the TEM
same procedure was followed as mentioned above for TNT. The 4.4 X 10- micrograph (Fig. 1a), the spherical shape and well dispersed NCQDs are
6
M concentration of each analog was assessed for their effect on the PL formed. The particle-size histogram (inset of Fig. 1a) was attained by
emission intensity of NCQDs and NCQDs/Ab. statistical analysis of approx. 100 QDs, which demonstrates that the size
of CQDs lies in a range from 3.2 to 6.8 nm, with an average diameter of
5.69 nm. Hence, the smaller avg. diameter and the narrow size distri­
2.6. Analysis of TNT in spiked samples
bution of NCQDs might be owing to the high pressure and consistent
heat energy during hydrothermal treatment.
Firstly, the suspended particles were removed from the tap water
The diffused rings can be visualized in a selected area electron
using vacuum filtration through a 0.22 mm membrane. The known
diffraction (SAED) pattern which points to the poly-nanocrystalline
concentration of as-prepared TNT dilutions from TNT stock solution was
nature of the NCQDs. The calculated interplanar distances were found
subjected to the filtered tap water. Thereafter, the sensing materials,
to be 4.49 A◦ for NCQDs which is associated with the (0 0 2) plane of
NCQDs, and NCQDs/Ab were added to the TNT spiked tap water sam­
graphitized carbon. The elemental composition of NCQDs is assessed by
ples. The final TNT concentration in the spiked water samples was
EDS measurement as shown in the inset (lower) of Fig. 1b. The presence
attained as 10 X 10-8 M, 30 X 10-8 M, and 50 X 10-8 M for both the NCQDs
of nitrogen in EDS data confirmed the successful synthesis of amine-
and NCQDs/Ab sensing materials. All these samples were evaluated for
functionalized CQDs from ABDC.
the PL emission output to assess the %Recovery and %RSD (n = 3).
The optical properties of as-synthesized NCQDs are studied from the
DLS zeta sizer, RAMAN, FTIR, UV–visible, and PL emission spectro­
2.7. Quantum yield calculation photometers. The hydrodynamic diameter of as-synthesized NCQDs was
observed to be 24.57 nm (Fig. 2a). The size difference in TEM and DLS
To ensure the QY of the as-synthesized NCQDs, the reference dye data can be credited to the fact that the most compact state of particles is
solution was prepared using Quinine sulfate (QS) in 0.1 M H2SO4. The studied by TEM whereas DLS is followed in a solvated state. Different
known QY (Φ) of QS is 54% [32]. The inner filter effect was decreased by non-covalent interactions (e.g. H-bonding, van-der Waals interactions,
adjusting the absorbance of NCQDs and QS solutions < 0.1. π-πstacking, etc.) are possible between the edge groups (–COOH, –OH),
The following Equation was used to determine the QY of NCQDs: amine groups of NCQDs, and the solvent molecules. These surface
functionalities are involved in the formation of the outermost solvation
Ix Ast È x
Φx = Φst ( )( )( )2 (1) layer with the solvent resulting in the large particle size data.
Ist Ax È st
At different pH values, the surface zeta potential of NCQDs was also
where I and A correspond to the fluorescence integral intensity and monitored by DLS (Fig. 2b) to validate its stability and applicability for
absorbance, respectively. The refractive index (η) of both the solvents, different applications. It can be observed that the as-synthesized NCQDs
0.1 M H2SO4, and water was 1.33. The subscripts ‘x’ and ‘st’ are the possess a positive surface charge (+38 mV) which confirms their higher
sample and standard, QS. dispersibility in water and stability. At pH ranging from 2 to 6, the
NCQDs bear a high positive charge showing excellent stability at this pH
3. Results and discussion range. Although, beyond this pH range (>pH 6), there was a noticeable
decrement in their surface charge and so the dispersibility. It suggests
3.1. Characterization of as-synthesized NCQDs that numerous amino ion groups are present on NCQDs’ surfaces. The
oxygen-rich and cationic functionalities improved the hydrophilicity
The bright yellow-colored solution of the NCQDs was procured from and stability in an aqueous medium, manifesting these NCQDs for
ABDC via a simple hydrothermal synthesis route at 180 ℃ for 12 hrs. sensing application in aqueous systems.
The as-utilized starting material, ABDC is a cheap, easily accessible, and The defects in the NCQDs were analyzed by RAMAN spectroscopy
available source for the synthesis of highly luminescent NCQDs. More­ (Fig. 2c). In the RAMAN spectrum, two prominent peaks named the D-
over, the exploitation of ABDC for NCQDs synthesis eliminates the band at 1357 cm− 1 and the G-band at 1589 cm− 1 were observed. These

