Ultrasensitive detection of Ag+ and Ce3+ ions using highly fluorescent carboxyl-functionalized carbon nitride nanoparticles

The fluorescence quenching of carboxyl-rich g-C3N4 nanoparticles was found to be selective to Ag+ and Ce3+ with a limit of detection as low as 30 pM for Ag+ ions. A solid-state thermal polycondensation reaction was used to produce g-C3N4 nanoparticles with distinct green fluorescence and high water solubility. Dynamic light scattering indicated an average nanoparticle size of 95 nm. The photoluminescence absorption and emission maxima were centered at 405 nm and 540 nm respectively which resulted in a large Stokes shift. Among different metal ion species, the carboxyl-rich g-C3N4 nanoparticles were selective to Ag+ and Ce3+ ions, as indicated by strong fluorescence quenching and a change in the fluorescence lifetime. The PL sensing of heavy metal ions followed modified Stern–Volmer kinetics, and CNNPs in the presence of Ag+/Ce3+ resulted in a higher value of K app (8.9 × 104 M−1) indicating a more efficient quenching process and stronger interaction between CNNP and mixed ions. Sensing was also demonstrated using commercial filter paper functionalized with g-C3N4 nanoparticles, enabling practical on-site applications.


Introduction
The proliferation of environmental contaminants due to largescale industrial development and urbanization has led to an urgent need to monitor potentially harmful heavy metal ions throughout the environment.Laboratory-based techniques such as atomic absorption spectroscopy, inductively coupled plasma mass spectrometry, and high-performance liquid chromatography (HPLC) are accurate, sensitive, and reliable methods to detect and identify the presence of contaminants in water samples [1,2].However, the high cost of equipment, time-consuming analysis, and intricate sample preparation limit the practicality of these methods, especially for on-site field applications.As such, there is a significant need for highly accurate and sensitive chemical sensors that are simultaneously simple to use and cost-efficient while allowing for rapid on-site detection.
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(i) electrochemical sensors [3,4], which monitor changes in electrical parameters such as voltage or impedance due to the presence of metal ions, and (ii) optical sensors [5], which depend on changes in optical properties such as color, refractive index or fluorescence emission.Recently, a third category namely electrochemiluminescence sensing which combines features of the first two, is becoming prominent [6][7][8].Fluorescence-based metal ion sensors have received considerable research interest due to their simplicity, low cost, rapid detection, and practicality.Their primary working principle involves the observation of an enhancement or quenching of fluorescence intensity after exposure to the analyte in question.Minute changes in intensity can be detected with miniaturized sensitive photodetectors while larger intensity changes can sometimes be detected by the naked eye with minimal equipment [9,10], especially when the fluorophore is hosted on filter paper [11], making it highly compatible for rapid on-site applications.
A wide variety of materials have been investigated for use as fluorescent probes, including small organic molecules [12], carbon quantum dots [13], inorganic quantum dots [14,15] and hybrid nanocomposites [16].Among these materials, graphitic carbon nitride (g-C 3 N 4 ) stands out because of its high fluorescence, compatibility with flexible substrates, stability against photobleaching, thermal resilience, and low-cost and green fabrication process [1,[16][17][18].Consequently, g-C 3 N 4 has been widely investigated as a fluorescent probe for heavy metal ions, organic species, and biomolecules [1,16,18].Despite its promising properties, conventional bulk form g-C 3 N 4 is typically unsuitable for use in the fields of chemical sensing, bioimaging, and biomedicine due to its insolubility in water, low PL quantum yield, and macroscopic particle size which results in particle sedimentation and larger polydisperse particles that are difficult to disperse uniformly in aqueous media [19].Previously, it has been reported that water-dispersible [20][21][22], nanostructured forms of carbon nitride [10,23,24] possess a higher surfaceto-volume ratio [25] which translates to improved sensitivity and detection speed due to an increase in active sites for reactions to occur and thus have been used as fluorescent nanoprobes and as photocatalysts [10,26].For instance, g-C 3 N 4 nanostructures synthesized by our group were applied as co-sensitizers for photoelectrochemical water splitting and CO 2 photoreduction [27][28][29][30].
One avenue towards increasing the water solubility of g-C 3 N 4 is surface functionalization [17] with hydrophilic groups.Several works exist that utilized carboxyl-rich carbon nitrides for catalysis [27,[31][32][33].However, there are a limited number of studies investigating its capabilities as a fluorescence-based metal ion sensor.Shiravand and coworkers developed carboxyl-rich g-C 3 N 4 through the hydrothermal oxidation of g-C 3 N 4 nanosheets [34].Using this synthesis route, they created carbon nitride with a blue fluorescence emission of 367 nm that can selectively detect Hg 2+ and Fe 3+ ions.However, they also reported that the synthesis route was energy-and time-intensive in addition to the technical challenges of poor yield and low stability [35].Additionally, the PL emission wavelengths of the nanostructures derived from bulk g-C 3 N 4 were all lower than those of their bulk due to the quantum confinement effect, and the emission peaks occurred at wavelengths shorter than 450 nm and even returned to the ultraviolet (UV) range, which is disadvantageous for bioimaging and sensing applications [36].Achieving g-C 3 N 4 nanoparticles with a long PL emission wavelength, large Stokes shift and high PL quantum yield is still challengingproblems that we have attempted to solve in this work.We report the synthesis of water-soluble, carboxylated g-C 3 N 4 nanoparticles and their use as a fluorescence-based probe for Ag + and Ce 3+ ions.The material was synthesized using a facile yet high-yield synthesis route based on a solid-state thermal polycondensation method [27].This method produces highly water-soluble g-C 3 N 4 nanoparticles that exhibit strong fluorescence emission in the green visible range.The material demonstrates high selectivity towards Ag + and Ce 3+ and can detect changes in metal ion concentrations as low as 30 pM.Furthermore, its water solubility enables easy deposition onto commercial filter paper, enabling the possibility of practical naked-eye detection [37,38].

