Novel Metal-Free Fluorescent Sensor Based on Molecularly Imprinted Polymer N-CDs@MIP for Highly Selective Detection of TNP.

In this article, we designed a fluorometric sensor based on nitrogen-passivated carbon dots infused with a molecularly imprinted polymer (N-CDs@MIP) via a reverse microemulsion technique using 3-aminopropyltriethoxysilane as a functional monomer, tetraethoxysilane as a cross-linker, and 2,4,6-trinitrophenol (TNP) as a template. The synthesized probe was used for selective and sensitive detection of trace amounts of TNP. The infusion of N-CDs (QY-21.6 percent) with a molecularly imprinted polymer can increase the fluorescent sensor sensitivity to detect TNP. Removal of template molecules leads to the formation of a molecularly imprinted layer, and N-CDs@MIP fluorescence response was quenched by TNP. The developed fluorescence probe shows a fine linear range from 0.5 to 2.5 nM with a detection limit of 0.15 nM. The synthesized fluorescent probe was used to analyze TNP in regular tap and lake water samples.

Introduction

Natural pollution has become more prevalent in recent years, and this problem now poses a serious threat to the public. As a result, enrichment and identification of toxic mixtures in the atmosphere are critical for human safety and well-being.[1,2] 2,4,6-Trinitrophenol (TNP), 2,4-difluoronitrobenzene, 2,4,6-trinitrotoluene, bisphenol-A,[3]m-nitrobenzene (m-DNB), 2,4-dinitrotoluene, and other nitroaromatic compounds are considered to be the most hazardous pollutants;[4] picric acid [TNP–TNP] is one of the ecological toxins because of its high-water dissolvability, poor biodegradability, and toxicity.[5,6] TNP is a typical reagent, a delegate of nitroaromatic compounds, and it is used to make firecrackers, amazing explosives for landmines, a fungicide in clinical and rural settings, a material intermediate in the synthetic industry, color, and narcotics. TNP has the potential to have a major impact on human health by causing severe skin inflammation, dizziness, and other serious medical problems.[7,8] TNP can be detected using a variety of methods, including surface plasmon reverberation-based immunosensors,[9] gas chromatography,[10] infrared and Raman spectroscopy,[11] liquid chromatography,[12] proton transfer reaction - mass spectrometry,[13] a fluorescent method,[14] and an electrochemical method,[15] based on observations of its inherent hazards. Among these methods, the fluorescent method has proven to be one of the most impressive because it is portable, sensitive, and simple.

Copolymerization of functional monomers and cross-linkers in the presence of a template molecule produces molecularly imprinted polymers (MIP), which are specially designed synthetic receptors.[16] After the template is removed, binding sites are exposed, allowing the target molecule to be bound with high specificity and affinity.[17] MIP are permeable materials, and molecular imprinting technology (MIT) is commonly used for selective enrichment and to make it easier to prepare MIPs with their unique identification sites by remembering the shape, size, and functional groups of templates.[18] MIT is one of the most effective techniques for synthesizing artificial recognition systems using a simple, inexpensive, and effective template polymerization technique.[19] The resulting MIP have been used as a selective material for adsorption of environmental analytes from complex samples due to their enhanced selectivity, structure predictability,[20] thermal stability in acidic and simple environments,[21] and ability to recognize target analytes.[22] MIP can be synthesized using several methods, in which most researchers are using bulk polymerization and precipitation polymerization to make conventional MIPs.[23] In addition to their traditional use in the areas of separation and enrichment from complex samples, MIPs are widely used for molecular accreditation to develop important and selective evaluation sites such as fluorometric evaluation methods, electrochemical evaluation methods, surface plasmon resonance,[24] and quartz crystal microbalance assay methods.[25] The infusion of carbon dots (CDs) with MIP has been widely applied because of the high sensitivity of CDs and high selectivity of MIP.[26]