Fig. 1. a) The TEM micrograph of as-synthesized NCQDs; [Inset: the size distribution of NCQDs]; b) SAED pattern of NCQDs; [Inset: the calculated d-spacing (in A◦ )
from the SAED rings (up) and the EDS data of NCQDs (down)].

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S. Devi et al. Microchemical Journal 181 (2022) 107607

Fig. 2. a) Hydrodynamic size of NCQDs and NCQDs/Ab; b) the surface charge on as-synthesized NCQDs at different pH values; c) the RAMAN spectrum of NCQDs;
and d) the FTIR spectra of NCQDs and NCQDs/Ab.

are commonly attributed to the disordered D and crystalline G charac­ presence of different-sized NCQDs might be another reason for this ab­
teristics of the NCQDs respectively [33]. The ID/IG i.e. intensity ratio of sorption behavior.
the D-band and G-band was found as 0.92, indicating a large portion of After being dialyzed for 5 hrs, the absorbance was decreased dras­
crystalline carbon in the NCQDs structure. This data confirms the for­ tically which can be credited to the excessively crossing impurities or
mation of NCQDs in the solution. ions from the solution in the NCQDs present in the dialysate. It could be
The presence of specific bonds was recognized by FTIR spectroscopy hindering the absorption of the main material. But, the presence of a
using vibrational states in the chemical structure of the materials and rather low-intensity absorbance fingerprint of NCQDs denotes the
illustrated in Fig. 2d and S5. The broad and strong peak present at 3442 presence of monodispersed and uniformly sized NCQDs in the dialysate.
cm− 1 is due to the intermolecular hydrogen bond O–H stretching vi­ Therefore, it can be stated that the dialysis time should be lowered from
brations and N–H stretching vibrations of primary amines in CQDs 5 hrs. The next dialysis was performed for 2.5 hrs and observed for the
(Fig. S5). The medium peaks in the range 3000–2840 cm− 1 are related to absorbance spectrum of dialysate. Very refined and resolved absorption
the C–H stretching vibrations. In Fig. 2d, the absorption peak located at peaks can be observed in the absorption spectra of NCQDs dialyzed for
1631 cm− 1 is assigned to the C = C stretching of cyclic alkene in NCQDs. 2.5 hrs. Hence, the dialysis time of 2.5 hrs was finalized for the synthesis
The medium intensity peaks at 1422 cm− 1 can be assigned to the O–H of ABDC-derived NCQDS. The absorption peak in the ultraviolet region
bending vibration of the carboxylic acid in NCQDs. The medium ab­ corresponds to the absorption of the aromatic-π system which designates
sorption peak located at 1383 cm− 1 and 1121 cm− 1 are attributed to the the successful synthesis of CQDs from ABDC [34]. The absorbance
stretching vibrations of C–N of amines in NCQDs [33]. The peaks at 802 maximum at wavelength, λmax 225 nm is assigned to the π- π* transition
cm− 1 and 874 cm− 1 are derived from the di- or tri-substituted C–H of the π-orbitals of aromatic carbon C = C bond. This absorption
bending vibrations. Therefore, the FTIR data demonstrate the presence behavior in the lower UV region is caused by polycyclic aromatic hy­
of hydroxyl, carboxylic, and N-containing groups in the structure of as- drocarbon (PAHs) having large energy band gaps such as two or three-
synthesized NCQDs from ABDC. It could be due to the hydrothermal ringed PAH [35]. The absorbance peak at wavelength 258 nm in­
treatment which tends to facilitate the ABDC subjection to dehydration, dicates the n– π* transition of carbonyl (C = O) groups of CQDs. The
decomposition, polymerization, and carbonization resulting in specific absorbance peak at 345 nm shows the presence of n– π* tran­
numerous carbon skeletons. sition of C-N bond in NCQDs which confirms the formation of amine-
The light absorption and emission properties of as-synthesized functionalized NCQDs from ABDC. The unshared/lone pair of elec­
NCQDs were studied through UV–visible and PL emission spectropho­ trons containing the amine group, an auxochrome, lead to the absorp­
tometry as shown in Fig. 3. Initially, the effect of dialysis procedure and tion at a higher wavelength i.e. 345 nm. This is the NCQDs structural
dialysis time was finalized from the absorption spectral study of NCQDs derived absorbance phenomenon. Under the UV-illumination, these
(Fig. 3a). The NCQDs after centrifugation and before dialysis was NCQDs exhibited an intense blue-colored emission.
observed to show a broad absorption in the wavelength range from 250 The PL emission spectrum of NCQDs at an excitation wavelength of
nm to 440 nm. It can be possibly due to the presence of various 347 nm is shown in Fig. 3b. At λex 347 nm, the NCQDs show the highest
unreacted byproducts or impurities left even after centrifugation. The emission at λem 445 nm. The PL emission property of NCQDs as dialyzed