Synthesis of carbon nitride
Carbon nitride nanoparticles (CNNPs) were synthesized by modifying previously published recipes utilizing a solid-state thermal polycondensation reaction methodology.Briefly, 0.79 g of citric acid and 1.8 g of urea were first crushed in a mortar.To conclude the thermal condensation synthesis, this solid was transferred to a 23 ml teflon-lined stainless-steel autoclave and held there for two hours at 160 °C.After thoroughly cleaning the synthesized carbon nitride using ethanol and deionized (DI) water, the finished product underwent a porous hydrophobic polytetrafluorethylene (PTFE) membrane filtration to remove the unreacted molecules and large-size granules.The processed CNNP solution was subjected to freeze drying to obtain the clean and greencolored carbon nitride powder (figure 1(a)) which was then stored for further use.

Characterization
The morphology of each sample was examined using a Zeiss Sigma field emission scanning electron microscope (FESEM) with an accelerating voltage of 5 kV.The Raman spectra were acquired, spanning the Raman shift range of 900-1800 cm −1 , using a 532 nm excitation laser and a Renishaw inVia Qontor Confocal Raman microscope.Employing a Perkin Elmer Lambda-1050 UV-vis-NIR spectrophotometer, the transmission spectra of carbon nitride in liquid suspensions were obtained.FTIR data was acquired using a Nicolet 8700 FTIR spectrometer (ThermoFisher) with a resolution of 0.09 cm −1 .Around 2-3 mg of each sample was used to prepare ∼250 mg KBr pellets by hydraulic pressing.The FTIR spectrometer was armed with a deuterated triglycine sulfate (DGTS) KBr detector, and the data was recorded in absorbance mode in the frequency range 450-4000 cm −1 .Steady-state photoluminescence spectra were recorded using a StellarNet spectrometer (SILVER-NOVA) with a UV-enhanced CCD detector.A 405 nm LED was chosen as the excitation source.X-ray diffraction (XRD) was used to determine the crystallinity and phase structure of the material.A Rigaku Ultima IV equipped with a Cu-K radiation source (40 kV, = 0.15418 nm) which includes a scintillation counter detector and a monochromator was used to record all XRD diffractograms within the 2θ range of 2°−90°.CHNS combustion analysis was carried out using an organic elemental analyzer CHNS/O (Flash 2000, Thermo) equipped with a TCD detector having a detection limit of <100 ppm.Zeta potential measurements were performed a temperature of 25 °C using a Nano-ZetaSizer instrument from Malvern Instruments.The collection of data was done in triplicate to retain statistical significance.A Leica ST8 stimulated emission depletion (STED) laser confocal microscope with a FLIM module was used to perform fluorescent lifetime imaging microscopy (FLIM).The FLIM module measures fluorescence lifetime by time-correlated singlephoton counting (TCSPC).A small amount of solubilized carbon nitride nanoparticles (pristine and mixed with Ag + ) sandwiched between a microscopic slide and glass cover slip was used for analysis.A 470 nm excitation wavelength was generated by a pulsed white light laser source using pulse frequencies of 20 and 10 MHz corresponding to pulse durations of 50 and 100 ns respectively.Based on a specified emission wavelength range, photons were detected using a Leica HyD hybrid photodetector.