Semiconductor quantum dots (QDs) and their peculiar fluorometric properties have received a lot of attention in recent years. However, due to the toxic nature of the materials used in their manufacture, the applications of these types of QDs are limited.[27,28] CDs have attracted a lot of attention recently because of their superior properties to traditional semiconductor QDs.[29−31] CDs are a form of carbon nanomaterial that has been widely used as a fluorescent material for the identification of multiple analytes, such as contaminants in the environment.[32,33] CDs have received a lot of attention because of their outstanding properties, such as fluorescence, good stability, ease of synthesis, biocompatibility, low toxicity, easy surface functionalization, cost feasibility, safety, and environmentally friendly nature.[34,35] CDs can be made using a variety of techniques such as chemical and thermal oxidation of carbon compounds,[36,37] graphite laser ablation,[38] one-step microwave synthesis,[39] magnetic hyperthermia,[40] hydrothermal processes,[41] and electrochemical oxidation.[42] CDs have traditionally been used to identify various analytes like metal ions and pollutant drugs because they can act as an optical sensor or sensor material.[43] They have also been used in pharmaceutical and environmental studies as a fluorophore. In this work, Cissus quadrangularis (C. quadrangularis), which is a perennial herb which belongs to the Vitaceae family, is used as a carbon precursor. This medicinal plant can be found in a variety of locations throughout India. The phytochemical constituents of C. quadrangularis are steroids, flavonoids, triterpenoids, Vitamin C, stilbene derivatives, and so forth.[44] The stem of C. quadrangularis is traditionally used for the treatment of gastritis, bone fractures, skin infections, constipations, eye diseases, piles, anemia, asthma, irregular menstruation, burns, and wounds.[45] Due to the availability of rich bioactive components, C. quadrangularis extracts are widely used for many biological activities, such as analgesic, antimicrobial, antioxidant, anti-inflammatory, and antipyretic. Besides the above merits, C. quadrangularis stem extract was selected as a novel carbon precursor for the synthesis of nitrogen-passivated carbon dots (N-CDs). On the other hand, MIPs are not sensitive enough to adsorb target molecules. As a result, carbon dots have been infused with MIP to improve selectivity, sensitivity, and anti-interference abilities.

Using a reverse microemulsion method, the author Wang et al. produced CD/Fe3O4@MIP magnetic fluorescent composite material where CDs and Fe3O4 act as a co-nucleus and MIPs as unique recognition sites.[21,46] The current study highlights on the preparation of CDs with various excitation and emission wavelengths via a hydrothermal method. MIPs and NIPs based on synthesized CDs were formed using the reverse microemulsion method. The process on the whole is simple, step-by-step polymerization with no chemical decomposition of target molecules, which makes it more meritorious when compared to other methods. To study N-CDs, N-CDs@MIPs, and N-CDs@NIPs, spectroscopic techniques such as transmission electron microscopy (TEM), energy-dispersive system, Fourier transform infrared (FT-IR), X-ray diffraction (XRD), UV–vis, fluorescence spectroscopy, Raman, scanning electron microscopy (SEM), and thermogravimetric analysis (TGA) were utilized. For selective and sensitive detection of TNP, the composite was used due to the specific recognition capacity of MIP and the enhanced sensitivity of CDs. Finally, the prepared composite N-CDs@MIP was able to detect TNP from real water samples in a short period of time.

Results and Discussion

Characterization of the N-CDs, N-CDs@MIP, and N-CDs@NIP

The detailed morphology of N-CDs, N-CDs@MIP, and N-CDs@NIP was distinguished by TEM. The TEM images of N-CDs are given in the Supporting Information (Figure S1). The N-CDs@MIP TEM images with lattice fringes presented in Figure S2a,b show the quasi-spherical structure. As envisioned, N-CDs@MIP has a large particle size than the N-CDs after the polymerization. This indicates that N-CDs have been coated on the MIP successfully. Additionally, Figure S2c depicts the selected area electron diffraction outline of the N-CDs@MIP, which confirms the amorphous character of the composite by the formation of clear circles.