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S. Devi et al. Microchemical Journal 181 (2022) 107607

Fig. 3. a) UV–Vis absorption spectra of NCQDs during various synthesis steps; b) the excitation and PL emission spectra of NCQDs; the PL emission spectra of NCQDs
dialyzed for 2.5 hrs (c) and 5 hrs (d) at different excitation wavelengths.

for 2.5 hrs and 5 hrs are shown in Fig. 3c and 3d respectively. At exci­ antibodies on the surface of NCQDs. The broad absorption peaks located
tation wavelengths ranging from 330 nm to 370 nm (with 10 nm in­ at 3416 cm− 1 (Fig. S5) and 1316–1269 cm− 1 are attributed to the C-N
terval), the NCQDs (2.5 hrs) delivered the high-intensity emission at stretching vibrations in aromatic amines of NCQDs/Ab. The absorption
λem 445 nm (λex 350 nm). Conversely, the NCQDs (5 hrs) did not peaks at 1197–1091 cm− 1 are due to the C-O and C-N stretching vi­
provide a noteworthy emission. Even though, their highest emission was brations of tertiary alcohol and C-N of amines respectively. The FTIR
also observed at λem 445 nm (λex 350 nm). The PL emission data of data confirmed the successful conjugation of NCQDs with anti-TNT Abs.
NCQDs prepared at different dialysis periods is also found in complete The PL emission behavior of S10.3 (or NCQDs/EN) is monitored with
agreement with the absorbance properties. It can be stated that the an increase in anti-TNT Abs concentration ranging from 0.025 μg/mL to
dialysis time should be fixed at 2.5 hrs to achieve the highly luminescent 12.8 μg/mL Abs in the final solution (Fig. 4a). It can be discerned that at
NCQDs from ABDC. The effect of pH, time UV light irradiation, and ionic high Abs concentration the PL emission intensity was achieved back
strength of solvent on NCQDs PL property is shown in Fig. S6. while at lower Abs concentration, there was a decrease in the PL emis­
sion intensity of NCQDs/EN S10.3. From Fig. 4b, it can be perceived that
3.2. EDC/NHS optimization of as-synthesized NCQDs at lower Ab concentration there was a red shift in λem from 442 nm to
447 nm, which was realigned back to 443 nm at higher Ab concentra­
For the conjugation of NCQDs with anti-TNT Abs, the NCQDs were tion. Beyond 3.2 μg/mL, anti-TNT Ab concentration, there was satura­
initially subjected to EDC/NHS treatment. The EDC/NHS/NCQDs best tion in the emission wavelength shift. The possible reason behind this
concentration ratio was optimized to eliminate the unwanted dimin­ behavior can be the dilution factor at lower Abs concentration resulting
ishing effect of EDC/NHS on NCQDs Pl emission properties as shown in in a sharp, instant decrease in the PL emission intensity and the redshift
Fig. S7 and S8. in emission wavelength. While at higher Abs concentration, the Abs
present in the sample exceed the dilution factor and the PL intensity was
attained back. Therefore, further experimentation for TNT detection was
3.3. Characterization of NCQDs/Ab
carried forward with NCQDs/ 12.8 μg/mL Abs.