Results and discussion
The carbon nitride powders synthesized using urea and citric acid had a distinctive green color in contrast with the pale yellow color of bulk unfunctionalized g-C 3 N 4 powders.According to the SEM image of the polymeric carbon nitride in figure S1, the solid agglomerates are several micrometers in size.Figures 2(b) and (c) show the well-dispersed carbon nitride suspension in DI water and its fluorescence under irradiation from a 365 nm UV black light.
The absorption spectrum of the synthesized CNNPs (figure 3(a)) showed a prominent and extended peak between 250 and 335 nm which has been linked to π-π * electronic transitions of C=C and C=N bonds of sp 2 hybridized carbons in the tri-s-triazine units [39].In response to n-π * electronic transitions of aromatic nonbonding orbitals for C=O and C=N bonds, a second broad peak develops between 365 and 430 nm [40].The distinctive features of the CNNPs produced using our method are their strongly redshifted absorption edge, which extends to 550 nm (figure 3   emission maxima is in excess of 100 nm.Significant Stokes shifts are widely favored to reduce self-absorption and light scattering in optical materials such as fluorescent probes [41], organic light-emitting diodes [42], organic lasers [43], etc.Here, we use highly fluorescent carbon nitride nanoparticles to detect the presence of heavy metal ions in aqueous media.For CNNP, the distinctive Bragg reflections of graphitic carbon nitride are noticeable in the XRD pattern (figure 3(b)).The peak at a 2θ value of 13.2°is due to reflections from the (100) plane caused by the in-plane structural encapsulating motif of tri-s-triazine units, while the peak at a 2θ value of 27.1°is due to the (002) plane resulting from the interlayer assembly of carbon nitride nanosheets along the hexagonal caxis [10,32].In addition, amorphous carbon is known to produce a broad diffraction peak at a 2θ value of 25.2°.XRD peak at 25.2°for CNNP suggests the formation of carbon-rich carbon nitride due to the existence of amorphous graphitic carbon in the carbon nitride framework [44].The incorporation of an amorphous graphitic framework presents an extension of conjugation length which results in a red shift in the absorption and PL emission.Additionally, CHNS combustion analysis (table 1) supports the carbon-rich carbon nitride system that results in a 1.93 C/N ratio as opposed to the 0.75 C/N ratio observed in bulk g-C 3 N 4 (figure S2, ESI).We believe that the carbon-rich carbon nitride structure forms through carbonization of excess −COOH groups.The FTIR spectrum for the unadulterated CNNP sample is displayed in figure 3(c).The stretching feature of the C=O (carbonyl bond) seen in the −COOH groups of carbon nitride is responsible for the peak at c.a. 1705 cm −1 .The C=N stretch, C−N stretch, and triazine ring (C 3 N 3 ) deformation have been identified as the causes of the peaks around 1615, 1465, and 1400-980 cm −1 respectively [45,46].Raman spectroscopy confirmed the synthesis of a layered graphitic framework and provided insight into the structure of the material [17,27].The Raman spectra of carboxylated carbon nitride displayed two prominent characteristic peaks at ∼1340 cm −1 and ∼1560 cm −1 , corresponding to the D band and graphitic G band (figure 3(d)) which are characteristic of the sp 2 hybridized framework.The D band is caused by sp 3 carbon defects, whereas the G band is the result of the in-plane vibration of sp 2 carbons in graphitic carbon nitride [47].
Using the DLS technique, the average particle size distribution value for the CNNPs in DI water suspension was found to be 95 nm (figure 4(a)).CNNPs in the DI water were found to have an average zeta potential value of −15 mV (figure 4(b)).An exceptionally durable colloidal suspension is indicated by such a strong negative surface charge [48].Such characteristics minimize the variance in PL intensity and lifetime by lowering the aggregation and sedimentation of particles suspended in the solvent [49,50].Additionally, the CNNP exhibited outstanding photostability.No evident photobleaching was seen after 20 min of continuous photoexcitation with UV light (365 nm irradiation) (figure 4(c)), which is a testament to the resilience of CNNP-based sensors.Furthermore, the PL intensity of CNNP was found to slightly decrease as the temperature was increased up to 50 °C.Thermal energy facilitates non-radiative relaxation mechanisms [51] such as vibrational relaxation or energy transfer to quenching species.When the non-radiative processes become more dominant, it reduces the efficiency of radiative recombination and leads to a drop in fluorescence intensity.Thermally induced defect states in carbon nitride also have the potential to function as more effective non-radiative recombination channels and lower the intensity of photoluminescence.Once the solution had cooled to room temperature, the PL intensity instantly returned to its normal level.