It is exemplified from N-CDs@MIP and N-CDs@NIP SEM images (Figure a,b); a thin and uniform distribution of the silica layer formed on the surface of N-CDs is one of the benefits of the reverse microemulsion method. Furthermore, the variation in chemical composition was analyzed using energy dispersive X-ray (EDX) spectrometry. In Figure c, the emergence of carbon (24.64%), oxygen (41.47%), nitrogen (8.00%), and silicon (25.89%) signals show that these are the main elements in the N-CDs@MIP. EDX mapping images of the N-CDs@MIP were taken to evidently assess their assembly structure. The results of the mapping are shown in Figure d.

Figure 1

(a,b) SEM image of N-CDs@MIP and N-CDs@NIP, (c) EDX spectrum of N-CDs@MIP, (d) elemental mapping of N-CDs@MIP.

N-CDs@MIP and N-CDs@NIP are light yellow owing to excellent dispersibility in aqueous solution. To ensure the hydrophilicity of N-CDs@MIP, the contact angle measurement with water was conducted. From Figure a, we see that the N-CDs@MIP shows greater contact angle on comparing with N-CDs@NIP as depicted in Figure b. The mean contact angles of N-CDs@MIP and N-CDs@NIP are 64.8°[47] and 6.5°, respectively. This result shows that N-CDs@MIP is hydrophilic due to the presence of amino groups in the surface of the composite.[48]

Figure 2

Contact angle of (a) N-CDs@MIP and (b) N-CDs@NIP.

To unearth the successful chemical modifications in each synthesized procedure, the FT-IR spectra of N-CDs, N-CDs@MIP (after the removal of template), and N-CDs@NIP were obtained and are presented in Figure , respectively. Based on the observations, there occurs a strong and broad absorption peak at 3339 cm–1 which belongs to stretching vibrations of −OH and −NH groups.[19] The peak at 1640 cm–1 is related to C=C stretching of the aromatic hydrocarbons (sp2 carbon). The bands at 1473 and 1341 cm–1 are assigned to C–N and C–H stretching vibrations, respectively. As a result, FT-IR spectra show that hydroxyl and amine groups are present on the surface of the synthesized N-CDs. The peak at 1052 cm–1 corresponds to asymmetric stretching vibrations of the Si–O–Si (siloxane) group.[49] The bands at 759 and 432 cm–1 show the existence of Si–O vibrations and Si–O–Si bending vibrations. Thus, the characteristic peaks which appeared indicate that the MIP is successfully grafted on N-CDs. It is obvious that the N-CDs@MIP and N-CDs@NIP FTIR spectra show similar absorption peaks, indicating that the polymer has been effectively synthesized and the template molecule had been removed.

Figure 3

FT-IR spectra of (a) N-CDs, (b) N-CDs@MIP, and (c) N-CDs@NIP.

X-ray photoelectron spectroscopy (XPS) was utilized for surface elemental analysis of the N-CDs@MIP. Figure a depicts the percentage of elemental composition of C, N, Si, and O, and Figure b displays the survey spectrum of N-CDs@MIP which shows four noticeable peaks with the binding energies of 285.2, 399.1, 101.9, and 531.8 eV that correspond to the C 1s, O 1s, Si 2p, and N 1s signals, respectively.[50] This demonstrates that the surface of the N-CDs@MIP was fundamentally made out of carbon, oxygen, silica, and nitrogen. The high-resolution spectrum of C 1s in Figure c exhibits four peaks at 284.7, 285.5, and 288.9 eV which are assigned to C=C/C–C, C–N, and C=O, respectively. Despite the two peaks of O 1s (Figure d) at 532.6 and 533.8 eV ascribed to the existence of C=O and C–O. Figure e depicts the convoluted spectrum of Si 2p at 102.7 eV due to Si–O vibrations. Furthermore, as shown in Figure f, two peaks exist in the N 1s spectrum at 401.9 and 399.47 eV which are attributed to C–N and N–H bonds, respectively. This information was in accordance with the FT-IR results, and it further confirms that the nitrogen atom has been effectively passivated into CDs. All peaks indicated that functionalized N-CDs@MIPs were generated successfully.