The hydrodynamic size of NCQDs was found to be increased from

24.57 d.nm to 107.09 d.nm. 3.4. TNT sensing
(Fig. 2a). The possible reason is the presence of a polymeric molecule
and antibodies on the surface of NCQDs that lead to the large increment Both the materials i.e. NCQDs and NCQDs/Ab were inspected and
in the overall hydrodynamic size of NCQDs/Ab. The FTIR spectra of compared for their TNT chemosensing and biosensing efficiency. The
NCQDs/Ab exhibited several additional peaks than those of NCQDs schematic illustration of the TNT sensing study using the respective
(Fig. 2d). Specifically, the absorption peaks at 1631–1600 cm− 1 are materials is shown in Fig. S9.
likely related to the C = C and C = O stretching of α,β-unsaturated ke­
tone. The intensified absorption peak at 1383 cm− 1 is ascribed to the 3.4.1. TNT chemosensing using NCQDs
increased C = C and C-N stretching vibrations due to the presence of For TNT detection using the NCQDs as sensing material, the PL

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S. Devi et al. Microchemical Journal 181 (2022) 107607

Fig. 4. a) The PL emission behavior of EDC/NHS treated NCQDs (NCQDs/EN) (S10.3) with different concentrations anti-TNT Abs; b) Variation in PL emission
intensity and λem of NCQDs/EN with increasing concentration of Abs; and PL emission spectra of NCQDs (c) and NCQDs/Ab (d) with an increase in TNT
concentrations.

emission spectra of as-prepared NCQDs were monitored in the presence The linear regression relation of the plot I0-I vs [TNT] ranging from
of increasing TNT concentration. The TNT concentration was varied 4.4 X 10-6 to 4.4 X 10-4 M (R2 = 0.996) as evidenced in Fig. 5a, can be
from 4.4X 10-6 M to 2.2 X 10-3 M as shown in Fig. 4c. It can be spotted presented as:
that with the increase in TNT concentration, there is a successive
ΔI = I0 − I = 7.29x105 C 4
decrease in PL emission intensity of NCQDs (λex 347 nm, λem 445 nm).
The theoretical interpretation of the TNT detection capability of NCQDs here C represents the TNT concentration in M.
in terms of detection limit (LOD), percentage quenching (Q%), While from 4.4 X 10-4 to 2.2 X 10-3 M TNT (R2 = 0.985), the rate of
Stern–Volmer constant (KSV), and the association constant (Ka) of CQDs change in PL intensity differed as:
and TNT by Benesi–Hildebrand method is performed and illustrated in
Fig. 5a, b, c, and d respectively. The Q% in the NCQDs emission intensity ΔI = I0 − I = 1.26x105 C 5
was demonstrated using the following relation [36]: Therefore, this NCQDs-based chemosensing nanoprobe delivered a
[
(I0 − I)
] very low LOD value of about 1.39 X 10-5 M. Under the optimized pa­
Q% = × 100 2 rameters, the TNT mediated quenching in the NCQDs PL emission is
I0
perfectly explicit by the stern-Volmer relation (Fig. 5c) as represented
Where I0 and I are the PL emission intensity of NCQDs in the absence here [37]:
and the presence of TNT. The resultant Q% was found to be proportional
I0
to the TNT concentration. More than 75% quenching was observed at = Ksv (Q) + C 6
1.6 X 10-3 M and 2.2 X 10-3 M TNT concentration in NCQDs solution I
(Fig. 5b). The relation between the change in PL emission intensity (I0-I) In this equation, the I0 and I stand for initial and final PL emission
at λem 445 nm was plotted against the increase in TNT concentration (in intensity of NCQDs in the absence and presence of analyte TNT
M) in NCQDs to determine the detection limit (LOD) of this NCQDs respectively; ‘Ksv’ denotes the stern-Volmer constant which represents
based chemosensing platform (Fig. 5a). The LOD was calculated using the affinity between the sensing material, NCQDs, and the target analyte
the following equation [37]: molecule, TNT (i.e. donor/acceptor affinity); ‘Q’ represents the TNT
concentration, and ‘C’ is a constant (C=~1). The linear regression
k.SD
LOD = 3 equation (R2 = 0.99) for the change in PL emission of NCQDs against
b
TNT concentration can be represented as:
Where k = 3; SD denotes the standard deviation in the variation in PL
I0
emission intensity of the blank at 445 nm; ‘b’ depicts the slope of the − 1 = 1.52x103 Q 7
I
regression line in the plot I0-I vs [TNT] in M. A definite linear decrease in
NCQDs emission intensity was witnessed in the presence of TNT. The Ksv is found to be 1.52 X 103 M− 1
in the concentration range of