Metal ion fluorescence sensing
The ability of CNNPs to detect a broad spectrum of metal ions was investigated.Synthesized CNNPs were dissolved in DI water at a concentration of 50 μg ml −1 .Metal ion stock solutions were made with the necessary concentrations of Ag, Al, Ca, Cd, Ce, Co, Cu, Eu, Mg, Na, Pb, Sr, Y, and Zn salts.
To achieve the desired ion concentration in the final volume of the combined solution, the correct volume of analytes was added to the CNNP solution.To obtain the highest emission intensity, the final reaction mixture was thoroughly mixed and held at room temperature for 15 min.Following that, the solution was transferred to a plastic cuvette, and PL spectra were collected.Figure 5 shows the PL response of the CNNP solution in the presence of various metal ions.It can be seen that all tested metal ions produce a certain degree of PL quenching, likely owing to interactions between the strongly negative surface charge and positively charged metal ions.However, the most significant quenching is produced by the Ag + -containing solutions (>25% reduction in PL intensity) and Ce 3+ -containing solutions (∼ 10% reduction in PL intensity).As such, we can conclude that the CNNP is most selective to Ag + and Ce 3+ ions.
In view of the strong PL quenching, broad photoexcitation, and large Stokes shift, we developed CNNP-based sensors for the luminescence-based detection of Ag + and Ce 3+ .To adhere CNNP to the cellulose fibers of the filter paper, it was first submerged in the CNNP-DI solution.Following the addition of a high concentration of Ag + , the pigmentation of the test zones in the filter paper changed to a light brown shade, and they fluoresced when exposed to UV light (365 nm irradiation, as shown in figure 6).In this filter paper-based configuration, 30 μM acted as the limit of detection, as below that concentration visual changes due to quenching becomes harder to observe.Our sensing probe has the benefit of being more affordable due to its usage of standard filter paper as the substrate [11,38].