Figure 4

(a) Elemental composition of N-CDs@MIP, (b) XPS survey spectrum of N-CDs@MIP, and deconvoluted spectra of C 1s (c), O 1s (d), Si 2p (e), and N 1s (f).

The XRD profile in Figure shows the nature of N-CDs@MIP and N-CDs@NIP. Furthermore, the XRD patterns of the MIPs and NIPs were alike with a broad hump around 2θ = 21.4° degree corresponding to the plane C(002) which can be clarified by their comparative chemical compositions, and it also imitates the amorphous nature of N-CDs.[51] Another small peak appeared at 43.5° related to the C(100) plane which represents the spacing of graphitic lattice carbon.

Figure 5

XRD pattern of N-CDs@MIP and N-CDs@NIP.

The thermal behavior of N-CDs@MIP and N-CDs@NIP was analyzed utilizing TGA in a stream of nitrogen. As shown in Figure , the thermogravimetric curve of N-CDs@MIP was almost the comparable as that in N-CDs@NIP signifying the complete expulsion of template molecules in the MIP layer. The initial weight loss from 25 to 100 °C corresponds to the loss of water molecules. The first thermal decomposition occurs at 200 °C, which was due to the presence of ethanol in the N-CDs@MIP during the template removal.[52] Nearly 40% of weight loss occurred because the material has been decomposed and the mass of polymers has been diminished forcefully from ∼400 to ∼450 °C due to the breakage of the carbon skeleton of the polymers. A further increase in temperature shows no weight loss for both the N-CDs@MIP and N-CDs@NIP.[53]

Figure 6

TGA curve of N-CDs@MIP and N-CDs@NIP.

Raman spectrum of N-CDs@MIP is shown in Figure S4. The D band at 1353 cm–1 is identified as sp3 hybridized vibrations, and the band at 1545 cm–1 represents the G band due to sp2 hybridized vibrations. To determine the degree of disorder, the Raman intensity ratio is utilized, which can be calculated using the formula ID/IG. In our present work, the ID/IG ratio is 1.03 which shows that N-CDs@MIP is amorphous in structure.[54]

Figure S5a shows the absorbance spectrum of the as-prepared N-CDs in aqueous medium and extract of C. quadrangularis which serves as the carbon source which was estimated in the range of 200–800 nm using the UV–vis spectrum. The adsorption peak of C. quadrangularis stem extract showed up at 266 nm, indicating the π–π* transition of polyphenols in the extract.[55] The peak appearing around 271 nm is related to the π–π* transition of aromatic conjugated C=C, and the shoulder peak at 310 nm is due to the C=O chromophore of different functional groups in the structure of the N-CDs.[56] The inset figure shows the blue luminescence of synthesized N-CDs when they are irradiated under UV–vis light at 365 nm.

The fluorescence spectral response for N-CDs is shown in Figure S5b. To confirm, the N-CDs show excitation at 320 nm and an emission wavelength of 408 nm. As per the spectrum in Figure S5c, the fluorescence intensity increases gradually at 320 nm and it starts to reduce under the excitation wavelength λext range of 335–425 nm due to the shifting of wavelength toward the bathochromic shift.[57] The maximum emission peak appeared at λem 408 nm with an excitation wavelength (λex) of 320 nm with a maximum concentration of 100 μL as shown in Figure S5d.

The fluorescence spectra of N-CDs@NIP (black line), N-CDs@MIP before TNP addition (red line), and N-CDs@MIP after TNP addition (blue line) are shown in Figure . It can be seen that the excitation wavelength is maximum at 320 nm with an emission wavelength around 408 nm. Further experiments will proceed with these values. The fluorescence response of N-CDs@MIP without TNP is stronger when compared with N-CDs@NIP.