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S. Devi et al. Microchemical Journal 181 (2022) 107607

Fig. 5. a) The relation between the change in PL intensity of NCQDs in the presence of TNT at different concentrations for LOD determination; b) Q% measurement of
NCQDs intensity with TNT presence; c) stern-Volmer plot between the change in PL emission intensity of NCQDs and TNT concentration; and d) Benesi–Hildebrand
plot to measure the association constant between the NCQDs and TNT.

4.4 X 10-6 M to 17.6 X 10-4 M (Fig. 5c). The association constant of the concentration, 1.32 X 10-6 M. It can be credited to the high specificity of
NCQDs with TNT is found by Benesi–Hildebrand equation (Fig. 5d) by the bioreceptor molecules, anti-TNT antibodies for its analyte.
following the equation [38]: Furthermore, the LOD of NCQDs/Ab immunoprobe respecting TNT is
also assessed using the relation between I0- I at λex 347 nm, and λem
1 1 1
= + 8 443 nm (Fig. S9b). Here, the I0 and I are the initial and final PL emission
I0 − I I0 − Ib ka (I0 − Ib )[TNT]
intensities of NCQDs/Ab in the absence and the presence of TNT. A sharp
where Ka represents the apparent association constant; I0 and Ib linear decrease in NCQDs/Ab intensity is witnessed in Fig. S9b. The LOD
stand for the PL emission intensity of NCQDs in the absence and pres­ of the NCQDs/Ab probe is also measured using the above-mentioned
ence of TNT excess, and ‘I’ represents the observed PL emission intensity equation (3) and found to be 1.07 X 10-9 M. Their linear regression
of NCQDs in the presence of different TNT concentrations. The 1/(I0-I) equation for the LOD value of the NCQDs/Ab/TNT system can be
value is plotted with 1/[TNT] and the intercept/slope value was illustrated as (R2 = 0.988):
measured to find the Ka. The final association constant is obtained as 2
ΔI = I0 − I = 5.54x108 C 9
X 103 M− 1 which depicts the high sensitivity of NCQDs for TNT.
where ‘C’ represents the TNT concentration (M). Therefore, it can be
3.4.2. TNT biosensing using NCQDs/Ab stated that the NCQDs-based biosensing probe is much more highly
Aligning to the principle of biosensing based on the formation of sensitive than that of the NCQDs-based chemosensing nanoprobe.
antigen–antibody complex, the TNT detection efficiency of anti-TNT Abs The Ksv and Ka of NCQDs/Ab in the presence of TNT at λex 347 nm
conjugated NCQDs-based immunosensing probe was explored. Sustain­ and λem 443 nm are also measured and shown in Fig. S9c and S9d,
ing the same, it can be perceived from the Fig. 4d, that the PL emission respectively. The Ksv is measured using the above mention equation (6)
intensity of NCQDs/Abs is found to decrease sequentially with the in­ and the linear regression relation can be presented thereof as (R2 =
crease in TNT concentration from 4.4 X 10-8 M to 1.32 X 10-6 M at λex 0.99):
347 nm, λem 443 nm. Consequently, the increased Q% in the PL emis­
I0
sion intensity of NCQDs/Abs (at λex 347 nm) with increased TNT − 1 = 8.6x105 Q 10
I
amount was also measured using equation (2) and is delivered in
Fig. S9a. The net Q% is observed to be increased from about 7.8% at 4.4 here the I0 and I are also the initial and final PL emission intensities
X 10-8 M TNT to 80 % at 1.32 X 10-6 M TNT. Superior to the TNT of NCQDs/Ab in the absence and the presence of TNT. The resultant Ksv
mediated quenching in NCQDs emission of about 75% at 1.6 X 10-3 M was found to be 8.65 X 105 M− 1 which is very high (hundred times) than
TNT, here about 80% quenching is reported even at the lower TNT that of the NCQDs/TNT system. It depicts the higher affinity of NCQDs/

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S. Devi et al. Microchemical Journal 181 (2022) 107607

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