Sensing mechanism
According to the zeta potential measurement, CNNPs have negatively charged surfaces.This results in a strong electrostatic attraction between CNNPs and positively charged metal ions, which when present in close proximity, produces a donor-acceptor pair that likely quenches the fluorescence of CNNPs through partial or complete electron transfer to Ag + ions, which are well-known electron scavengers.The other possibility which consists of the CNNP exciton being dissociated by Ag + ions, is less likely owing to the high exciton binding energy in carbon nitride.Likewise, Ce 3+ ions are mild hole scavengers which are oxidized to Ce 4+ ions by capture of a single hole.
To assess the effectiveness and sensitivity of CNNPs for metal ion detection, various Ag + and Ce 3+ concentrations were tested (figure 7).The CNNPs demonstrated significant selectivity for Ag and Ce ions because of the more potent complex formation with surface functional groups.Adsorption is encouraged close to CNNPs' negatively charged surface because Ag and Ce ions are cationic species.An electrostatic attraction exists among Ag + /Ce 3+ ions and carbon nitride nanoparticles.As the concentrations of Ag + and Ce 3+ ions increase, the photoluminescence intensity decreases rapidly.
Figure 7 demonstrates the facile detection of Ag + metal ions down to a concentration of 30 pM by the CNNP-based fluorescent probe sensor.Very little quenching was seen for the CNNP solution when a 30 pM Ce 3+ quencher was introduced, while 30 nM Ce 3+ ions resulted in noticable quenching of the PL intensity.As the concentration of Ag + ions increased to roughly 30 mM, the photoluminescence spectrum for CNNP solution was slightly red-shifted (∼7 nm), but the PL spectrum blue-shifted (∼3 nm) for Ce 3+ ions.The small red-shift in the CNNP absorption upon interacting with Ag + ions is consistent with the formation of a ground-state complex [24].The Ce 3+ induced blue-shift in the optical absorption is attributed to a small decrease in conjugation length either due to a change in aggregation of the CNNPs or due to a greater intra-nanoparticle localization of photoexcited charge.As evident in figure S4 and table S1 in supporting information, the time-resolved PL of CNNPs has two components-a short duration 1.06 ns lifetime component and an intermediate duration 4.38 ns lifetime component.These two components are attributed to direct radiative recombination of electron-hole pairs and intersystem crossing-mediated radiative decay of electrons from antibonding π * to lone pair (LP) orbitals respectively [52,53].The longer duration component attributed to inter-sheets recombination is absent for CNNPs formed in this study.Exposure to Ag + ions produces a drastic decrease (approximate halving) of the shorter lifetime component from 1.06 to 0.58 ns while no specific trend is observed for the longer-  lived lifetime component (compared to CNNPs) (table S1 in supporting information).Ce 3+ ions produce a roughly 0.3 ns decrease in the lifetimes of both the short and longer-lived lifetime components.The overall lifetime of CNNPs exposed to Cu 2+ ions is the shortest among the three ions that exhibit significant quenching of the CNNP photoluminescence.Such a specific change in the time-resolved PL of CNNPs due to various metal ions could itself be a source of selective identification.
In figure 8, the fluorescence of the CNNP solution gradually decreases with increase in concentration of the Ag + and Ce 3+ ion solutions.The insets in figure 8   the initial fluorescence intensity, (I) is the fluorescence intensity of CNNP solution with diverse concentrations of quencher, and (K app ) is the apparent Stern-Volmer constant [M-1], and this is the crucial parameter that indicates the sensing capability of a fluorescent sensor device.Here, quencher constant K D = K q × τ, and K S = [I−M]/[I][M], where K q is the bimolecular constant and τ is the average lifetime of the fluorophore molecule before quenching.K q exhibits the efficiency of quenching, for dynamic quenching, the maximum scattering collision quenching constant of various quenchers is ∼10 10 Lmol −1 s −1 for carbon nitride [62].From the slope, the K app has been assessed and the value is 46.37 × 10 3 M −1 for CNNP_Ag + , 29.6 × 10 3 M −1 for CNNP_Ce 3+ , and 89 × 10 3 M −1 for CNNP_Ag + /Ce 3+ , respectively.The higher value of K app for the CNNP_Ag + /Ce 3+ system indicates that the quenching process is more efficient in mixed media and there is stronger interaction between the CNNP and the Ag + /Ce 3+ .