Figure 7

Fluorescence spectra of N-CDs@NIP (black line), N-CDs@MIP (red line), and N-CDs@MIP after the addition of TNP (blue line).

Mechanism of N-CDs@MIP for TNP

There are two techniques used to formulate passivated CDs infused with MIP, the StÖber and reverse microemulsion methods.[19,54] The StÖber method is more difficult than the reverse microemulsion method, and the distribution of particle size cannot be controlled with the exactness of the reverse microemulsion method.[58] Hence, in our current work, we made use of the reverse microemulsion method. Here, cyclohexane as a continuous phase and Triton X-100 as a surfactant are used to stabilize the microscale water droplets which cover the fluorescent N-CDs@MIP. Due to the adsorption of hydrolyzed tetraethoxysilane (TEOS) in the cyclohexane/water interface, using ammonia as a catalyst initiates the formation[59] and later development, and MIPs were obtained with the copolymerization of the functional monomer and cross-linker on the outer layer of N-CDs. The formation of imprinted sites is displayed in Figure . According to Figure , template molecules interact through the hydrogen bond and van der Waals force interaction with functional monomer 3-aminopropyltriethoxysilane (APTES). The template molecule can be rebound to N-CDs@MIP by means of noncovalent interactions after expulsion of template molecules which are responsible for fluorescent nature of N-CDs@MIP.

Figure 8

Formation of imprinted sites by N-CDs@MIP.

Conditions for Optimization

Optimization of the following parameters such as (a) pH value, (b) ionic strength, and (c) solvent for N-CDs@MIP dispersion is the best way to perceive the optimal detection condition for TNP with no potential inferences. These optimization conditions yield excellent results. The fluorescence intensity of the N-CDs@MIP can be influenced greatly by the values of pH, which is fundamentally because the pH value affects the charge of the template molecule as well as the association of N-CDs@MIP and the template (TNP). According to Figure a, the sensor response for the corresponding template molecule is at pH = 6.0, and this optimum pH value is chosen for further experimentations. Low pH values lead to a lack of hydrogen bonds between template molecules and APTES which decreases the sensor response.[43] Likewise, the sensitivity response decreases due to the occurrence of surface defects at high pH values.[19]Figure b shows the effect of ionic strength on the fluorescence intensity of N-CDs@MIP. Insignificant changes were observed by adding different concentrations of NaCl solution. This result depicts that the fluorescence intensity remains stable at different ionic strengths. Therefore, NaCl is not needed in further experiments. The results obtained from Figure c show that the N-CDs@MIP response is high while dispersing in water compared to other solvents. The sensitivity of N-CDs@MIP will be reduced in a lower amount due to the signal to noise ratio.[19]

Figure 9

Fluorescence intensity of the N-CDs@MIP based on the effect of pH (a), effect of concentration of NaCl (b), and effect of solvent (c). (d) FL response of N-CDs@MIP in the presence of TNP and metal ions.

N-CDs@MIP Fluorometric Sensing

To investigate the fluorescence quenching ability, sensitivity, and linear dynamic ranges of N-CDs@MIP and N-CDs@NIP, various concentrations of TNP ranging from 0 to 100 μM are introduced, and fluorescence intensities were recorded under finest conditions (excitation wavelength is 320 nm, emission wavelengths is 408 nm). By increasing the concentration of TNP, the intensity of the N-CDs@MIP decreases gradually; this is envisioned in Figure a. This aspect can be credited due to the distinct engraved cavities delivered by TNP in N-CDs@MIP in the course of the synthesis process. Because of vague adsorption of TNP on the outer layer of N-CDs@NIP, TNP additionally can be adsorbed on composite material, and it has some quenching effect on the fluorescence of N-CDs@NIP.[59]

Figure 10

(a) Fluorescence spectra of N-CDs@MIP with different concentrations of TNP, (b) resolution of fluorescence against TNP concentrations, (c) linear relationship between the fluorescence intensity and TNP concentration (0–2.5 nM), and (d) FL spectra of N-CDs@MIP with TNP and coexisting compounds. Error boxes display the standard deviation for three individual tests.

The Stern–Volmer equation (eq ) is used to describe the relationship between the fluorescence intensity and concentration of the template molecule in this system.where is the initial fluorescence intensity before the addition of the template, is the fluorescence intensity with the addition of the template, is the Stern–Volmer quenching constant, and is the concentration of TNP. The calibration plot (Figure b,c) from the Stern–Volmer equation infers that the N-CDs@MIP shows a good linear response toward TNP obtained with the concentration range from 0 nM to 100 μM. The regression equation for N-CDs@MIP is F0/F = 0.9841 + 0.0735[c], and 0.9985 is the correlation coefficient. The limit of detection (LOD) is found to be 0.15 nM. Simultaneously for N-CDs@NIP, F0/F = 0.9639 + 0.0243[c] is the regression equation and the correlation coefficient is 0.9787 with a LOD of 1.7 μM as shown in Figure . The LOD of N-CDs@NIP is significantly higher than that of the N-CDs@MIPs. These results show that N-CDs@MIPs are highly sensitive to TNP and can be reliably used for the determination of TNP. In Table , the comparison table for the detection of TNP is displayed.

Figure 11

(a) Fluorescence spectra of N-CDs@NIP with different concentrations of TNP, (b) resolution of fluorescence against TNP concentrations, (c) linear relationship between the fluorescence intensity and TNP concentration (0–8 nM), and (d) FL spectra of N-CDs@NIP with TNP and coexisting compounds. Error boxes display the standard deviation for three individual tests.

Table 1

Comparison Table for TNP Detection with Reported Methods

materials	linear range	detection limit (nM)	references
N-GQDs	0–4 μM	420	(50)
Tb-CDs	500 nM–100 μM	200	(60)
probe HBN	0–45 μM	57	(61)
P doped CDs	0.2–17.0 μM	16.9	(62)
Zn(II)-MOF	0–25 μM	4.47	(63)
N-CDs@MIP	0.5–2.5 nM	0.15	current study

In order to appraise the selectivity of the imprinted material, the imprinting factor (IF) was calculated. The IF can be determined as the proportion of MIP and NIP. The IF was calculated to be 3.05, which proves that the quenching efficiency is enlarged for the spectral sensitivity of TNP. The obtained results reveal that N-CDs@MIP is more selective for TNP.

Selectivity of N-CDs@MIP

The selectivity of N-CDs@MIP was determined for TNP, phenolic compounds, nitro compounds, and heavy metal ions such as Fe2+, Cu2+, Co2+, Ni2+, and Mn2+ ions for quenching N-CDs@MIP using a similar concentration of each substance. The fluorometric response of N-CDs@MIP is greater for TNP when compared with that for other phenolic and nitro compounds as shown in Figure d. Even the fluorometric response of N-CDs@NIP has no variations for those compounds. N-CDs@MIP fluorescence has to be explored with metal ions because it may interfere in the detection.[64]Figure d shows that no interference of metal ions was present for N-CDs@MIP as well as N-CDs@NIP. From this, it is evident that N-CDs@MIP has greater potential in practical application of detecting TNP in real water samples.

Application of N-CDs@MIP in Real Water Samples

To authenticate the applicability of the designed fluorescent probe, N-CDs@MIP was utilized for TNP detection in regular tap water and lake water. The preserved samples were heated in order to eliminate the chlorine content. After that, samples were spiked with standard addition of TNP in the concentration range of 2–6 nM for recovery studies. Various concentration ranges of TNP are displayed in Table ; from this it is evident that the recovery range is 95.5–100.8%. This indicates that the accuracy of N-CDs@MIP in detecting TNP is prompt in real water samples.