Conclusion
Water-soluble carboxyl-rich carbon nitride nanoparticles were used as fluorescent probes to selectively detect Ag + and Ce 3+ ions in aqueous media.Using a solid-state thermal polycondensation method, we synthesized graphitic carbon nitride nanoparticles with an average size of 95 nm and a green fluorescence emission (λ max = 540 nm).The carboxyl-rich nature of the g-C 3 N 4 nanoparticles was confirmed through CHNS combustion analysis, Raman spectroscopy and FTIR analysis.The fluorescence of carboxyl-rich g-C 3 N 4 nanoparticles is selective to Ag + and Ce 3+ ions in aqueous media and can be used to detect Ag + concentrations as low as 30 pM and Ce 3+ concentrations as low as 30 nM.Ag + appears to quench the emission of carbon nitride nanoparticles through the creation of a ground-state complex.The strong fluorescent quenching using g-C 3 N 4 -functionalized commercial test paper demonstrates the potential for rapid, on-site detection of heavy metal ions.

Figure 1 .
Figure 1.Schematic illustration of highly fluorescent carbon nitride nanoparticles as fluorosensor.
(a)), and their green emission peak, which is located at approximately 540 nm.The effective Stokes shift between the absorption and

Figure 2 .
Figure 2. (a) Macroscopic image of green carboxylated carbon nitride powder (b) Well-dispersed carbon nitride suspension in water and (c) green fluorescent carbon nitride suspension in water excited by a 365 nm UV lamp.

Figure 3 .
Figure 3. Clockwise from top left (a) Absorption and emission spectra of CNNP in DI water.The excitation wavelength was set to 405 nm (b) XRD pattern (c) FTIR spectrum of CNNP and (d) Raman spectrum of CNNP.

Figure 4 .
Figure 4. (a) Dynamic light scattering spectra, (b) Zeta potential of carbon nitride nanoparticles, and (c) effect of continuous UV irradiation for 20 min, and heating (20 °C-50 °C) environments (inset) on the fluorescence intensity of CNNP.

Figure 5 .
Figure 5. Exclusive PL sensitivity of carbon nitride nanoparticles following exposure with (a) and (b) 1 μM metal ion solutions and (c) 1 μM of other metal ions the influence with 1 μM Ag + .The error bar describes the standard deviation of three experiments for each interfering species.

Figure 6 .
Figure 6.Macroscopic images of the test paper with different concentrations of Ag + ions under (a) UV light irradiation (365 nm) and (b) Ambient lighting.

Figure 7 .
Figure 7. Quenching of luminescence intensity of CNNP fluorophore solution in the presence of a different concentration of (a) Ag + ions, (b) Ce 3+ ions.
displays the intensity ratios (I 0 /I) of the untreated CNNP and quenched fluorescence (I) versus quencher concentration.The classic Stern-Volmer equation, I 0 /I = 1 + K SV [M] predicts a linear dependence of I 0 /I on the concentration of the quencher, if the quenching constant is a true constant [54].If the rate constant for the quenching reaction is dependent on the quencher, then a nonlinear Stern-Volmer plot is obtained.It has been known for many years that certain quenching reactions lead to curved Stern-Volmer plots [55].Both positive curvature and negative curvature have been observed [56-58].Here, we are observing negative curvature Stern-Volmer plots for all three tests, and is associated with the change in absorption and fluorescence spectrum of the fluorophore (blue shift and red shift) [59].Deviation from the Stern-Volmer equation in such reactions is explained by the existence of multiple fluorescing states or by compound formation [60].The resulting complex can have its own unique properties, such as being non-fluorescent and having a unique absorption spectrum.The fluorescence of the sample is reduced since the quencher is essentially reducing the number of fluorophores which can emit photons [57].The condition of heterogeneous population of fluorophores also results in a negative deviation from linearity [61].The modified form of the Stern-Volmer equation arises when both static and dynamic quenching (association constants K S and K D ) occur for the same fluorophore and is given as I 0 /I = 1 + K app [M], where K app = (K D + K S ) + K D K S [M] = (I 0 /I) − 1 [57].Here, [M] is the molar concentration of the quencher, (I 0 ) is

Figure 8 .
Figure 8.Effect of (a) Ag + (b) Ce 3+ , and (c) mixed ion concentrations on the fluorescence spectrum of CNNP.Insets show the PL intensity ratio of CNNP versus quencher concentration.The error bars denote the standard deviation of three experiments.(d) The concentration of metal ions ranging from 10 to 50 μM and their visual color effects are displayed.