Table 2

2,4,6-Trinitrophenol Detection in Regular Tap Water and Lake Water

sample	spiked (nM)	detected ± SD	recovery (%)
		TNP	TNP
tap water	2	1.91x ± 0.37y	95.5
	4	4.03x ± 0.44y	100.8
	6	5.93x ± 0.31y	98.9
lake water	2	2.01x ± 0.36y	100.5
	4	3.99x ± 0.44y	99.8
	6	5.89x ± 0.82y	98.2

Conclusions

In this research work, fluorescent N-CDs@MIP composite material was successfully synthesized by a reverse microemulsion method using N-CDs as fluorophores and MIPs as binding sites for sensing TNP. This approach has the advantages of being less expensive, simple, self-supporting of organic diluent, convenient, and a low LOD. The synthesized N-CDs@MIP shows a good linear range with a 0.15 nM LOD for TNP. These fluorescent probes were applied to detect TNP in real water samples. From the recovery values, it is evident that N-CDs@MIP can selectively monitor and detect trace amounts of TNP; this shows that it can also be utilized in ecological monitoring and evaluation fields.

Experimental Section

Reagents and Materials

All materials utilized in the current investigation were of scientific quality and were utilized without refining. Deionized water (DI) was used for the purpose of package and solution preparation. TNP (, >98%), p-nitroaniline (>98%), Triton X-100, m-nitroaniline (>98%), hydroquinone (>99%), APTES, catechol (>98%), TEOS, cyclohexane, n-hexanol, ammonia solution, sodium chloride, copper(II)sulfate, manganese(II)acetate, ferrous sulphate, cobalt(II)acetate, nickel(II)sulfate, acetic acid, acetonitrile, acetone, methanol, and ethanol were acquired from Southern India Scientific Corporation (SISCON). A laboratory centrifuge Remi R-20C (8 × 50 mL), microsample tubes, a magnetic stirrer, a polytetrafluoroethylene-coated stainless-steel autoclave, and glasswares were acquired from Vijaya Scientific Company.

Characterization

A Fluoromax-4 spectrometer (HORIBA JOBIN YVON) was used for obtaining fluorescence spectra. To study the functional groups, FT-IR spectra (at 4000–400 cm–1) were obtained on an ALPHA-T-FT-IR spectrometer. A double-beam UV–vis spectrophotometer (LI-2800, Lasany) was used for recording adsorption spectra, and the scanning range is 200–800 nm. The average diameter and morphology were measured utilizing HRTEM (JEOL JEM-2100). HRTEM images with the help of ImageJ software were analyzed for particle size distribution analysis of C-dots. FESEM by an FEI Quanta FEG200 was performed for determining the surface structure. XRD was carried out in the range of 0–80° for obtaining wide-angle patterns by “BRUKER” to test the nature. XPS on a PHI versa probe III was performed to study the chemical composition. The Raman spectrum was recorded on a micro-Raman spectrometer (HORBIA France, LABRAM HR Evolution). A contact angle meter (HOLMARC Opto-Mechatronics) was used to measure the static contact angle.

Synthesis of N-CDs

The stem of C. quadrangularis which belongs to the family Vitaceae were was from Jawadhu hills, Tiruvannamalai district, Tamil Nadu, India and confirmed by Dr. M. Ganesan, Asst. Prof [S.G], IIISM, SRMIST. Fluorescent N-CDs from C. quadrangularis belonging to Vitaceae family (Tamil name: Pirandai) were inferred through a simple one-step hydrothermal method. Initially, 50 gm of C. quadrangularis stem was washed a few times with water to remove the impurities and made into juice with 100 mL of DI. Then, 48 mL of the extract was mixed with 2 mL of aqueous ammonia solution under continuous stirring where C. quadrangularis acts as a carbon source and ammonia solution is used as the nitrogen source. This combination was filled in a 100 mL polytetrafluoroethylene-coated stainless-steel autoclave placed in a hot air oven for 10 h at 200 °C. After the reaction is over, the autoclave was cooled to room temperature. The obtained colored solution (dark brown) reveals the development of N-CDs. To isolate any nonresponsive materials from the N-CDs, the solution was centrifuged at 4500 rpm for 20 min. The supernatant of the centrifuged part was taken out. Finally, clear and pure brown colored N-CDs were obtained and stored at 4 °C for further experiments.

Synthesis of N-CDs@MIP and N-CDs@NIP

The synthesis of N-CDs@MIP and N-CDs@NIP was based on a reported method with slight modifications.[21] In the reverse microemulsion method, momentarily, cyclohexane was used as a continuous phase, n-hexanol was used as a cosurfactant, and Triton X-100 was used as a surfactant. First and foremost, 20 mL of cyclohexane together with 4.5 mL of Triton X-100, and 3.6 mL of n-hexanol were added and stirred with a magnetic stirrer for 15 min. Then, 220 μL of TEOS and 120 μL of ammonia were added into 2 mL of the N-CDs mixture sequentially and stirred for 2 h. In the following stage, 20.4 mg of the template molecule TNP in 1 mL of n-hexanol and 220 μL of APTES functional monomer was added to the previous solution and stirred in a closed container for 11 h at room temperature. Subsequently, the breakdown of the microemulsion process was led by addition of 30 mL of acetone and stirring for 5 min. The reaction solution was then precipitated by centrifuging at 4500 rpm for 8 min. Then, N-CDs@MIP was washed using ethanol, and removal of the template molecule was performed by washing the resulting polymer with a mixture of ethanol–acetic acid (9:1, v/v) multiple times till no TNP could be distinguished by the UV–vis spectrophotometer (Figure S6). Eventually, N-CDs@MIPs were dried overnight under vacuum at 50 °C. Likewise, the N-CDs@NIPs were synthesized following a similar procedure but in the absence of the template molecule TNP similar to Scheme .

Scheme 1

Synthesis of N-CDs and N-CDs@MIP with and without TNP

Measurement of Quantum Yield

The fluorescent N-CDs quantum yield (%) was examined using eq . The selected standard reference is quinine sulfate with 54% of quantum yield because of its similar excitation and emission wavelengths to the synthesized N-CDs. Quinine sulfate with an excitation wavelength of 370 nm in 0.1 M H2SO4 and N-CDs in DI were dissolved.where and are the quantum yields for N-CDs and quinine sulphate, while and stand for fluorescence emission intensities of the reference and sample, respectively, , is the measured absorbance, and denotes the refractive index of the solvent used.[55]

Fluorescence Analysis of N-CDs@MIP for TNP

The N-CDs@MIP or N-CDs@NIP with a concentration of 7.3 μg/mL was first dispersed in DI and sonicated to form the working solution. In a 3 mL colorimetric fluorescence tube, 500 μL of water was added with 0.6 mL of working solution with phosphate buffer solution. A fluorescence spectrometer was used to measure the mixed solution fluorescence intensity at room temperature. The whole spectra were scanned to find the excitation wavelength range to evaluate the composite material fluorescence properties. Then, fluorescence spectroscopy is utilized to measure excitation and emission at different excitation (320–425 nm) wavelengths. After that, solutions of N-CDs@NIPs and N-CDs@MIPs before addition and after addition of TNP were also measured. All the fluorescence measurements were executed with the emission wavelength recorded over the range of 300–600 nm.

Selectivity of N-CDs@MIP to 2,4,6-Trinitrophenol

The selectivity of both N-CDs@MIP and N-CDs@NIP was evaluated for TNP, p-nitroaniline, m-nitroaniline, catechol, hydroquinone, and heavy metal ions such as Fe2+, Cu2+, Co2+, Ni2+, and Mn2+ions. Each was added individually to the working solutions to measure the fluorescence intensity under the same conditions as the previous section experiment.

Real Water Analysis

We hoped that our proposed fluorometric sensor (N-CDs@MIP) would be capable of detecting TNP in real water samples such as lake and tap water. The samples were collected near SRMIST Chennai, India. The collected lake and tap water samples were pretreated and filtered to remove insoluble impurities. Different concentrations of TNP were spiked with pretreated real water samples to measure the fluorescence intensity of N-CDs@MIP.